The Role of Sumoylation in Early Development of Xenopus Laevis And

THE ROLE OF SUMOYLATION IN EARLY DEVELOPMENT OF XENOPUS LAEVIS AND

REGULATION OF 5S RIBOSOMAL RNA GENES

A Dissertation

Submitted to the Graduate School

of the University of Notre Dame

in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

Michelle M. Bertke

Paul W. Huber, Director

Graduate Program in Chemistry and Biochemistry

Notre Dame, Indiana

April 2014

THE ROLE OF SUMOYLATION IN EARLY DEVELOPMENT OF XENOPUS LAEVIS AND

REGULATION OF 5S RIBOSOMAL RNA GENES

Abstract

Michelle M. Bertke

The 5S rRNA gene-specific transcription factor, TFIIIA, interacts with the SUMO

E3 ligase, PIAS2b, and with one of its targets, the transcriptional corepressor XCtBP.

PIAS2b and XCtBP are present on the oocyte, but not somatic, 5S rRNA genes up through the gastrula-neurula transition, as is a limiting amount of TFIIIA. Histone H3 methylation, coincident with the binding of XCtBP, also occurs exclusively on the oocyte genes. Immunohistochemical staining of embryos confirms occupancy of some fraction of the oocyte genes by TFIIIA that become positioned at the nuclear periphery shortly after the midblastula transition. SUMOylation can be inhibited through injection of mRNA encoding the adenovirus protein Gam1, which decreases the levels of the E1 activating enzyme by triggering its proteolytic degradation. Gam1-induced decrease in

SUMOylation activity relieves repression of the oocyte 5S rRNA genes and is correlated with a decrease in methylation of H3K9 and H3K27. These results reveal a novel

Michelle M. Bertke function for TFIIIA as a negative regulator that recruits histone modification activity, through the CtBP repressor complex, exclusively to the oocyte 5S rRNA genes, resulting in their terminal repression.

SUMOylation deficient embryos also exhibit a range of important developmental defects including failure of the blastopore and neural tube to close, shortened axis, fused eyes, and perturbed heart development. Embryos injected with Gam1 mRNA or water (control) were taken for microarray analysis at three developmental time points: early gastrula, late gastrula, and early neurula. A bioinformatics analysis of this data was conducted using the MetaCore® suite of programs, BiNGO, DAVID, and the Gene

Ontology database. Functional enrichment analysis of the differentially expressed genes demonstrates that SUMOylation regulates the expression of genes that span several different biological processes during early embryogenesis. Bioinformatics analysis provides evidence that, in some cases, SUMOylation generates two pools of a given transcription factor that control different subsets of genes. Although SUMOylation impacts a large variety of processes, certain signaling pathways appear to be particularly sensitive to the loss of this modification and can account for the observed phenotypes.

Pathways enriched for differentially expressed genes were identified using the extensive

MetaCore® database and include; non-canonical Wnt signaling and regulation of cytoskeleton remodeling (shortened axis and open blastopore), regulation by Yin Yang 1

(heart defects), Twist/Snail regulation of the epithelial to mesenchymal transition (open blastopore and neural tube), and Ets-1 regulation of transcription factors E2F1/E2F4

(heart defects and open blastopore).

To my husband Jeffery for believing in me particularly when I did not.

ii CONTENTS

Figures ...... i v

Tables ...... vi

Acknowledgments...... viii

Introduction ...... 1

Materials and Methods ...... 20

Results: Regulation of the 5S Ribosomal RNA Genes ...... 47

Discussion: Regulation of the 5S Ribosomal RNA Genes ...... 68

Results and Discussion: Role of SUMOylation in Early Development of Xenopus laevis. 77

Appendix A ...... 190

References ...... 293

iii

FIGURES

Figure 1. An internal view of the early stages of Xenopus laevis development ...... 7

Figure 2. Conjugation of SUMO protein to a target...... 15

Figure 3. Procedure for Keller sandwich explants...... 36

Figure 4. Diagram of nucleus isolation for ChIP assays...... 39

Figure 5. Depletion of SUMOylation activity prevents repression of oocyte 5S rRNA genes...... 49

Figure 6. TFIIIA levels in Gam1-injected embryos...... 51

Figure 7. Expression of Gam1 in embryos eliminates E1 enzyme activity...... 53

Figure 8. SUMOylation of TFIIIA...... 56

Figure 9. Occupancy of the 5S rRNA genes during early development...... 61

Figure 10. Localization of TFIIIA to the nuclear periphery...... 66

Figure 11. Developmental defects of Gam1-injected embryos...... 81

Figure 12. Volcano plots and comparative histogram of genes measured on microarray.87

Figure 13. Comparison of biological replicates from Gam1 and H2O injected embryos. . 91

Figure 14. Comparison of microarray and qRT-PCR data ...... 96

Figure 15. Functional enrichment analysis...... 138

Figure 16. Heat maps of differentially expressed genes for three developmental time points...... 141

Figure 17. Syn-expression cluster analysis of differentially expressed genes...... 147

Figure 18. SUMO regulation of Wnt pathway leading to defects in cytoskeleton remodeling...... 156

Figure 19. SUMO regulation of Twist/Snail which controls the epithelial to mesenchymal transition...... 169

Figure 20. SUMO regulation of Ets-1 and control of gastrulation and heart development...... 175

Figure 21. Gene expression levels (log10) from microarray data compared to previously measured mRNA levels for genes regulated by Ets-1...... 182

Figure 22. Gam1 disrupts convergence and extension in Keller sandwich explants...... 187

TABLES

Table 1. Developmental stages of X. laevis from early blastula to late neurula...... 5

Table 2. Forward and reverse primers used for qRT-PCR analysis ...... 29

Table 3. Top 30 transcription factors which regulate the largest number of differentially expressed genes at early gastrula along with their associated gene ontology processes...... 102

Table 4. Top 30 transcription factors which regulate the largest number of differentially expressed genes at late gastrula along with their associated gene ontology processes...... 107

Table 5. Top 30 transcription factors which regulate the largest number of differentially expressed genes at early neurula along with their associated gene ontology processes...... 113

Table 6. Top 30 transcription factors which regulate the largest number of non- differentially expressed genes at early gastrula along with their associated gene ontology processes...... 118

Table 7. Top 30 transcription factors which regulate the largest number of non- differentially expressed genes at late gastrula along with their associated gene ontology processes...... 122

Table 8. Top 30 transcription factors which regulate the largest number of non- differentially expressed genes at early neurula along with their associated gene ontology processes...... 126

Table 9. Compiled list of transcription factors, identified to regulate differentially expressed genes from all three time points, analyzed for potential consensus and non-consensus SUMOylation sites...... 134

Table 10. Top transcription factors controlling the largest number of genes from each of the eight syn-expression cluster groups...... 148

Table 11. Process enrichment analysis for each of the eight syn-expression cluster groups performed with MetaCoreTM and BiNGO...... 152

Table 12. Genes from the Wnt and frizzled (Fzd) family down regulated (percentage) at one or more of the time points in development...... 158

Table 13. Differentially expressed genes (up-regulated or down-regulated) controlled by the transcription factor YY1 at each of the three time points...... 167

Table 14. Differentially expressed genes regulated either directly or indirectly by Ets-1.177

Table A.1 Genes differentially expressed (p<0.05) between Gam1 mRNA and H2O injected embryos at early gastrula...... 191

Table A.2 Genes differentially expressed (p<0.05) between Gam1 mrna and H2O injected embryos at late gastrula...... 198

Table A.3 Genes differentially expressed (p<0.05) between Gam1 mrna and H2O injected embryos at early neurula...... 234

vii

ACKNOWLEDGMENTS

First and foremost, I would like to thank my boss Dr. Paul Huber for all of his advice and support during my graduate school career. I feel incredibly grateful to have worked under such an amazing and knowledgeable advisor. My experience at Notre

Dame has been wonderful at times and trying at times. Paul has helped me through it all, guiding me in my scientific and personal growth and I can only begin to thank him for that. I would like to also thank Dr. Patricia Clark, Dr. Holly Goodson, and Dr. Robert

Schulz for serving on my committee and for all their scientific expertise over the years.

Thank you to the Freimann Life Science Center for all their help taking care of the frog colony and their technical advice. Additional thanks to the Genomics and Bioinformatics

Core, specifically Dr. Erliang Zeng and Dr. John Tan, for their invaluable assistance with the microarray data analysis.

I could not have made it though my years at Notre Dame without the help from everyone on the 4th floor of Stepan Chemistry Hall, specifically all the past and present members of the Huber lab. Thank you all for your scientific knowledge and many coffee breaks which kept me sane. Additional thanks go to the Henderson group for making me an honorary member, allowing me to use their group room, and for many fun content hours. Many thanks to Dr. Allen Oliver for his support, his advice, and passing on his knowledge and his whiskey. To all the past and present graduate student

viii

members of the Chem Demo Team, along with Sarah West and Karen Morris, thank you for helping me to find my true passion and for bringing the joy of science to hundreds of kids.

I would not be where I am today without the support of my parents, Marlene and Michael, who always made sure I knew I could do anything and to Matt and

Meredith for always showing me who I would like to be when I grow up. Additionally, to my second family Barb, Nick, and Jenn Dixon for always checking up on me and welcoming me home. Thank you to my married family of the Bertkes and the Osterfelds for being the best in-laws a person could ask for. I must thank my South Bend family of the Bells and Banets, specifically Angie, Aaron, and Sophia, for opening their home and their hearts to me and Mr. and Mrs. B for many amazing tailgates and Oasis bus trips.

Last but not least, thank you to my wonderful husband Jeffery, the original Dr.

Bertke. Thank you for your continuous support, advice, and reassurance throughout my graduate school career and beyond.

INTRODUCTION

Xenopus laevis provides an exceptionally useful model organism for studying early embryonic development. The fate maps of X. laevis are well established as are signaling pathways associated with known developmental processes. Additionally, one cell embryos are approximately 1 mm in diameter and can be easily manipulated through microinjection and microsurgeries. These features allow for specific and directed manipulation of early developmental changes.

The developmental regulation of the X. laevis 5S ribosomal RNA (rRNA) genes has long been used as a model for differential transcription control. Studies of these genes have provided the first evidence for the active role of chromatin in gene expression (1). There are two major families that encode for 5S rRNA, the oocyte and the somatic (2, 3). Understanding the mechanism controlling the expression of the somatic 5S gene over the oocyte 5S gene has provided insight into gene regulation as a whole. During the gastrula neurula transition in development, the oocyte genes are permanently repressed and only the somatic remain actively transcribed. The permanent repression is attributed to positioning of nucleosomes denying access of the transcription factor complex (TFIIIA, TFIIIB, and TFIIIC) to the promoter of the oocyte but not the somatic 5S rRNA genes (4–6).

SUMOylation is the process of attaching an 11 kDa polypeptide (Small Ubiquitin like Modifier) to a target protein (7, 8). This post-translational modification can alter many aspects of a protein including stability, protein-protein interactions, localization, and DNA binding (9–11). Transcription factors are a major target of SUMO modification and conjugation to SUMO is used to regulate genes at different points in development and in different tissues. SUMOylation controls various aspects of development in many model systems. Knockdowns of SUMOylation pathway components in zebrafish and mice lead to embryonic lethality in some studies (12–15), while others observed no defects in SUMO-1 deficient mice and suggested that SUMO isoforms had the ability to compensate for one another (16, 17). Additionally, knockdowns in Drosophila lead to defects in metamorphosis and knockdowns in S. cerevisiae leads to cell cycle arrest at

G2/M (18, 19). Experiments in Xenopus have shown that embryos deficient in SUMO-1 have defective neural tubes and shortened axis (20).

Previous studies in the laboratory have shown that TFIIIA is phosphorylated on serine-16 and this modification can account for the decreased expression of the oocyte- type gene that occurs at the MBT (21). However, phosphorylation of TFIIIA did not affect its binding to either promoter indicating it function is most likely to alter interactions with additional proteins. In order to investigate this, a yeast-two hybrid was carried out using TFIIIA as bait and identified PIAS2b, a SUMO ligase, and CtBP, a co- repressor regulated by SUMOylation as candidates.

The goal of this project was to understand the role of SUMOylation in developing

Xenopus embryos, specifically focusing on regulation of the 5S rRNA genes. This work 2

sought to identify the role of SUMOylation in the initial repression of the oocyte type 5S rRNA genes thus leading to permanent repression in late stage embryos and somatic cells along with studying the role of SUMOylation in global regulation of early Xenopus genes.

Early development in Xenopus laevis: Fertilization through neurulation

Patterning of the frog embryo begins before fertilization during oocyte maturation. At this time several factors are localized to the animal (Zic2, Xgrhl1) or vegetal (Vg1, VegT, Wnt11) regions of the oocyte (22). This maternal inheritance and localization is important for proper germ layer formation prior to the mid blastula transition since there is no zygotic transcription taking place in the embryo during this period of rapid cell division.

At the time of fertilization, sperm entry causes a rotation of the cortex so that the sperm entry point is roughly opposite of the future dorsal side of the now fertilized embryo (23). The dorsal and ventral positioning of the embryo has a rough correlation to the pigmentation observed but is not absolute. Generally, the future dorsal side of the embryo is lighter in color (vegetal) and the future ventral side is darker (animal) (24,

25).

The first Xenopus laevis embryonic cell division occurs 1.5 hours post fertilization

(hpf) and bisects the vegetal and animal pole. Subsequent cell divisions occur at

approximately 0.5 hour intervals and occur synchronously until cycle 13 (mid-blastula transition- MBT, 6 hpf) when gap phases are introduced and the cell cycle extends to 50 minutes (26). At this point the cells of the animal hemisphere begin to divide more rapidly than the cells of the vegetal hemisphere creating a difference in size with smaller cells forming at the former and larger cells at the latter. During the MBT zygotic transcription is activated and the embryo no longer relies on its maternal transcripts for protein production (27–29).

About 9 hpf, the embryo begins gastrulation during which cell movement and several patterning events occur (Table 1). The embryo can now be roughly divided into three regions; the animal cap, which is fated to become ectodermal tissue, the marginal zone which is prospective mesoderm and neural tissue, and the vegetal mass which will become the endoderm (30, 31). The marginal zone can be further divided into non- involuting marginal zone (NIMZ) and involuting marginal zone (IMZ) based on the degree of cell migration. The first observable evidence of gastrulation is the contraction of a subset of cells, termed the bottle cells, at the vegetal pole of the embryo. The contraction of these cells forms a densely pigmented area, the blastopore lip, which is where invagination of the first cells will take place (Fig. 1) (32). The cells migrate inward losing connections with the outer layer of epithelial cells and travel along the inside of the embryo through connections with the blastocoel roof. The IMZ forms the prospective roof and floor of the archenteron, eventually fusing into a tube. At the same time that the cells are migrating inward, the outer layer of cells are

TABLE 1

DEVELOPMENTAL STAGES OF X. LAEVIS FROM EARLY BLASTULA TO LATE NEURULA.

undergoing epiboly, elongating and spreading out across the embryo forming the future ectoderm (33).

The convergence and extension process is the driving force of embryonic patterning and is responsible for proper positioning of the germ layers and tissues in the adult frog (34). The NIMZ converges towards the end of gastrulation, closing the blastopore. The NIMZ continues to converge toward the dorsal midline and extends in the anterior-posterior direction forming the neural folds and later the neural tube.

Additional convergence and extension occurs with the IMZ moving toward the dorsal midline and extending in the anterior-posterior direction forming the prospective notochord and somites, though the mechanisms of the two are thought to differ (32).

At the end of gastrulation, cells of the three germ layers, ectoderm, endoderm, and mesoderm, have migrated to their positions and neurulation begins (Fig. 1) (34). As mentioned above, cells of neural folds converge toward the dorsal axis of the embryo to form the neural tube and future spinal cord. The anterior-posterior axis of the embryo also begins to elongate and the embryo begins to grow in size. Up until this point, the embryo has remained roughly 1 mm in diameter only slightly increasing in size throughout gastrulation.

Proper coordinated cellular movements early on are critical to the development of the embryo. Any defects in early development will be propagated and result in severe phenotypes. For example, failure of the blastopore to close during gastrulation leads to a failure of the neural tube to close and a shortened axis phenotype (35).

Figure 1. An internal view of the early stages of Xenopus laevis development. The blastula, stage 8, is characterized by the presence of the fluid filled blastocoel. As early gastrula, stage 10, cell migration begins and the blastopore lip forms. During gastrulation, the mesoderm and endoderm cells are moved into the interior of the embryo and the ectoderm covers the exterior. At the neurula stages, 14-24, the embryo begins to elongate and the notochord along with the neural tube is formed. These structures are the earliest components of the central nervous system of the tadpoles. Images adapted from Principles of Development by Lewis Wolpert and Cheryll Tickle, 4th edition, Oxford University Press, 125-128.

Additionally, improper involution of the presumptive endoderm and mesoderm leads to defects in the circulatory and digestive system in later stage tadpoles.

Many pathways controlling left–right, dorsal-ventral, and anterior-posterior patterning have been determined. Patterning begins during oocyte formation and many gradients of signaling molecules such as bone morphogenic protein (BMP), fibroblast growth factor (FGF), and Wnt ligands are established in this early stage. BMPs are members of the transforming growth factor beta (TGFβ) super family of signaling molecules and are essential in dorsal-ventral patterning of mesodermal cells. Over- expression of BMP4 in developing embryos leads to ventralized embryos lacking dorsal structures such as a notochord and neural tube. Conversely, inhibition of BMP4 causes dorsalization of embryos. Therefore, it is evident that a balance must be maintained between BMP4 expression and inhibition in order to properly induce mesodermal cells

(36–38). In the case of neural cells, BMP4 inhibition alone is not sufficient to induce cell fate, other signaling involving FGF must be active (39). TGFβ signaling is also important for cell cycle control as the embryo enters gastrulation and the length of the cell cycle increases. This cell cycle control introduces gap phases during gastrulation and beyond, which provide the embryo vital time to carry out proper patterning.

In addition to directing cell fate, signaling molecules are important for directing cell movements and proper placement of the germ layers. Specifically, platelet derived growth factor alpha (PDGFα) and its receptor PDGFαR are expressed on the roof of the blastocoel and the involuting mesoderm. This interaction is an important force for convergence and extension and is necessary for directing those cells fated to be 8

mesoderm toward the interior of the embryo. Additionally, knockdowns of these proteins cause failure of blastopore and neural tube to close, a common phenotype when patterning is disrupted in early development (40, 41).

Differential regulation of the oocyte and somatic 5S ribosomal RNA genes

5S rRNA is a structural component of the eukaryotic ribosome and in Xenopus laevis is coded by two major multigene families along with several trace and pseudogenes (2, 3). There are 20,000 copies of the major oocyte per haploid genome and are expressed throughout oogenesis and briefly following the mid-blastula transition. They are found in tandem repeats of approximately 1,000 copies located near the telomere of all chromosomes. The somatic-type genes are expressed throughout development and are located in a single cluster of 400 copies on chromosome 9 (42, 43).

Transcription of the 5S rRNA genes by RNA polymerase III requires the formation of an initiation complex comprised of TFIIIA, TFIIIB, and TFIIIC. TFIIIA is recruited to the promoter first and binds with equal affinity to both promoters. TFIIIA is solely dedicated to the transcription of the 5S rRNA genes and is extremely abundant in oocytes with approximately 1012 molecules per cell (44–46). 5S rRNA is mass produced during this period and stored in the cytoplasm in an RNP storage particle with TFIIIA (47). By the end of oogenesis 98% of the total pool of TFIIIA is in this RNP complex in the cytoplasm.

Although levels of TFIIIA decrease at the end of oogenesis there are still 107 molecules per cell at the MBT, representing an approximately 100 fold excess of the factor over the number of 5S genes (46, 48). By the gastrula neurula transition the oocyte gene is permanently repressed and the only 5S rRNA present is transcribed from the somatic gene (1).

The promoters of the oocyte and somatic genes only differ in six nucleotides and bind the same set of transcription factors. Because of this excess and equal affinity of

TFIIIA for each promoter (49), decreasing levels of the transcription factor cannot fully explain the differentially expression of these two genes (50). The transcriptional advantage of the somatic gene is attributed to this six nucleotide difference as it promotes increased recruitment of TFIIIC and a more stable transcription complex (51–

53). Binding of TFIIIA is necessary to recruit TFIIIC, the next factor to bind, which forms a stable transcription complex and recruits TFIIIB (54). TFIIIB is itself a complex composed of TATA-binding proteins (TBP) and TBP associated factors. The complete transcription complex is necessary for RNA polymerase III recruitment (55).

Nucleosomes are DNA packaging structures composed of two copies of four core histone proteins, H2A, H2B, H3, and H4, around which the DNA is wrapped twice. H1 linker histones bind between the nucleosomes in order to fold the DNA into 30nm chromatin fibers (56). The final repressed state of the oocyte-type genes occurs during the period in which the maternally inherited histone H1 variant, H1M, is replaced by the adult H1A subtype (57–59). There is substantial evidence that H1A-dependent positioning of nucleosomes in somatic cells ultimately determines the differential 10

expression of the two types of 5S rRNA genes (5, 6, 59–65). In the case of the somatic

5S rRNA genes, H1A binds to the 5' side of the nucleosome, leaving much of the internal promoter sequence exposed; in contrast, H1A binds to the 3' side of the nucleosome positioned on the oocyte-type genes, preventing TFIIIA from binding (5, 63, 64). This distinct arrangement of nucleosomes relies on the sequences that flank the two types of genes, which are A:T-rich in the case of the oocyte genes and G:C-rich in the case of the somatic genes (62, 65). Core histone modifications such as phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination can alter the accessibility of promoters and act as repressive markers (66). Specifically, hyperacetylated lysines on

H3 and H4 are common markers for actively transcribed genes while hypermethylated lysines are associated with repressed genes (67, 68). While there is a detectable difference in the acetylation of histone H4 associated with somatic (hyperacetylated) compared with oocyte (hypoacetylated) 5S genes in adult (kidney) cells, this modification does not appear to influence the binding of TFIIIA (5). Therefore, histone modifications may sustain rather than establish the disparate activity of the two gene families in somatic cells.

TFIIIA is phosphorylated on serine 16 by casein kinase 2 (CK2) beginning at oogenesis and is stimulated by progesterone-induced maturation of the oocyte to an egg (21, 69). A mutant form of TFIIIA that mimics constitutive phosphorylation (S16E) has been shown to repress oocyte 5S rRNA transcription, but supports somatic-type 5S rRNA transcription. Previous experiments with template exclusion assays demonstrated that transcription complexes containing the S16E variant can form on the oocyte-type 11

genes, but are inactive. Thus, this post-translational modification of TFIIIA can account for the decreased expression of the oocyte- relative to the somatic-type genes that is measured when zygotic transcription begins at the MBT and provides a mechanism for initial repression of the former when levels of TFIIIA are still high (70).

Phosphorylation of TFIIIA may disrupt protein-protein interactions with other factors in the polymerase III transcription complex as serine-16 is in the β-strand of the first zinc finger and is exposed in the DNA bound conformation and mutations at this position do not affect DNA or RNA binding affinity (71, 72). To test this directly, a yeast two-hybrid screen of a Xenopus cDNA library was carried out using the first four fingers of TFIIIA as bait. This screen identified PIAS2b, a SUMO ligase, and XCtBP, a transcriptional co-repressor regulated by SUMOylation, as candidates and were confirmed using pull-down assays. Preliminary chromatin immunoprecipitation (ChIP) assays detected the presence of these proteins as well as SUMO-1 and SUMO-2/3 exclusively on the oocyte-type genes. Additionally, the ChIP assay revealed that some amount of TFIIIA remains associated with the oocyte 5S rRNA genes throughout development, indicating an active role in establishing the repressed state of these genes. Immunohistochemical staining of embryos with anti-TFIIIA antibody reveal a pattern around the nuclear periphery that corresponds to the observed subnuclear arrangement of telomeres in somatic Xenopus cells (43). This provides further evidence that TFIIIA is not entirely displaced from this gene family and ultimately acts as a negative regulator of oocyte-type 5S rRNA gene transcription.

Small ubiquitin like modifier in Xenopus laevis development

Unlike ubiquitin, which solely marks a protein for degradation, SUMO has a plethora of effects on its target protein including stability, localization, protein-protein interactions, and DNA binding activity (9–11). Transcription factors are a major target of

SUMO modification (73). It was once thought that SUMO modification was responsible for repressing the activity of transcription factors, however recent evidence points to a much more varied role for SUMOylation. In some cases, contradictory evidence has been found for certain transcription factors leading to the idea that SUMO attachment can either function to repress or activate a specific transcription factor depending on the tissue type or time point in development (74, 75). Initially, SUMOylation of the

SMAD4 transcription factor was shown to stabilize the protein and increase transcriptional activity (76). However, additional studies indicated that SUMOylation had a repressive effect on certain target genes (77). This one example (out of many) illustrates the difficulty in assigning a general and global role for SUMOylation in all tissue types throughout development. Additionally, only a small percentage of a protein is SUMOylated at any given time, often leading to difficulties in detecting the

SUMOylated form, and supporting the hypothesis that different pools of transcription factors exist utilizing SUMO as a developmental switch.

The gene for SUMO (SMT3) was first discovered in Saccharomyces cerevisiae in

1995 (78). Since that initial report, genes encoding for SUMO homologues have been found ubiquitously throughout eukaryotic organisms. Invertebrates (D. melanogaster 13

and C. elegans) contain one isoform of SUMO. The genomes of higher organisms, such as vertebrates and plants, code for multiple SUMO genes. X. laevis contains three

SUMO proteins, SUMO-1, SUMO-2, and SUMO-3. All forms are expressed throughout the entire animal and are present from early stages of development to maturity.

SUMO-2 and -3 are 95% identical and most often referred to as SUMO-2/3. However,

SUMO-1 and SUMO-2/3 share only about 50% amino acid similarity (79). Additionally,

SUMO-2/3 has the ability to form SUMO chains as it contains a SUMO consensus motif

(80); whereas SUMO-1, lacking this motif, cannot and is sometimes used to terminate chains of SUMO-2/3 on certain proteins. Humans contain a fourth SUMO protein that is expressed only in kidney, lymph node, and spleen. The physiological use for this SUMO isoform has yet to be identified (81).

Covalent attachment of SUMO to target proteins occurs in a series of enzymatic steps (Fig. 2) (7). Initially, the C-terminal amino acids of the SUMO precursor need to be cleaved by a SUMO specific hydrolase to reveal a di-glycine motif. The number of amino acids to be cleaved ranges from 2-11 and varies between organism and SUMO isoform.

The mature SUMO is then covalently linked to an activating enzyme (E1). The E1 enzyme is a heterodimer consisting of SAE1 (AOS1) and SAE2 (UBA2) subunits. This reaction is ATP dependent and requires the formation of a SUMO-adenylate bond before SUMO is transferred to a catalytic cysteine residue of the SAE2 subunit through its C-term glycine. Next, the carboxy terminus of SUMO forms another thioester bond with a cysteine residue on the E2 conjugating enzyme (Ubc9 in Xenopus). From this point, SUMO can be immediately transferred to the target protein and forms an 14

isopeptide bond between the terminal glycine of SUMO and the ε-amino group of the acceptor lysine on the target. In many instances, there is a need for an E3 ligase to confer specificity on the final step. E3 ligases are also used to preferentially attach

SUMO-1 versus SUMO-2/3 to certain proteins.

Figure 2. Conjugation of SUMO protein to a target. The pathway involves the pre- processing of the immature SUMO during transfer to the E1 activating enzyme. SUMO is transferred to E2 which catalyzes addition to target lysine residues with specificity often conferred by the E3 ligase. The conjugation is a reversible process and specific proteases can remove SUMO from its target.

This process is reversible; another set of enzymes are the SUMO proteases often called SENPs (sentrin-specific protease) from the rarely used name for SUMO, sentrin

(82). SUMO proteases were the last enzymes of the SUMO pathway to be characterized and were first identified in yeast as Ulp1 and Ulp2 (83). Deletion of Ulp1/Ulp2 in yeast

prevents cell cycle progression, an outcome seen with mutation or deletion of Ubc9,

Uba2, or Smt3 (84). In addition to removal of SUMO from target proteins, SENPs are responsible for cleaving the precursor SUMO protein to expose the di-glycine motif necessary for conjugation. SUMOylation is a dynamic process involving many cycles of conjugation and de-conjugation controlling the activity of proteins. In the case of the androgen receptor, rapid cycling between SUMOylated and non-SUMOylated forms is important for repression of certain androgen receptor genes during cellular stress (85).

In mammalian cells, SENPs themselves are primarily controlled though subcellular localization (86) with different forms of the protein localized to different compartments.

While this is not the only control mechanism, it potentially explains some of the specificity seen between different SENPs and SUMO isoforms.

The site of SUMO attachment is often found in a specific consensus motif ψKxE

(where ψ is a hydrophobic amino acid and x is any amino acid). While this is a general consensus sequence, SUMO has also been shown to be attached to lysines found in non- consensus sequences. In addition to this consensus sequence, a phosphorylation dependent SUMO motif (PDSM) has been described (87). This motif, ψKxExxSP, is comprised of a consensus sequence with a proline directed phosphorylation site separated from the SUMO consensus site by any two amino acids. In target proteins, phosphorylation at the PDSM is necessary for SUMOylation as the former enhances binding of the enzymes for the latter. The presence of this motif highlights the importance of the cross-talk that can occur between protein regulatory mechanisms.

This crosstalk is not unique to phosphorylation; examples exist in which SUMOylation, 16

acetylation, and/or ubiquitin are used in various combinations to control the activity of a protein. For example, one study showed that the DNA binding activity of tumor suppressor p53 is inhibited by SUMOylation. However, the binding capability can be restored if p53 is additionally acetylated (88). In some instances, such as the transcription factor Sp1, SUMO modification recruits factors which increase ubiquitination thus leading to protein degradation (75). In other cases, SUMOylation acts as an antagonist to ubiquitin by being covalently attached to the ubiquitin acceptor lysine thereby increasing protein stability (76).

SUMO or SUMO modified proteins can interact non-covalently with proteins that contain SUMO interacting motifs (SIM) (89, 90). The SIM sequence is more varied than that of the SUMO consensus attachment site; however it is characterized by hydrophobic interactions between the SIM containing protein and SUMO. Additionally, many SIMs contain a cluster of acidic residues adjacent to the hydrophobic amino acids that contribute to the strength of the interactions. In the case of the Kruppel-like factor

4 (KLF4), both the SUMO consensus sequence and the SIM are necessary for its transcriptional activity and mutations made in either domain abolish the activation of this factor (91).

SUMOylation knockout studies have been integral in describing the importance of this process in the development of many organisms. Knockdown of the E2 enzyme in

S. cerevisiae (Ubc9) leads to cell cycle arrest at G2/M (18). Depletion of maternal smt3 mRNA encoding the single SUMO isoform in Drosophila leads to decreased hatching and of those larvae that do hatch, all die in the first instar (19). Drosophila lacking the smt3 17

gene hatch but die early in the second instar and recent studies in Drosophila point to a differential need for SUMO in dividing and non-dividing cells, highlighting the fact that

SUMO may act as an important developmental regulator (92). Similarly, deletion of

Ubc9 in zebrafish and mice leads to early embryonic lethality (12, 13) although the findings in mice are debated. Less severe phenotypes (such as cleft palate) are seen when mice are heterozygous for mutant SUMO and heart failure is seen in mice with decreased levels of SUMO protein (14, 15, 93). Previous studies of Xenopus injected with a morpholino against SUMO-1 showed tadpoles with slowed or complete failure of the blastopore to close and shortened axis indicating a role for SUMOylation in development and early embryonic patterning (20).

Disruption of the SUMOylation pathway in model systems can be accomplished through traditional methods such as morpholino injection against an enzyme in the pathway or creation of transgenic knockouts. Recently, a novel method was described using the expression of an adenoviral protein, Gam1. Expression of Gam1 in viral infection reprograms host cells by blocking transcriptional repression and apoptosis by increasing the expression of protective genes (94). Gam1 was first identified in a screen for anti-apoptotic genes but was found to have an effect on the amount of SUMOylated proteins in infected cells. Gam1 binds to the SAE1 subunit of the E1 enzyme and causes the protein to be degraded through the ubiquitin pathway (95, 96). Since E1 is currently the only known activating enzyme of the SUMO pathway, its Gam1-mediated degradation effectively blocks conjugation of all SUMO isoforms to target proteins.

The results presented in this thesis provide evidence for the role of SUMOylation in the terminal repression of the oocyte 5S rRNA genes that occurs at the gastrula- neurula transition. SUMO 1 and SUMO 2/3, along with repressive chromatin markers, are found solely on the promoter of the oocyte gene and not on the somatic form. Lack of SUMOylation also causes reactivation of the oocyte 5S rRNA genes indicating a necessary role of SUMOylation in the active repression of these genes. SUMOylation is also involved in a large number of developmental processes including cell cycle control, cell migration, and epithelial to mesenchymal transition. In particular, microarray data indicate that SUMOylation controls the expression of many genes involved in these important processes. Additionally, lack of SUMOylation causes severe developmental defects and disrupts the convergence and extension necessary for proper patterning and axis elongation.

MATERIALS AND METHODS

Embryo preparation

Spawning females were injected with 500 units of human chorionic gonadotropin (HCG) at least 12 hours prior to spawning to induce egg laying. The following morning, eggs were spawned into a dry Petri dish for in laboratory fertilization. Testes were isolated from a male, which was placed on ice to anesthetize

o and stored at 4 C in high salt MBS (0.7 mM CaCl2, 108 mM NaCl, 1 mM KCl, 1 mM

MgSO4, 5 mM HEPES, pH 7.8, 2.5 mM NaHCO3 ) for up to a week. Approximately 1/4 of a testis was minced well in the Petri dish containing the eggs with a clean razor blade.

Minced testis and eggs were mixed together with 3 mL 1/3 MMR (0.1 M NaCl, 2.0 mM

KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM HEPES, pH7.8) and left to fertilize at room temperature. After 10 minutes, fertilizing eggs were flooded with additional 1/3 MMR.

After 30 minutes fertilized eggs were washed with 2% cysteine for no more than 5 minutes in order to remove the jelly coating. This prepares the embryos for microinjection and manipulation. After incubation with the cysteine, fertilized eggs were washed 8 times with 1/3 MMR. Fertilized eggs can be left to develop at room temperature or stored at 18oC in order to slow cell divisions.

Oocyte Preparation

Females were anesthetized with tricane and oocytes were removed and placed

- in a beaker containing 200 mL OR2 (82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 5 mM

HEPES, pH 7.8). Oocytes were treated with 500 uL (2.5 mg collagenase) liberase blendzyme (Roche) in 20 mL fresh OR2- at 37oC with gentle shaking for 30 minutes.

Oocytes were then washed 4 times with OR2- and 4 times with OR2+(82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 5 mM HEPES, 1 mM MgCl2, 1 mM CaCl2) and were stored in

OR2+ at 18oC.

RNA synthesis

Capped mRNA was synthesized for microinjection into one cell embryos.

Plasmids used for RNA synthesis were pCS2p+MTGFP, pWTGam1 (Wildtype Gam1), pMutGam1 (Mutant Gam1), and pTFA (TFIIIA). Plasmid (20ug) was linearized with the appropriate restriction enzyme (Gam1-Xho1, GFP-Not1, TFA-Bam H1) overnight at 37oC.

Digested plasmid was extracted twice with phenol, pH 8.0, and twice with 24:1 chloroform: isoamylalcohol. The DNA was precipitated with 1/10 volume 3 M sodium acetate, pH 5.2, and 2.5 volumes ethanol overnight at -20oC. Precipitated DNA was spun

at 16,000 rcf at room temperature for 30 minutes. The DNA pellet was washed with ice cold 70% ethanol and air dried for 5 minutes. DNA was re-suspended in 20 uL H2O (all

H2O used was DEPC treated and sterilized).

mRNA was synthesized by mixing the following components: 2.5 ug linearized plasmid, 1x transcription buffer, 10 mM DTT, 2.5 mM capanalog, 50 units RNasin, 200 units RNA polymerase, and 0.5 mM each UTP, ATP, CTP, GTP in a total volume of 50 uL.

The reaction was incubated at 37oC for 90 minute, followed by the addition of another

200 units of polymerase and incubation for 90 additional minutes. The reaction mixture was extracted twice with phenol pH 4.5 and twice with 24:1 chloroform: isoamylalcohol.

The mRNA was precipitated with ethanol and re-suspended in 50 uL of H2O. mRNA was run though a NucAwayTM spin column (Ambion, #AM10070) per manufacturer instructions to remove unincorporated nucleotides. mRNA concentrations were determined using a NanoDrop spectrometer (Thermo Scientific), and samples were stored at -80oC until use.

Microinjection of capped mRNA

Fertilized embryos were prepared as previously described and were maintained in 1/3 MMR until microinjection. At the time of microinjection, they were transferred to a small Petri dish, with mesh on the bottom, containing 1/3 MMR supplemented with

3% Ficoll. Needles, pulled from capillary tubes (3.5” Drummond #3-000-203-G/X) using

a Sutter Instrument micropipette puller (heat-500, pull-120, velocity-90, time-200), were backfilled with mineral oil and the tip of the needle was clipped very small. A small amount of mRNA (1-3 uL) was placed on a small piece of Parafilm and loaded into the needle. Amounts of mRNA injected into each embryo depended on the specific experiment and the concentration of each mRNA sample but injection volumes ranged from 5-30 nL. Embryos were injected in the animal hemisphere. Embryos were transferred from the microinjection dish to plastic Petri dishes containing 1/3 MMR and allowed to develop at room temperature.

SUMOylation Assays

Gam1 mRNA was synthesized from pWTGam1 cut with Xho1 and using T7 polymerase. Gam1 mRNA was injected into one cell embryos which were allowed to develop to the indicated stage (mid-blastula, late-blastula, early-gastrula, and mid- neurula). Ten embryos per time point were collected and homogenized in 11 uL SUMO reaction buffer (Boston Biochem, #K-710) and spun at 16,000 rcf for 5 minutes.

Supernatant was removed and added to the remaining assay components to a final concentration: 60 uM SUMO-1, 5 uM Ubc9, 5 uM E2-25K (substrate peptide), 25 mM

Mg-ATP for a total reaction volume of 25 uL. Control reactions contained all assay reagents to the following concentration: 60 uM SUMO-1, 1X reaction buffer, 500 nM

SAE1/SAE2, 5 uM Ubc9, 5 uM E2-25K (substrate peptide), 25 mM Mg-ATP for a total

reaction volume of 20 uL. Reactions were incubated at 37oC for four hours. SDS loading dye was mixed with the reactions and vortexed briefly in order to stop the reaction.

In order to control for changing protein concentrations in the developing embryos, SUMOylation assays were also carried out with a constant amount of protein from embryo extracts. mRNA was injected as described above and samples were taken at the indicated time points. Twenty embryos per time point were homogenized in 22 uL of SUMO reaction buffer (Boston Biochem) and spun at 16,000 rcf for 5 minutes.

Supernatant was removed and the protein concentration was determined by Bradford assay using a 10 uL aliquot of the supernatant. SUMOylation assays were conducted as described above using 25 ug of whole cell extract per assay.

Proteins were separated by SDS polyacrylamide gel electrophoresis and analyzed by western blot. Samples were boiled for 5 minutes and 15 uL of the reaction volume was loaded on to a 4% stacking/ 6% separating polyacrylamide gel and run for 2 hours at

15 milliamps (through the stacking gel) and 25 milliamps (through the separating gel).

Proteins were transferred (transfer buffer: 25 mM Tris pH 7.5, 192 mM glycine, 20% methanol) to a nitrocellulose membrane (Osmonics, #EP4HY00010) at 4oC overnight at

40 volts.

Nitrocellulose membranes were blocked for 30 minutes at room temperature using 3% dry milk in TBS (20 mM Tris, pH 7.5, 0.5 M NaCl). Membranes were then washed with TTBS (0.05% Tween-20 in TBS) for 5 minutes. Antibody specific to the E-

25K peptide was applied in 1:500 dilution in TTBS containing 3% dry milk and incubated at room temperature for 30 minutes. The membrane was then washed twice with TTBS 24

for 5 minutes each. Goat anti-rabbit IgG: alkaline phosphatase fusion was applied at a dilution of 1:3000 in TTBS containing 3% dry milk and incubated at room temperature for 30 minutes. The membrane was then washed with TTBS twice for 5 minutes each and subsequently washed once with TBS for five minutes. The membrane was developed with the addition of AP buffer (100 mM Tris pH 9.5, 100 mM NaCl, 5 mM

MgCl2, 330 ug/mL NBT, 165 ug/mL BCIP) until visualization of the precipitate occurred.

The membrane was then washed thoroughly with water and stored protected from light.

Sample preparation for microarray analysis

One cell embryos were injected with either 0.5 ng Gam1 mRNA in a total volume of 9.2 nL or with H2O as a control. Embryos were allowed to develop in 1/3 MMR at room temperature until the desired time points. Samples were taken at early gastrula

(9 hpf), late gastrula (13.5 hpf) and early neurula (16.5 hpf). Twenty embryos from each time point were homogenized in 500 uL proteinase K buffer (100 mM Tris pH 8, 150 mM

NaCl, 12.5 mM EDTA pH 8.0, 1% SDS, 200 ug proteinase K) and incubated at 37oC for one hour. Samples were then extracted twice with an equal volume of phenol, pH 4.5, and twice with 24:1 chloroform: isoamylalcohol. Samples were purified using an RNAEasy spin column (Qiagen, #74104) according to manufacturer’s directions and precipitated with 2.5 volumes of ice-cold ethanol. The concentration and purity of the samples was

determined using a NanoDrop spectrometer. RNA samples were stored at -80oC until submission to the Genomics and Bioinformatics Core Facility (GBCF). Prior to microarray data collection, the RNA integrity number (RIN) for each sample was determined by the

GBCF using an Agilent 2100 BioAnalyzer.

Microarray Data Collection

Total RNA (50-500 ng) was used for cDNA synthesis according to the GeneChip®

3’ IVT Express Kit for Affymetrix microarray chips. The cDNA was then converted to double-stranded DNA and used as a template for transcription of amplified RNA (aRNA) which was labeled with a biotin-conjugated nucleotide. aRNA samples (15 ug) were subsequently purified and fragmented to produce probes for hybridization onto

Xenopus laevis 2.0 GeneChip® 3’expression arrays. Three biological replicates were carried out for Gam1 and H2O injected samples at each time point utilizing a total of 18 microarray chips. Analysis of the microarray chips was carried out with the Affymetrix

GeneChip® System which includes a GeneChip® Hybridization Oven 640, a GeneChip®

Fluidics Station 450, and a GeneChip® Scanner 3000 7G. Microarray chips were stained with a streptavidin phycoerythrin (SAPE) solution and fluorescence intensity of each probe was measured and exported to a probe intensity data (.cel) file.

Affymetrix .cel files were analyzed using the Bioconductor software package

(http://www.bioconductor.org/). The mean fluorescence intensity was derived from a

log2 transformation of the data and normalized using the quantile normalization method. A Student’s t test was used to determine if there was a significant difference between the Gam1 and H2O embryo gene expression values. A p value less than 0.05 was used as a cut off for differential expression. The fold change, between control and treated samples, for each expression value was also calculated. Gene lists for those genes differentially expressed (p<0.05) were compiled for bioinformatics analysis. Since the Xenopus laevis genome was not sequenced at the time of analysis, there are several probes on the 2.0 chip which do not have identified names or functions. Thus, only those annotated genes were used for bioinformatics analysis.

Real time polymerase chain reaction

Samples of RNA (30 ug) in a final volume of 50 uL were incubated at room temperature with 20 units of RNA-free DNaseI for 15 minutes before inactivating the enzyme by addition of 1 uL of 25 mM EDTA. Samples were heated for 10 minutes at

65oC and placed on ice before they were run over an RNAEasy spin column (Qiagen) following the manufacture’s protocol. RNA was exchanged into 30 uL of H2O giving an approximate final concentration of 1 ug/uL.

RNA samples (4 uL) were mixed with 1 uL of random hexamers (Promega,

#C118A) and heated at 70oC for 5 minutes. Samples were then placed on ice for at least

5 minutes. Reverse transcription reactions for each sample contained 5 uL of RNA annealed to random hexamers, 1x reaction buffer, 3 mM MgCl2, 0.5 mM dNTPs, 10 units 27

RNasin (Promega, #N251A), and 1 uL GoScript reverse transcriptase (Promega, #A501C) in a final volume of 20 uL. The reaction mixture was placed in a Thermal Cycler with program parameters: 25oC for 10 min, 42oC for 50 min, 70oC for 15min.

Dilution calculations of the reverse transcription (RT) reaction were based on a proportional amount of cDNA being created from the total RNA added to the reaction thereby producing a solution with a cDNA concentration of 200 ng/uL. RT samples were diluted with H2O to give a final cDNA concentration of 5 ng/uL. An aliquot of 50 uL of cDNA was mixed with an additional 200 uL of H2O to give a final working concentration of 1 ng/uL which was used in SYBR green PCR reactions. An additional 50 uL aliquot of each 5 ug/uL RT sample was taken in order to make standard curve dilutions for testing primer efficiency. A four point standard curve was created with 10 fold dilutions from the 5 ng/uL cDNA arbitrarily termed: 1000 (undiluted), 100, 10, and 1. A standard curve was generated for each primer pair in order to test the primer efficiency.

A master mix containing 25 uM of both forward and reverse primers (Table 2) and 12.5 uL SYBR Green Master Mix (Applied Biosystems, #4309155) was prepared for each sample. The master mix was mixed thoroughly, quick spun, and 15 uL was added to each well of a 96 well plate. cDNA (10 uL) was added to each well with master mix and the plate was spun down to ensure proper mixing of all reaction components.

Samples were run with the following program parameters: 50oC for 2 minutes, 95oC for

15 minutes, 95oC for 15 seconds, 60oC for 30 seconds, 72oC for 30 seconds (last three steps repeated for 45 cycles), 95oC for 15 seconds, 60oC for 1 minute, ramping

temperature for 20 minutes, 95oC for 15 seconds. The ramping temperature function was used to produce melt curves for calculation of primer efficiency.

TABLE 2

FORWARD AND REVERSE PRIMERS USED FOR QRT-PCR ANALYSIS

Gene Symbol Forward sequence (5’- 3’) Reverse sequence (5’- 3’) Gsc GCT GGC AAG GAG AGT TCA TC TTC CAC TTT TGG GCA TTT TC Foxc1 GAT CAC CCT GAA TGG CAT CT GGC TTC TTG TCG TCT CT GG Chrd AAC TGC CAG GAC TGG ATG GT GGC AGG ATT TAG AGT TGC TTC Xbra AGC CTG TCT GTC AAT GCT CC ACT GAG ACA CTG GTG TGA TGG Ccng1 TTC TGG AAA AGT TGG GTT GG CAG AGG GCT TAG CTT TGG AA Dmrta AGC ATA AGG CTC TGG GAA CA GAT GCA GCA GAG GTC TAG GG Wnt8b TGG GGT GGA TGT AGT GAC AA CAC CAT GAC ATT TGC AGG TC Xpo CGG CTA TAC AGG CAA ATG GT CAA CCA TCT GTG GGT CAC TG Krt GGC CCC TTA ACA TTA CG CA AAA GGC TGA GCT GG CAC AT Rpl8 GTT GCT GGA GGT GGT CGT AT GAT GGT TGA GGG CTT ACC AA Gapdh CTT TGA TGC TGA TGC TGG AA GGG GTT GAC AGG TGA CAA GT

Microarray data analysis: MetaCore®

Gene lists were uploaded to the MetaCore® software suite of programs using the official gene symbol to identify each gene. Only annotated genes were used for each analysis and genes were group differently depending on the analysis. Genes were grouped into lists based on differential expression at each time point (EG, LG, EN), not-

differential expression at each time point (EG, LG, EN), or common expression patterns across all three time points (Cluster 1-8). MetaCore® was used to analyze the gene lists for (i) transcription factor networks, (ii) functional enrichment, and (iii) pathway analysis each using an algorithm with specific well-defined instructions for creating the network based on established interactions.

(i) The Transcriptional Regulation algorithm adds a transcription factor as a seed node and uses the provided gene list to build a network of interactions around the given factor. This algorithm was used to determine which transcription factors can be associated with the largest number of genes in the created lists. These networks are generated using an auto-expand algorithm in which preference is given to those objects with the most connectivity to the seed node. Additionally, connectivity is directionally indicated and each direction is considered a separate interaction. This provides a list of over-connected transcription factors, providing a unique network for each factor. All gene lists were analyzed using this algorithm.

(ii) Enrichment analysis consists of mapping genes, from activated gene lists, onto gene IDs present in functional ontologies provided in MetaCore®. Mapping is based on statistical relevance of the matches found and utilizes both MetaCore® classification and public ontologies (or controlled vocabularies) such as Gene Ontology

(GO). Three GO functional ontologies are present in MetaCore® (biological processes, molecular function, and cellular localization) each represented by a hierarchical structure made up of terms and sub-terms corresponding to a list of genes. The mapping of uploaded gene lists onto known ontologies can be used to categorize genes 30

with unknown relatedness into groups and thus reveal their functions or common characteristics. In order to remove the redundancies created by the MetaCore® enrichment algorithms, GeneGo IDs identified in the initial analysis were further categorized using GO Term classification software CateGOrizer (v. 3.218). As mentioned previously, gene ontologies are hierarchically organized and the CateGOrizer software allows the specific sub-terms to be grouped into their broader categories providing a more accurate enrichment picture.

(iii) Identified differentially regulated genes were also mapped on to known biochemical pathways to determine the biological significance of altered genes.

Pathway maps in MetaCore® are created by compiling known biochemical processes or signaling cascades into graphical images. These maps can be used to determine the interconnectedness of uploaded genes in a biological context. Additionally, pathway maps can be used to determine downstream gene targets not present on the microarray. Gene lists were uploaded into MetaCore® while pathways relevant to development (heart and nervous system), cell adhesion, cell cycle, cytoskeleton remodeling, and epithelial to mesenchymal transition were manually searched for any matching genes. Pathways containing differentially regulated genes were analyzed further for their connection to the observed developmental phenotypes.

Database of SUMO consensus motifs in Xenopus laevis proteins

A searchable database of SUMO consensus motifs in X. laevis proteins was created. Protein sequences obtained from Xenbase (http://www.xenbase.org) were scanned using the SUMOsp2.0 software located at http://sumosp.biocuckoo.org.

Briefly, this software locates potential lysines for SUMOylation within the protein sequence and separates them into two groups, Type I or consensus (ψKxE) and Type II or non-consensus. Non-consensus motifs are determined based on the degree of hydrophobic similarity to consensus sites and the degree of similarity to SUMOylation sites in proteins with identified non-consensus sites (97, 98). The software then scores each sequence based on previously reported SUMOylated proteins and displays a confidence value for each lysine. Thresholds can be set for this analysis where high stringency will return only those Type I motifs with high confidence values and medium stringency will identify lower, non-consensus motifs. The database can be searched for specific proteins, by name or accession number, to determine if they contain potential sites for SUMO modification.

Cluster analysis

Genes that were differentially expressed at each stage were pooled together and subject to network analysis. First, a co-expressed gene network was constructed using microarray data. Nodes in the network correspond to genes and edges represent

expression similarities (measured by Pearson correlation coefficient) between genes.

For two genes to be considered as co-expressed, their expression profiles needed to satisfy at least one of the following conditions: their correlation coefficient is higher than 0.5, and one gene is ranked as the top-10 most correlated gene of the other. The motivation was that genes involved in the same functional pathway are directly connected to each other or linked via short paths. After network creation, the nodes were clustered into dense modules using Qcut software (99). Eight gene modules were found by Qcut algorithm.

Keller sandwich explant assays

One cell embryos were injected with either Gam1 mRNA (2.5 ng or 5.0 ng) or an equivalent volume of H2O. Embryos were allowed to develop in 1/3 MMR until early gastrula stage (~9 hpf) when the blastopore lip was initially beginning to form. Embryos were maintained in 1X MBS buffer (88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4,

5 mM HEPES pH7.8, 2.5 mM NaHCO3) during the dissection. The vitelline membrane of each embryo was removed making sure to not pierce the animal cap above the blastopore lip (Fig. 3 A). The membrane was pierced by a jeweler’s forcep held in one hand on the ventral side of the embryo in order to avoid damaging the area needed for explants. A jeweler’s forcep in the other hand was used to hold the embryo in place being caution to not tear the embryos. Once a hole in the membrane forms the embryo naturally came out. If it did not, the vitelline membrane was slowly removed with the

forceps by pealing it away from the embryo. Devitellinated embryos were stored in 1X

MBS until dissection. Several devitellinated embryos were prepared before beginning dissection to avoid having to stop once starting the explants.

Two devitellinated embryos were placed animal pole side down to expose the blastopore lip. An eyebrow hair knife was held in one hand and a hair loop was held in the other. The embryo was positioned with the dorsal side of the blastopore lip facing the hand with the eyebrow knife. Using the hair loop to hold the embryo in place, a small cut was made beginning at the blastopore lip and extending anteriorly through the animal cap (Fig. 3 B).

An equivalent cut was made on the opposite side of the blastopore lip and the embryo was flipped over orienting the animal cap up. A horizontal cut (#3) was made to complete the dissection forming a rectangle which was then peeled away from the rest of the embryo (Fig. 3 B). The embryo was then flipped over again with the vegetal pole up and the eyebrow knife was used to cut downward along the blastopore lip completing the rectangle. The dissected rectangular piece of the embryo was trimmed to the desired size. The above procedure was completed for another devitellinated embryo and the dissected portion was trimmed to the same dimensions as the previous excised piece.

The two rectangular dissections were immediately placed inner sides together making sure to align the sides that were previously the blastopore lip of each respective embryo. The edges the sandwich were then trimmed so that the explants were exactly the same size and gently covered with a small portion of coverslip with vacuum grease 34

on one side, making sure not to squash the sandwich, to ensure the dissected portions remain in contact with each other. Explants were then cultured in the dissection dish for one hour allowing the sandwiches to adhere together. Explant sandwiches were then moved using a blunt Pasteur pipet (made from cutting a larger opening from the tip) from the dissection dish to a Petri dish containing 1X MBS supplemented with 50 ug/mL gentamicin with agarose coating the bottom and left to culture for an additional

14 hours.

Samples were photographed immediately after moving them to the Petri dish

(10 hpf) and again at 24 hpf using an Olympus SZX16 microscope. Scale bars on the

CellSens software were used to measure the length and width of the sandwich at the mentioned time points. Explants from water-injected or Gam1-injected embryos (2.5ng or 5ng) were compared to each other to determine the level of convergence and extension. Sample sets were analyzed for significant differences using a Student’s T-Test

(http://studentsttest.com/).

Figure 3. Procedure for Keller sandwich explants. (A) Removal of the vitelline membrane. The embryo is held in place with one pair of forceps (top) while the membrane is pierced with another (bottom) making sure to avoid the blastopore lip and surrounding area. (B) Dissection of the embryo. Two vertical cuts (#1 and #2) are made anteriorly from the blastopore lip. A third horizontal cut (#3) connects the first two and the dissected portion of the embryo is peeled away and removed from the remaining region.

Chromatin immunoprecipitation assay

Embryos were staged according to Nieuwkoop and Faber and collected at the following time points; late blastula, early gastrula, late gastrula, early neurula and late neurula. Embryos were collected, washed once with nucleus isolation buffer (10 mM

o Tris, pH7.5, 0.25 M sucrose, 3 mM CaCl2) and stored at -80 C. Frozen embryos were thawed on ice and homogenized in 260 uL of homogenization buffer (10 mM Tris, pH

7.5, 2.2 M sucrose, 3 mM CaCl2, 0.5% Triton-X) supplemented with EDTA-free protease inhibitor tablet (Roche, #11836170001) and 1 mM PMSF. Samples were placed in a centrifuge tube (Beckman, #349622) on a layer of 150 uL homogenization buffer and layered above with 1.6 mL nucleus isolation buffer supplemented with protease inhibitor and 1 mM PMSF (Fig. 4). Nuclei were isolated by centrifugation in a TLA 100.3 rotor at 75,000 rpm for 55 minutes at 4oC (acceleration 2, deceleration 2) using a

Beckman Optima table top centrifuge. Pellets were gently resuspended in 250 uL of nucleus isolation buffer using a plastic homogenizer, spun at 9,500 rpm for 10 minutes

(4oC) using a microcentrifuge. Supernatant was removed and pellets were resuspended in 360 uL of nucleus isolation buffer using a plastic homogenizer.

Samples were fixed by addition of formaldehyde to a final concentration of 1% and incubated on ice for 10 minutes followed by incubation at room temperature for 20 minutes. Formaldehyde was removed by centrifugation for 5 minutes at 9,500 rpm

(4oC) and pellets were resuspended in 250 uL of lysis buffer (50 mM Tris pH 8.1, 1% SDS,

10 mM EDTA) supplemented with protease inhibitor tablet and 1 mM PMSF. Samples 38

were chilled on ice for 10 minutes before chromatin was sheared by sonication using a

Fisher Scientific Sonic Dismembrator 100 with the power output set to 7. Twelve 15 second pulses, with 15 second pauses between each pulse, were used and samples were kept on ice at all times. Sonicated samples were centrifuged at 13,000 rpm in a microcentrifuge for 10 minutes (4oC) to remove insoluble material and supernatants were stored at -80oC.

Figure 4. Diagram of nucleus isolation for ChIP assays. Homogenized embryos for nucleus isolation were layered with homogenization buffer below and nucleus isolation buffer above in a 0.5 inch X 2 inch Beckman polycarbonate centrifuge tube (#349622) for use in TLA 100.3 rotor.

In order to normalize the amount of DNA in each immunoprecipitation assay,

DNA levels for each sample were quantified using the PicoGreen® fluorometric quantitation assay (Molecular Probes, #P7589) following the provided protocol. A 5 uL aliquot from each chromatin sample was incubated with 15 uL of 5 M NaCl and 280 uL

o of freshly prepared NS buffer (0.1 M NaHCO3, 1%SDS) and incubated at 65 C overnight in order to reverse crosslinking. Samples were moved to 42oC and treated with 10 ug of

RNase A (1 hour) followed by 15 ug of proteinase K (2 hours). Samples were extracted twice with phenol, pH 8.0, and precipitated with 1/10 volume of 3 M sodium acetate and 2.5 volumes of ice cold 100% ethanol. Precipitated DNA was spun at 13,000 rpm

(room temperature) for 30 minutes. Pellets were washed with 70% ice cold ethanol, air dried for 5 minutes, and dissolved in 20 uL of TE buffer. A standard curve (pg/uL: 0, 2.5,

5.0, 7.5, 25, 50) was generated from λDNA in a 96 well plate in a total volume of 100 uL.

DNA samples were diluted 100 fold by mixing 1 uL of DNA with 99 uL of TE buffer in a 96 well plate. Samples were mixed with 100 uL of PicoGreen dye diluted 200-fold in TE buffer and incubated at room temperature for 5 minutes. Sample fluorescence was read on a Spectra Max M5 microplate reader (Molecular Devices) using the PicoGreen protocol (excitation wavelength, 488nm; emission wavelength, 525nm). Each DNA standard was prepared in duplicate and each sample was prepared in triplicate. The standard curve was used to determine the DNA concentration of each chromatin sample.

For each immunoprecipitation, 0.1 ug of chromatin was combined with TSEI (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Triton-X, 2 mM EDTA supplemented with 40

protease inhibitors) to give a final volume of 160 uL. Chromatin samples were pre- cleared at 4oC with 40 uL of a 50% slurry of protein A-Sepharose beads containing 1 ug/uL carrier tRNA and 1 ug/uL of herring sperm DNA. Beads were removed by centrifugation at 600 rcf for 2 minutes at room temperature. Samples were combined with 5 ug of affinity purified (TFIIIA, PIASxβ) or commercially purchased (Santa Cruz biotechnologies: CtBP (sc-17759), SUMO-1 (sc-5308), SUMO-2/3 (sc-32873) or Cell

Signaling Technologies: Histone3Lysine9 (#9753), Histone3Lysine27 (#9756)) antibodies and incubated at 4oC overnight with rotation. Samples with no antibody added were also incubated at 4oC overnight to determine non-specific binding of beads. Pre-cleared chromatin (0.025 ug) was saved at -20oC and used as input to normalize PCR signals.

Equivalent immunoprecipitations (IP) were set up for each of the five timepoints.

Protein A-Sepharose beads (75 uL of a 12% slurry containing 1 ug/uL tRNA, 1 ug/uL herring sperm DNA, 1 ug/uL BSA in TSEI) was added to each IP reaction and incubated at 4oC for 2.5 hours. Beads were collected by centrifugation at 500 rcf for 2 minutes at room temperature initially and after each wash. Beads were washed twice with 1 mL of TSEI and twice with 1 mL of TSEII (50 mM Tris, pH 8.0, 500 mM NaCl, 0.1%

SDS, 1% Triton-X, 2 mM EDTA). Beads were incubated for 15 minutes at 4oC after the first round of each buffer wash. Beads were then washed once with immune complex wash buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA) and twice with TE. Samples were eluted by incubation at room temperature with 185 uL of freshly prepared NS buffer for 30 minutes. Beads were spun at 500 rcf for 2 minutes and supernatant was removed. Beads were incubated with an additional 100 uL of NS 41

buffer. Both supernatants were combined and mixed with 15 uL of 5 M NaCl and incubated at 65oC overnight. The saved input samples were treated in the same manner. DNA was isolated from all samples as described earlier however the pellet was dissolved in 50 uL of H2O.

PCR reactions (50 uL) contained the following: 10 pmol of each primer, 250 uM of dNTP mix, 1X Taq reaction buffer and 2.5 units of Taq DNA polymerase (Promega,

#M3008). MgCl2 concentration for the oocyte 5S amplification was 4 mM and for the somatic 5S was 2.5 mM. The oocyte 5S gene was amplified with primers ChIPO1 (5’ CCA

CAG TGC CGC TGA CAA G 3’) and ChIPO2 (5’ CAG CAG CAC CTT TTG GCT CC 3’) using 5 uL of DNA template from IP samples and 2.5 uL of 1:20 diluted input samples. The somatic gene was amplified with primers ChIPS3 (5’ GGC CCC AAC AAC GCA GCA C 3’) AND

ChIPS2 (5’ GCA GCT AGC TGT CTG GCT GTT G 3’) using 5 uL of DNA from IP samples and undiluted input samples. PCR programs were identical for both sets of primers (25 cycles: 1 minute at 94oC, 45 seconds at 60oC, and 1 minute at 72oC). A portion of each

PCR reaction (15 uL) was loaded and resolved by electrophoresis on a 1.5% agarose gel in 1X TBE.

In vivo 5S rRNA transcription assays

Eggs were collected, fertilized, and microinjected as previously described.

Capped TFIIIA mRNA was synthesized from pTFA linearized with Bam H1 using T7 polymerase. One cell embryos were injected with one of three samples: 0.08 uCi [α-32P]

UTP, 0.08 uCi [α-32P] UTP and 7 ng capped TFIIIA mRNA, or 0.08 uCi of [α-32P] UTP and 5 ng of capped Gam1 mRNA.

Injected embryos were cultured in 1/3 MMR until early gastrula (~10 hpf). Nine embryos from each sample set were collected and homogenized in 500 uL SETS (150 mM NaCl, 10 mM EDTA, 50 mM Tris, pH7.5, and 0.5% SDS). Samples were treated with

125 ug of proteinase K at 60oC for 30 minutes. Samples were extracted twice with phenol, pH 4.5, and twice with chloroform: isoamyl alcohol (24:1). Isolated RNA was precipitated with ethanol by adding 1/10 volume of 3 M sodium acetate and 2.5 volumes of 100% ice cold ethanol. The RNA pellet was resuspended in 25 uL of formamide dye (90% deionized formamide, 10 mM Tris, pH 7.8, 0.1% SDS, 0.1 mM

EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol) and heated at 85oC for 5 minutes. Three embryo equivalents of RNA was loaded onto a 10% polyacrylamide gel containing 7 M urea, separated by electrophoresis, and visualized using autoradiography. Developed films were scanned and saved as .jpg files. Image files were analyzed using ImageJ software in order to determine to ratio of 5S rRNA to tRNA in each loaded sample.

TFIIIA levels in Gam1- and H2O-injected embryos

Embryos (20) injected with either H2O or 5 ng of Gam1 mRNA from each time point (early gastrula, late gastrula, early neurula) were homogenized in 85 uL of NET-2

(50 mM Tris-pH 7.5, 150 mM NaCl, 0.05% NP-40) buffer supplemented with EDTA-free

protease inhibitor (Roche) and 1 mM PMSF. Embryo homogenate was spun at 13,000 rpm at 4oC for 5 minutes. Supernatant was removed and added to 15 uL of SDS loading buffer. Samples were boiled for 5 minutes before proteins from two embryo equivalents were separated using a 6%/ 10% polyacrylamide stacking/ separating gel.

Proteins were transferred to a nitrocellulose membrane overnight at 4OC and developed as previously described for western blotting with the following changes; the primary antibody, produced in rabbit, specific to Xenopus laevis TFIIIA was incubated on the membrane for 2 hours and the secondary was incubated for 1 hour. Protein bands from the scanned westerns were quantified using ImageJ in order to compare the levels of

TFIIIA between Gam1 embryos and H2O embryos.

Immunohistochemistry and confocal microscopy

Embryos injected with either 2.5ng of Gam1 mRNA or H2O were collected at the early gastrula (9 hpf) and mid neurula (20 hpf) according to Nieuwkoop and Faber

(1994) and were distributed approximately 30 embryos to a well in a 24-well culture plate. Embryos were fixed with 1 mL of MEMFA (0.1 M MOPS, pH 7.4, 2 mM EDTA, 1 mM MgSO4 and 3.7% formaldehyde) for 1 hour at room temperature with gentle shaking. The MEMFA solution was changed and embryos were fixed for another hour.

Samples were washed with 1 mL of TBSN (10 mM Tris, pH 7.5, 155 mM NaCl, and 0.2%

NP-40) five times for five minutes each.

Embryos were incubated, at room temperature, with 1 ml of bleaching solution

(1% H2O2, 5% formamide, 0.5X standard saline citrate) with gentle shaking. Bleaching was carried out for several hours (3-4) until pigment was adequately removed. Embryos were then washed five times with 1 mL of TBSN for five minutes each.

Fixed embryos were incubated with 5 ug of affinity purified antibody labeled with Alexa Fluor® 568 targeted against TFIIIA in 500 uL of TBSN containing 4% (w/v) BSA for 48 hours. All antibody incubations were carried out at 4oC and washes at room temperature with gentle agitation and protected from light at all times. Samples were then washed with 1 mL of TBS five times over a period of 36 hours, changing buffer approximately every 7 hours. DAPI (4', 6-diamidino-2-phenylindole) DNA stain was added to the samples to a final concentration of 300 nM and incubated for 10 minutes.

Samples were washed three times with 1 mL TBS for five minutes each. Samples were dehydrated with four five minute washes with 1mL of methanol. Embryos were then cleared with 1 mL of Murray’s solution (benzyl alcohol, benzyl benzoate, 2:1) and stored in Murray’s, protected from light, at 4oC until imaged.

Embryos were bisected with an eyebrow knife, mounted on slides with the anterior side of the embryo down, and covered with a cover slip. Samples were observed using a Nikon A1R-MP Confocal microscope using 60X oil immersion lens. Two different laser wavelengths were used for visualization of the antibodies: DAPI-

Excitation: 403.7nm, Emission: 450nm and Alx568- Excitation: 561.7nm, Emission:

595nm. For each sample the laser intensity was set between 5-11%. Collected scans were viewed and adjusted using the Nikon Imaging Software (NIS) Elements v. 4.0. 45

Background fluorescence was eliminated through adjustment of the laser intensity and gain during collection and adjusting the contrast and pixel intensity values post collection.

RESULTS:

REGULATION OF THE 5S RIBOSOMAL RNA GENES

SUMOylation is required for repression of the oocyte-type 5S rRNA genes

A yeast two-hybrid screen of a Xenopus laevis cDNA library identified two proteins that interact with a segment of TFIIIA that is specifically phosphorylated on serine-16 and is important for the differential regulation of the two types of 5S rRNA genes, major oocyte-type and somatic-type. The major oocyte-type genes are expressed during oogenesis and for a short time following the mid-blastula transition when they are permanently repressed. The somatic-type genes are expressed throughout oogenesis and embryogenesis (2, 3). Clones encoding the Xenopus ortholog of the SUMO ligase PIAS2b isoform and a Xenopus homolog of the co-repressor protein

CtBP, XCtBP, were unique isolates that gave particularly strong signals in β-galactosidase assays. Additionally, the interactions of PIAS2b and XCtBP with TFIIIA were confirmed using pull-down assays.

The interaction of TFIIIA with a SUMO ligase and with a transcriptional corepressor that is regulated by SUMOylation raises the question whether this post- translational modification plays any role in the regulation of the 5S rRNA genes. In

addition to these interactions, it was determined that the subcellular location of PIAS2b changes during development. The protein is restricted to the cytoplasm of the oocyte and becomes increasingly nuclear during the stage of development when the oocyte- type 5S rRNA genes are permanently repressed. To test whether SUMOylation regulates the repression of the oocyte-type genes, this activity was inactivated by the expression of the avian adenovirus protein Gam1. Gam1 was first identified in a screen for antiapoptotic genes (94, 101, 102) and it was found to bind to the SAE1 subunit of the

SUMO E1 activating enzyme thereby marking it for degradation though the ubiquitin pathway (95). Since E1 is the only known SUMO activating enzyme, its degradation prevents all SUMOylation activity in the infected cell. The advantage of this strategy, as opposed to an antisense methodology, is that the effect is immediate and eliminates both existing and de novo accumulation of the E1 enzyme.

One-cell embryos injected with mRNA encoding Gam1 express elevated levels of

5S rRNA by gastrula stage due to activation of the normally repressed oocyte-type genes

(48). This effect was compared to embryos injected with TFIIIA mRNA (Fig. 5) through co-injection of α-32P and mRNA for either Gam1 or TFIIIA. In this assay, any increase in

5S rRNA can be attributed to oocyte 5S rRNA as the oocyte genes, but not the somatic genes, are repressed at gastrula stage. The SUMOylation-deficient embryos exhibit a similar level of enhanced 5S rRNA synthesis, attributable to the oocyte-type genes, relative to control embryos injected with H2O. While similar, the levels of reactivation seen with the injection of Gam1 are lower than that seen with injection of TFIIIA mRNA.

This could be for two reasons. The SUMO knockdown achieved through Gam1, while 48

effective, is not complete and some residual SUMOylation activity exists in the developing embryos leading to partial repression of the oocyte-type genes.

Alternatively, TFIIIA levels could be altered in the Gam1 injected embryos leading to a change in transcription of the 5S rRNA genes. Even though it is a less pronounced effect, this result implicates SUMOylation activity in the establishment of the repressed state of the oocyte 5S rRNA genes.

Figure 5. Depletion of SUMOylation activity prevents repression of oocyte 5S rRNA genes. (A) One-cell embryos were injected with TFIIIA mRNA (7 ng), which supports transcription of the oocyte-type genes after the MBT, or Gam1 mRNA (5 ng). RNA was isolated at early gastrula stage analyzed by electrophoresis/autoradiography. Lane 1, control embryos injected with 0.08 uCi [32P] UTP only; 2, injection with TFIIIA mRNA, 3, injection with Gam1 mRNA. (B) Autoradiographs for 4 experiments were scanned and 5S rRNA quantitated relative to tRNA using ImageJ software. Error bars indicate the standard deviation. 49

Western blots were used, with antibody specific to X. laevis TFIIIA, to rule out the possibility that the increase in oocyte-type 5S rRNA transcription was due to an increase in TFIIIA protein. Figure 6 A shows that there is no increase in levels of the TFIIIA protein in Gam1-injected embryos at various stages in development. On the contrary, when multiple experiments are completed, quantified, and normalized to the earliest time point (late blastula) there seems to be a small decrease in the amount of TFIIIA present in later stages of Gam1-injected embryos (Fig. 6 B). This provides evidence that the decrease in SUMOylation activity, not increased levels of TFIIIA, is responsible for the increase in transcription of the oocyte-type 5S rRNA genes.

Verifying the knockdown of SUMOylation activity in Xenopus embryo extract

SUMOylation tests with a verified SUMO substrate were used to confirm that E1 enzyme activity was diminished in Xenopus embryos injected with increasing amounts of Gam1 mRNA (0.1 ng, 0.5 ng, or 1.0 ng). Whole embryos were homogenized in lysis buffer containing protease inhibitors and extract was used as the source of E1 enzyme activity in an in vitro assay. E1 activity was measured by the ability of a 25 kDa polypeptide, E2-25K, containing a SUMO consensus motif to be SUMOylated as seen by a shift in molecular weight from 25 kDa to ~36 kDa. Control reactions show that the polypeptide was SUMOylated in the presence of E1 enzyme, E2 enzyme, SUMO-1, and

ATP (Fig. 7) as indicated by the presence of two bands on the western blot. However,

Figure 6. TFIIIA levels in Gam1-injected embryos. (A) Western blot developed with antibody specific to TFIIIA was used to analyze extracts from Gam1 and H2O (control) injected embryos at late blastula, early gastrula, or early neurula. Lanes 1, 3, 5 injected with Gam1 mRNA (5 ng), lanes 2, 4, 6 injected with H2O. (B) Westerns from 3 experiments were scanned and quantified using ImageJ software. TFIIIA levels in Gam1 and H2O injected embryos were normalized to late blastula. Error bars indicate the standard deviation. when the E1 enzyme was absent from the reaction, E2-25K no longer is SUMOylated confirming the necessity of E1 in the conjugation of SUMO to this target.

To test the activity of E1 enzyme in Gam1-injected embryos, extract from homogenized control (water-injected) or Gam1-injected embryos was used as the source of E1 enzyme in the in vitro SUMOylation assays. Embryos were collected at four time points, mid-blastula, late blastula, early gastrula, and late neurula. These times were chosen to span critical stages in development including the mid-blastula transition

(when zygotic transcription begins) and the gastrula-neurula transition (when organogenesis begins) (26, 50). When reaction components (E2, SUMO-1, E2-25K, ATP) were mixed with extract from water-injected embryos, a slower migrating band corresponding to SUMOylated E2-25K is detected at all time points (Fig. 7) indicating that extract from control embryos contains enough E1 enzyme activity to support

SUMOylation.

However, embryos injected with any of the three amounts of Gam1 show a lack of E1 enzyme activity at the mid-blastula time point observed as a loss of SUMOylation of the target peptide. The SUMOylation activity of the extract recovers at the late blastula time point for all amounts of injected Gam1. SUMOylation activity at this time point can be explained by the activation of zygotic transcription at the mid-blastula transition (26). This marked increase in levels of mRNA and subsequent protein translation could cause a temporary increase in E1 restoring SUMOylation activity transiently. Shortly after the MBT, the SUMOylation activity of extracts decreases in a dose dependant manner. Embryos injected with 0.1 ng of Gam1 continue to support

SUMOylation of the target peptide through early gastrula and late neurula. However, extract from embryos injected with 0.5 ng or 1.0 ng show a loss of E1 activity by late gastrula and a recovery of the activity by the late neurula stage.

The results of this assay confirm that injection of Gam1 into one cell embryos is a rapid and effective way to inhibit SUMOylation at the earliest time points in development. In addition, while the embryo recovers its ability to carry out

SUMOylation by the late neurula stage, the effects of SUMOylation depletion are 52

Figure 7. Expression of Gam1 in embryos eliminates E1 enzyme activity. E1 activity in extract prepared from in Gam1 or H2O (control) injected embryos was measured by western blot using a 25 kDa SUMO substrate peptide. The SUMOylated substrate peptide is ~36 kDa. Lane 1, peptide incubated with E1 (SAE1/SAE2) enzyme, E2 (Ubc9) enzyme, SUMO1, and ATP; 2, peptide incubated with E2 (Ubc9) enzyme, SUMO1, and ATP; 3, peptide incubated with extract from H2O (control) embryos; 4, peptide incubated with extract from Gam1 (0.1 ng) injected embryos; 5, peptide incubated with extract from Gam1 (0.5 ng) injected embryos; 6, peptide incubated with extract from Gam1 (1.0 ng) injected embryos. Reactions in lanes 3-6 also contained E2 (Ubc9) enzyme, SUMO1, and ATP. Extract was prepared from embryos at the indicated stage. Positions of molecular weight markers are indicated.

propagated well into the late tadpole stages. This demonstrates that processes controlled by SUMOylation may begin early and are important for proper patterning of the later stages of development.

SUMOylation of TFIIIA

An important question is whether TFIIIA is a target of SUMOylation. While there is no consensus sequence (ψKxE, where ψ is a hydrophobic residue and x is any amino acid) in TFIIIA, a possible site (CKEE46) is close to the major site of phosphorylation at serine-16. Detection of SUMOylated TFIIIA using immunoprecipitation of endogenous protein from early neurula embryo extract followed by analysis using either western blots or mass spectrometry was attempted. Neither approach was conclusive. Weak bands that potentially correspond to SUMOylated TFIIIA (at ~50 kDa) were detected on western blots and peptides corresponding to SUMO-1 and SUMO-2/3 were detected in the mass spectra of immunoprecipitated TFIIIA. It is well established that the steady- state levels of SUMOylated protein are generally low, typically less than 5% (103–105); nonetheless, the results from both analyses were not sufficient to establish that there is a pool of SUMOylated TFIIIA in embryos. TFIIIA can be SUMOylated (Fig. 8) as shown by in vitro reactions. TFIIIA protein was mixed with SUMOylation reaction components (E1 and E2 enzymes, ATP, and SUMO-1) and resolved on a polyacrylamide gel. Western blots were developed with antibody specific to Xenopus TFIIIA and an increase in molecular weight is seen for SUMOylated TFIIIA. While in vitro TFIIIA SUMOylation is

evident, it is not possible at this time to say whether the transcription factor is an actual target of this modification in vivo.

Figure 8. SUMOylation of TFIIIA. In vitro SUMOylation of TFIIIA (38 kDa) was tested using purified E1 and E2 enzymes. Products of the reactions were analyzed by a western blot developed with TFIIIA antibody. Lane 1, TFIIIA with SUMO1 and E1 and E2 enzymes; 2, TFIIIA alone. The positions of molecular weight markers are indicated.

Occupancy of the 5S rRNA genes during embryogenesis

The effect of Gam1 on the transcription of the oocyte 5S rRNA genes implicates

SUMO in the terminal repression of these genes. To explore this effect in more detail, chromatin immunoprecipitation (ChIP) assays were used to test for the presence of

SUMO, PIAS2b, and XCtBP on the 5S rRNA genes. The ChIP assays were used to monitor the promoter occupancy of the oocyte and somatic genes at time points following the resumption of transcription at the MBT, specifically late blastula, early gastrula, late gastrula, early neurula, and late neurula. These time points also span the reprogramming that occurs at the gastrula-neurula transition. Both SUMO-1 and

SUMO-2/3 are present on the oocyte 5S rRNA genes at late blastula and maintain a constant level through all time points (Fig. 9). There are several potential targets of

SUMOylation that can account for this result, including PIAS2b (106), CtBP (107, 108), histone proteins (109, 110), and additional co-repressors/chromatin modifiers

(reviewed in Garcia-Dominguez et al. 2009). The detection of both SUMO-1 and SUMO-

2/3 may indicate that there are multiple targets associated with the oocyte genes; however, some proteins may be modified by more than one isoform (11) and poly-

SUMO-2/3 chains present on some proteins can be terminated by SUMO-1 (112).

Contrary to current models of regulation the Xenopus 5S rRNA genes, the ChIP assays revealed an approximately constant amount of TFIIIA associated with the oocyte- type genes at all time points. There is a similar amount of PIAS2b, indicating that the transcription factor immediately recruits the ligase upon binding or that the two bind as 57

a preformed complex (Fig. 9). TFIIIA and PIAS2b remain bound to the oocyte 5S rRNA genes through late neurula when these genes are fully repressed. Prior work determined that there is a constant amount of PIAS2b protein present from stage II oocytes through the late neurula stage of embryogenesis. Sub-cellular localization of the protein changes drastically beginning at mid-blastula. The protein is primarily located in the cytoplasm in oogenesis and early embryogenesis however becomes increasingly nuclear at the mid-blastula transition and is primarily nuclear by early gastrula.

The ChIP assays were only used qualitatively, since the DNA content of early

Xenopus embryos changes substantially due to the loss of amplified rDNA that dissipates during the first 15 hours after fertilization (113, 114). Initially, the amplified, extrachromosomal rDNA is approximately 30 pg compared to 12 pg of total chromosomal DNA (115). Notwithstanding this complication, we estimate that less than

1% of the oocyte-type 5S rRNA genes are occupied by TFIIIA.

The CtBP proteins are transcriptional co-repressors that are found in a pool of free protein as well as components of large multisubunit complexes that contain histone modifying enzymes, chromatin associated proteins, and other co-repressors (116, 117).

Western blot analysis indicates that CtBP appears at the end of oogenesis and remains relatively constant throughout embryogenesis with a slight increase seen in the late gastrula and early neurula time points. Notably, CtBP co-repressor activity is controlled by SUMOylation with PIAS2b (PIASxβ) serving as a specific E3 ligase for this protein

(108). The ChIP experiment shows that XCtBP binding to the oocyte genes is transient. 58

In earlier experiments, initial binding wasn’t detected until the early neurula stage; however, repetitions show binding as early as early gastrula. In all experiments, XCtBP binding persists into neurula stage, but oftentimes declines at the last time point, late neurula. This behavior is consistent with the CtBP complex providing chromatin modifying activity exactly at the developmental stage, the gastrula-neurula transition, when there is a coordinated repression of several genes transcribed by RNA polymerase

III, specifically the oocyte-type 5S rRNA genes (118).

It has been demonstrated that, in the case of X. laevis oocyte 5S rRNA genes, histone acetylation does not override the repressive effect of the positioned nucleosome (5) which is different from the X. borealis genes where histone modification, specifically of H3 and H4, does impact TFIIIA access (119–123).

Nonetheless, there is some evidence for differences in chromatin structure between the two types of X. laevis 5S rRNA genes in somatic cells; histone H4 on the oocyte-type genes is hypoacetylated in contrast to the somatic-type that is hyperacetylated (124).

The CtBP complex carries several chromatin modifying activities including histone deacetylases, methylases, and a demethylase that contribute to its activity as a transcriptional corepressor. The two methylases, EHMT1 and EHMT2, target H3K9 and, more weakly, H3K27; both modifications are a signature of transcriptionally repressed chromatin. Additional ChIP assays, using antibodies specific for these repressive modifications, were used to determine whether they occur and, if so, whether they correlate temporally with the binding of XCtBP.

Dimethylation of H3K9 is first detected at late blastula and reaches a maximum at late gastrula; the less pronounced trimethylation of H3K27 is first detected at early gastrula and slowly increases during progression to early neurula (Fig. 9).

H3K9me2 seems to precede the appearance of XCtBP on the oocyte-type genes, which would indicate that this co-repressor complex is not responsible for this particular modification, while the appearance of H3K27me3 more closely parallels the appearance of XCtBP. It is equally possible that XCtBP fulfills some role other than histone modification. H3K9me2 and H3K27me3 are detected on the oocyte, but not somatic genes, which along with the differential acetylation of H4 (124), clearly distinguishes the chromatin structure of the two different 5S rRNA gene families.

The somatic 5S rRNA genes showed no evidence of occupation by any SUMO- modified proteins, PIAS2b, or CtBP; only the presence of TFIIIA is detect. As expected, the repressive chromatin marks, methylated H3K9 and H3K27, are absent from the somatic-type genes.

Since reduction of SUMOylation activity increases transcription of the oocyte- type genes (Fig. 5), additional ChIP assays were used to determine whether there is a corresponding change in the occupancy of the 5S rRNA genes in Gam1-injected embryos. With the repeated caveat that these ChIP assays are semi-quantitative at best, the amount of SUMO protein on the oocyte genes is decreased, although not eliminated. The presence of TFIIIA is mostly unaffected, but there is a small decrease in the amount of PIAS2b and XCtBP.

Figure 9. Occupancy of the 5S rRNA genes during early development. ChIP assays of embryos at the indicated stage of development tested for the presence of specified proteins or histone modifications on the oocyte or somatic 5S rRNA genes. LB-late blastula, EG-early gastrula, LG-late gastrula, EN-early neurula, LN-late neurula. Antibodies used: lane 1, control protein A-Sepharose beads alone; lane 2, SUMO-1; lane 3, SUMO-2/3; lane 4, H3K9me2; lane 5, H3K27me3; lane 6, XCtBP; lane 7, PIAS2b; lane 8, TFIIIA; lane 9, input DNA. Input oocyte DNA (0.019%) or somatic DNA (0.63%) was amplified to normalize the

amount of chromatin from each stage. (A) Oocyte 5S rRNA

genes. (B) Oocyte 5S rRNA genes from Gam1-injected embryos. (C) Somatic 5S rRNA genes. (D) Somatic 5S rRNA genes from Gam1-injected embryos.

There is a much more discernible decrease in H3K9me2 and, especially,

H3K27me3. Thus, the deficiency in SUMOylation has affected the chromatin structure of the oocyte-type genes, decreasing their repressive mark and accounting for their increased transcription. Interestingly, the ChIP assay shows a considerable loss, to nearly undetectable levels, of TFIIIA from the somatic 5S rRNA genes in the Gam1- injected embryos. This suggests that the reduction of SUMOylation activity allows the oocyte genes, which outnumber the somatic genes by 50:1, to compete for the declining amounts of TFIIIA. This phenomenon would also explain why the increased transcription of 5S rRNA genes in Gam1-injected embryos never reaches the same level as embryos injected with mRNA encoding TFIIIA.

Subnuclear localization of TFIIIA in Gam1-injected embryos

The current model for the terminal repression of the oocyte 5S rRNA genes has a positioned nucleosome excluding the binding of TFIIIA to the internal promoter (64, 125,

126). However, the ChIP assays demonstrate that some amount of TFIIIA is normally associated with the oocyte genes past the time when they become fully repressed. In X. laevis, the tandem repeats of the oocyte-type genes are found at the end of the long arm of most chromosomes and are distributed around the nuclear periphery of interphase somatic cells (43). Previous work has shown, at the mid-blastula much of the transcription factor is nuclear and the first indication of placement around the nuclear periphery is detectable. This arrangement is well established at early gastrula

(Fig. 10 A) and is retained though mid-neurula stage (Fig.10 B). The single repeat of the somatic-type genes cannot account for observed staining pattern. These images support the ChIP experiments that detect the presence of TFIIIA on the oocyte 5S rRNA genes during the same period. In addition, the robust methylation of histone H3K9 on the oocyte-type genes is consistent with two recent reports that this modification is required for positioning transcriptionally repressed chromatin at the nuclear periphery

(127, 128).

Since reduction of SUMOylation activity by Gam1 partially relieves repression of the oocyte-type genes, experiments were conducted to determine whether loss of this activity also affects the subnuclear localization of the 5S rRNA genes.

Immunohistochemical staining of Gam1-injected embryos did show changes in TFIIIA staining to various degrees relative to water-injected controls (Fig. 10 C-D). Staining of the nuclear periphery was decreased in some cases and the pattern of TFIIIA that does remain in this region appears aggregated into fewer, but in some cases larger, structures. It was concluded that the loss of SUMOylation does perturb the normal peripheral arrangement of the oocyte-type genes to various degrees. However, it is important to caution that the 5S rRNA repeats are close to the telomeres, whose positioning at the nuclear periphery also appears to require SUMOylation activity (129–

131). Therefore, at this time, it is not possible to judge whether the disrupted staining pattern of TFIIIA in Gam1 embryos arises from changes in the chromatin structure of the

5S rRNA genes, of telomeres, or both. The degree to which the heterochromatic

structure of the oocyte 5S rRNA genes may contribute to the subnuclear positioning of telomeres will be important to investigate.

Figure 10. Localization of TFIIIA to the nuclear periphery. TFIIIA antibody directly conjugated to Alexa Fluor 568 was used to immunohistochemically stain embryos at the indicated stage of development. Embryos were counter stained with DAPI to identify the nucleus. (A) nucleus of an early gastrula (stage 10) embryo. (B) nucleus of a mid-neurula (stage 19) embryo. (C) nucleus of an early gastrula embryo injected with Gam1. (D) nucleus of a mid-neurula embryo injected with Gam1 mRNA. The first column is superimposed TFIIIA and DAPI staining, the middle column is DAPI staining, and the last column is TFIIIA staining.

DISCUSSION:

REGULATION OF THE 5S RIBOSOMAL RNA GENES

Taking into consideration the increasing amounts of somatic histone H1 and the declining amounts of TFIIIA that occur simultaneously during the early stages of embryogenesis, the current model of repression of the oocyte-type genes is based on a simple competition between factor binding and nucleosome organization. There is a considerable amount of evidence that histone H1 positions a nucleosome over much of the internal promoter of the X. laevis oocyte 5S rRNA genes excluding TFIIIA, while the nucleosome that occupies the somatic 5S rRNA genes permits binding of TFIIIA and formation of an active transcription initiation complex (6, 59–62, 64, 65, 125, 126).

This model is also supported by the observation that siRNA-mediated reduction of H1 levels leads to a delay in repression of the oocyte-type genes (59, 61). Data presented in this thesis show that the differential repression of the oocyte 5S rRNA genes does not occur though a passive mechanism of competition, but rather that TFIIIA itself actively orchestrates this process which involves SUMOylation, the co-repressor XCtBP, and histone modifications that lead to subnuclear localization and ultimate repression of the oocyte-type genes.

ChIP assays have revealed that TFIIIA is associated with the oocyte-type genes well into neurula stage, although it is estimated that only a small fraction are occupied by the factor. The tandem repeats of the oocyte-type genes are found near the telomeres of most chromosomes and become localized to the nuclear periphery in somatic cells (43). The confocal images of embryos immunohistochemically stained with

TFIIIA antibody, showing its location at the nuclear periphery, provide a direct visual conformation of its association with the oocyte-type genes. Complete repression of the oocyte-type genes occurs at the gastrula neurula transition (GNT) (118); yet, there is still an excess of TFIIIA (~90 molecules/ 5S rRNA genes) over the total number of 5S rRNA genes at this developmental stage (46, 48). That is sufficient to bind the somatic and some fraction of the oocyte 5S rRNA genes. Additional evidence that TFIIIA is not limiting during this period comes from two observations. Ribozyme-depletion of histone H1 is sufficient to activate transcription of oocyte 5S rRNA genes at least through the GNT (59, 61) and Gam1 inhibition of SUMOylation activity has the same effect. In combination, these observations indicate that the formation of the repressive chromatin structure established by histone H1 depends on SUMOylation activity at the oocyte-type genes due to the ability of TFIIIA to recruit the SUMO ligase, PIAS2b, and the co-repressor, XCtBP to this subset of genes.

An outstanding question to still be addressed is how TFIIIA is able to function simultaneously as an activator and apparent repressor of transcription. It is firmly established that the A:T-rich sequences that flank the oocyte-genes dictate repression of transcription (62, 65, 132). Sera and Wolffe (1998) determined that the A:T-rich 69

sequence immediately downstream of the oocyte genes between nucleotide positions

123 and 144, the predicted binding site for histone H1, is the major determinant for positioning of the nucleosome. It is noteworthy that TFIIIA binds to the 5S rRNA genes in an extended conformation oriented with its N-terminal end at approximately nucleotide 96. Previous yeast two-hybrid experiments establish that PIAS2b and XCtBP bind to TFIIIA within the first three zinc fingers of the factor, suggesting that these two proteins are positioned near or at this A:T tract. PIAS2b, like nearly all members of the

PIAS family, possesses an SAP domain that has high affinity for A:T-rich sequences (133).

Through this DNA-binding domain, PIAS2b possibly stabilizes TFIIIA binding to a subset of oocyte genes during a period when much of the region is being organized into a repressive chromatin structure. Indeed, TFIIIA, occupying the binding site for the core histone octamer, along with PIAS2b, occupying the site of H1, may be integrated into the ordered array of nucleosomes that occupy the oocyte 5S rRNA gene repeats in somatic cells (4, 134). This proposal also has the potential to explain the absence of

PIAS2b from the somatic-type genes that are flanked by G:C-rich sequences. There could also be a SUMOylation independent role for PIAS2b, in addition to its E3 ligase activity, in converting TFIIIA to a repressor on the oocyte-type genes as has been seen in other transcriptional repressors (133, 135). The DNA-binding specificity of the transcription factor Msx1 is regulated by its interaction with PIAS1 in a SUMO- independent mechanism (136). The interaction of Msx1 with PIAS1, but not

SUMOylation of Msx1, is necessary for localization of Msx1 at the nuclear periphery placing it in proximity to target genes such as MyoD and thereby controlling its 70

transcriptional activity. Similarly, the specific interaction of FLI-1 with the SUMO ligase,

PIASxα, regulates its subnuclear localization and represses the transcriptional activity of

FLI-1 (137). This repression is not dependant on the SUMOylation state of FLI-1 but on the ability of PIASxα to recruit FLI-1 to an inactive complex.

XCtBP potentially accounts for much of the chromatin modifications found on the oocyte-type genes by virtue of its presence in a large complex that contains histone demethylase, histone methyltransferase, histone deacetylase, and SUMO ligase activities as well as an assortment of other transcriptional co-regulatory proteins (116).

SUMOylation of CtBP1 has been reported to control transcriptional repression with

PIAS2b serving as its SUMO ligase in HeLa cells (108). Previous studies in our laboratory have provided the first evidence for a direct interaction of PIAS2b with a member of the

CtBP family. In addition, the CtBP complex itself contains the polycomb protein,

Pc2/CBX4, which is another SUMO ligase that targets CtBP and promotes conjugation to either SUMO-1 or SUMO-3 (107). At least eight of the components of the CtBP complex can be SUMOylated, perhaps explaining our detection of both SUMO1 and SUMO2/3 on the oocyte-type genes (111).

The histone modifying activities in the CtBP complex can account for the presently known differences between chromatin structure of the oocyte versus somatic

5S rRNA genes. The deacetylases HDAC1 and HDAC2 can not only establish the hypoacetylated state of histone H4 on the oocyte 5S rRNA genes (124), but they are likely needed for the eventual methylation of H3K9 and H3K27 that can be accomplished with the methyltransferases, EHMT1 and EHMT2 that are also constituents of the 71

complex. The dimethylation of histone H3 on the oocyte-type genes reflects both their repressed state and also their localization to the nuclear periphery in somatic cells.

Consistent with the differential modification of histones on the two types of 5S rRNA genes observed here, a genome-wide analysis of human pol III genes revealed that histones flanking inactive tRNA genes are minimally acetylated, but are marked by

H3K9me3 and H3K27me2/3. Thus, just as for pol II genes, these histone modifications act as a signature of chromatin associated with repressed genes transcribed by pol III

(138).

There have been several observations linking transcriptional repression to the positioning of genes at the nuclear lamina (139) and there is now evidence that methylation of H3K9 plays an essential role in this process (127, 128). Transcriptionally silent regions found in lamina associated domains (LADs) are highly enriched for

H3K9me2 (reviewed in Towbin et al. 2013), consistent with the strong ChIP signal measured for this modification exclusively on the oocyte-type genes. Experiments in C. elegans indicate that mono or dimethylation of H3K9 is specifically required for perinuclear localization with trimethylation needed for transcriptional repression that may be reinforced by methylation at H3K27 (128), which was also found exclusively on the Xenopus oocyte-type genes. In combination, the results indicate that maintenance of the transcriptional repression of the oocyte 5S rRNA genes relies on H3K9me2- dependent localization to the nuclear periphery.

While the CtBP complex contains the methyltransferase activities needed to generate the H3K9 and H3K27 methylation observed, the H3K9 modification seems to 72

occur prior to the binding of XCtBP on the oocyte-type gene. Therefore, additional histone methyltransferases may be present which are independent of the CtBP complex.

Specifically, H3K9 methylation is often attributed to Suv39h1,2 transferase activity which is necessary for chromatin stability and segregation in mice (141). Additionally,

Suv29h1 modification of H3K9 is necessary for proper organogenesis in early Zebrafish development and may act as a tissue specific regulator of gene expression (142). This redundancy in enzyme activity provides an explanation for the presence of the

H3K9me2 signal on the oocyte-type gene prior to the binding of the CtBP complex.

The involvement of SUMOylation in the global repression of the Xenopus oocyte

5S rRNA genes has striking parallels with other complexes that promote the formation of heterochromatic domains (111, 143). The zinc finger protein Sp3 is converted from an activator to a potent repressor of transcription by conjugation to SUMO-1 (or SUMO-

2 in vitro) with PIAS1 serving as a specific ligase (144, 145). The modification results in the localization of Sp3 to the nuclear periphery in a pattern remarkably similar to that of

TFIIIA (144). The SUMOylated form of Sp3 seeds the formation of a repressive complex that establishes heterochromatic marks, including H3K9me3 (146), and that also contains at least one Polycomb protein (147).

The Polycomb group (PcG) proteins form multimeric complexes that maintain the repression of homeotic genes and other target loci. The complexes are able to mediate their effect over large distances, which depend on the formation of subnuclear structures referred to as PcG bodies that contain histone methyltransferase activity.

The PcG protein, Pc2, is a SUMO ligase that recruits CtBP and the SUMO conjugating, E2 73

enzyme, to PcG bodies and promotes the SUMOylation of CtBP (107). As noted above, in addition to being part of polycomb repressive complex 1, Pc2 (CBX4) is also a component of the CtBP repressor complex (116).

One of the first identified targets of SUMOylation was the PML protein (148) that multimerizes to form highly heterogeneous protein complexes called PML nuclear bodies (149). PML protein like many constituents of this subnuclear structure is both

SUMOylated and contains a SUMO interaction motif (SIM) that enables a network of complex, higher-order, protein-protein interactions. The PLM nuclear bodies have been implicated in several processes including transcriptional regulation (149). With these examples in mind, it is not surprising that the repression of the multiple tandem repeats of the oocyte-type genes uses a similar strategy for the formation of heterochromatin over a considerable distance that becomes spatially segregated at the nuclear periphery.

The H3K9 and H3K27 methylation detected in the ChIP assays not only accounts for transcriptional repression, but also the perinuclear organization of the 5S rRNA genes. The ability to decipher cause and effect in these large complexes (CtBP, Sp3,

PML) is impeded by the fact that many of the component proteins, like PML protein, are

SUMOylated and also possess a SIM. Thus, it is difficult to determine whether

SUMOylation activity recruits histone modification activity or vice versa. This paradox may actually explain the self-amplifying activity of complexes that have the ability to regulate gene activity over very large distances. Indeed, the periodic positioning of

TFIIIA within the tandem repeats of the oocyte-type genes would fit a model in which it recruits chromatin modifying activities that can act over an extended length. It is 74

important to note that the active participation of TFIIIA in directing the formation of the repressive chromatin structure and subcellular localization of the oocyte 5S rRNA genes does not eliminate the role of histone H1 in positioning nucleosomes, but rather adds another layer to establishing and/or maintaining the permanently repressed state of these genes.

ChIP assays have shown that some amount of TFIIIA remains associated with the

Xenopus oocyte 5S rRNA genes after they are permanently repressed. This observation can account for the cellular amount of TFIIIA during this stage of embryogenesis, which greatly exceeds the total number of somatic- and oocyte-type genes. It is also consistent with the ability to activate the oocyte-type genes by simply lowering the level of histone H1 (59, 61). The repression of the oocyte-type genes depends on

SUMOylation activity that can be traced to an interaction between the transcription factor and a SUMO ligase, PIAS2b, and one of its targets, XCtBP. It has not been possible to determine whether TFIIIA itself is SUMOylated in vivo. However, early studies of

TFIIIA isolated a larger form, approximately 40 kDa, of the protein in late stage embryos and adult tissue which did not support the 5S rRNA synthesis (46). The 40 kDa form is identical to the 37 kDa form in structure and researchers could not determine the functional differences. It is possible that this slower migrating form indicates a TFIIIA-

SUMO conjugate that acts as a repressor of 5S rRNA. The steady state level of SUMO modification of most proteins is generally low; this has been termed the “SUMO enigma” (104). Models to explain this phenomenon postulate that the modification is transient: a SUMOylated protein enters into some interaction and, once established, the 75

modification can be rapidly removed by one of several SUMO-specific proteases without disrupting the integrity of the complex (104). Our estimate that only a small fraction of the oocyte genes is occupied by TFIIIA combined with a short half-life for the modification may explain the difficulty in detecting the conjugated form of the transcription factor. However, it is equally possible that TFIIIA simply acts as a scaffold to bring together PIAS2b and its target XCtBP.

RESULTS AND DISCUSSION:

ROLE OF SUMOYLATION IN EARLY DEVELOPMENT OF XENOPUS LAEVIS

Global regulation of gene expression by SUMOylation

SUMOylation contributes to the differential regulation of the 5S rRNA oocyte- type and somatic-type genes in early X. laevis embryogenesis. In order to understand the broader role of SUMOylation in developing embryos; Gam1 was expressed in one cell embryos resulting in the loss of SUMOylation. Expression was achieved through microinjection of one cell embryos with capped mRNA encoding Gam1. Embryos were observed for developmental defects and changes in gene expression were determined through microarray and subsequent pathway analysis. Xenopus laevis is a well studied and important model system for early vertebrate development. Many similarities exist between frog and human development thereby allowing parallels to be drawn between research on Xenopus and treatments for human disease (150, 151). Processes in early development such as axis patterning, germ layer establishment, and organ formation are conserved throughout vertebrate species with only subtle differences. For example, the final amphibian heart is a three chambered structure, whereas the final human heart is four chambers; however, the earliest structures of the heart, including the heart

fields and heart tube (the structure that occurs prior to looping) are remarkably conserved among species (152).

SUMOylation knockdown causes reproducible developmental defects

Embryos were injected with two different amounts of Gam1, 5 ng or 0.5 ng.

Embryos injected with 5 ng of Gam1 did not survive past the gastrula-neurula transition

(GNT, ~15 hpf). In contrast, a mutant form of Gam1 (L258A, L265A) that cannot bind to the SAE1 subunit and therefore cannot disrupt SUMOylation did not cause a decrease in viability when injected with the same amount (5 ng). Embryos begin organogenesis at the GNT and a decrease in embryo viability at this stage indicates that SUMOylation plays an important role in orchestrating the necessary changes. This is not surprising since other model systems have also shown a requirement for SUMOylation activity for viability of embryos including zebrafish, Drosophila, and mouse (12, 13, 19, 153), although the studies in mice have yielded conflicting results (16, 17).

In order to examine the role of SUMOylation in developing embryos, a tenfold smaller amount of Gam1 mRNA, 0.5 ng, was injected. This experiment follows the observation that mice with a SUMO-1 haploinsufficiency survive embryogenesis longer and present distinct phenotypes including cleft lip and palate (14). As before, one cell embryos were injected with capped Gam1 mRNA and observed throughout development. Embryos were also injected with the same volume of H2O to serve as negative controls.

Figure 11 A shows Gam1-injected embryos as compared to water-injected embryos at the late gastrula stage in development (stage 12, ~13 hpf). Embryos injected with Gam1 or water develop normally, as compared to uninjected controls, until the gastrula stage. Normally, at the gastrula stage in development, the prospective mesoderm begins to involute and the embryo forms a blastopore (32, 35), which is a ring of contracting cells which eventually close and force the endodermal and mesodermal tissue into the interior of the embryo. Cell movements occur normally in embryos injected with water, however, embryos injected with Gam1 show delayed or incomplete closure of their blastopore. In some cases, blastopores of Gam1-injected embryos were able to close but at a much slower rate than water or uninjected controls.

This delayed closure was observed through time lapse photography of water and Gam1- injected embryos. In some instances, these embryos develop normally from this stage onward.

In other Gam1-injected embryos, the blastopore fails to close completely.

Embryos in which the blastopore fails to close do not develop normally and have cascading effects in the neurula stage of development. During neurulation, the neural tube forms from the convergence and extension of the outermost layer of cells on the embryo (33, 39). The posterior portion of the neural tube closes at or very near to the point of blastopore closure. Therefore, when the blastopore fails to close properly, the neural tube also fails to close properly and embryos display a spina bifida like phenotype in the late neurula stage (stage 24, 26 hpf) (Fig. 11 B). In the most drastic cases,

embryos injected with Gam1 present completely open neural tubes with failure of the neural folds to fuse together along the entire anterior-posterior axis (Fig. 11 C).

Embryos that are viable at the late neurula stage usually develop into free swimming tadpoles (>48 hpf). Gam1 injected embryos at this stage of development have shortened or bent axes (Fig. 11 D) due to the failure of the neural tube to close properly. Additionally, the majority of embryos display a certain degree of edema, or fluid collection, in the ventral regions of the embryo specifically around the heart and digestive system. In conjunction with edema and a shortened axis, embryos display defects in eye, heart, and gut development, including cyclopia (one eye) or eyes that are not fully separate (fused eye) (Fig. 11 E) and disordered digestive system.

Defects in the heart are more severe and include improper looping, disruption of chamber formation, and an absence of blood flow. Normal amphibian hearts contain three chambers, two atria and one ventricle, and looping of the outflow track to the right. Hearts of Gam1-injected embryos are absent of discernible chambers and seem to have not developed beyond a heart tube structure due to a lack of proper looping

(Fig. 11 E). Additionally, heart structures that have formed contain little to no blood past the stage in development when control matched embryos have begun producing blood. Surprisingly, these embryos are viable and have heart structures that continue to beat, although contractions are erratic. Defects in these later forming organs indicate a role for SUMOylation throughout the extent of development, not simply at the earliest stages.

Figure 11. Developmental defects of Gam1-injected embryos. (A) Page 82: Blastopores of late gastrula stage embryos injected with Gam1 (top left) close slower than those injected with H2O (top right) and cells of the yolk plug fail to migrate inward in Gam1 (bottom left) when compared to H2O injected embryos (bottom right). (B) Page 82: Late neurula stage embryos injected with Gam1 (left) display open neural tubes and severe edema on the dorsal side of the embryo compared to normal developing H2O injected controls (right). (C) Page 83: Two day old Gam1 injected embryos have shortened axes (lower left) and split tails (lower right) when compared to the elongation of the axis in control embryos (top). (D) Page 83: Free swimming tadpole stage embryos (6 days) injected with Gam1 (left) display severe edema and a shortened axis compared to H2O injected controls (right) of the same stage. (E) Page 83: Gam1-injected embryos also display eye defects (left) and defects in heart looping (middle) when compared to hearts of H2O injected embryos (right). Heart structures in control embryos are out lined indicating two upper chambers, atria (pink and red), and a lower chamber, ventricle (black). The heart tube structure of the Gam1 injected embryo is outlined in black with no discernible chambers.

Microarray analysis of Gam1-injected embryos spanning the GNT

It is clear from the phenotypes that knockdown of SUMOylation has a profound impact on early development of Xenopus. Previous studies using a morpholino against

SUMO-1 have shown similar phenotypes of a shortened or kinked axis (20). This study also tested the expression levels of a small number of known mesoderm marker genes

(Xbra, Gsc, Chd) induced by the activin/nodal pathway. It was found that morpholino- based knockdown of SUMO-1 not only disrupted axis elongation but specifically down- regulated the mesodermal cell markers and directly disrupted this important signaling pathway.

Even though Xenopus has been used as a model system for decades, many aspects of development are still largely unknown. An enormous amount of regulation at the level of transcription must occur in order to differentiate one cell type from another.

Differential expression of transcription factors cannot be the only mechanism for regulating target genes as certain transcription factors are ubiquitously expressed throughout the developing embryo yet still have tissue specific functions. Regulation of ubiquitously expressed transcription factors can be achieved through utilization of cofactors, cell specific alternative splicing, protein degradation, subcellular compartmentalization, and post translational modification including acetylation, phosphorylation, ubiquitination, and SUMOylation (8, 77, 88, 154–159). Cofactor binding and post translational modifications of transcription factors seemingly provide a

quicker molecular ‘switch’ between active and repressive states than protein degradation or alternative splicing and mounting evidence has established a role for

SUMOylation in the regulation of transcription factors (7).

Microarray analysis provides the benefit of measuring the expression levels of thousands of genes at one time allowing for rapid and global measurement of mRNA level changes (160). To understand the effect of Gam1 on development at the level of transcription, total RNA was collected from embryos injected with water or with 0.5 ng of Gam1 mRNA. RNA was isolated from embryos at three time points (early gastrula-EG, late gastrula-LG, and early neurula-EN) in order to span the gastrula-neurula transition.

As mentioned previously, this time point in development marks the beginning of many changes in transcription that will lead to organogenesis and patterning of the embryo.

Therefore, many genes are activated or repressed at this point in order to coordinate these precisely timed events.

Total RNA from each time point was analyzed with an Affymetrix GeneChip,

Xenopus 2.0. The collected data files were processed, using the Bioconductor software package, and normalized using quantile normalization methods (161). The data were subjected to Student’s t-test using a p-value threshold of less than 0.05 to determine those genes that were differentially regulated between the water and Gam1 mRNA injected embryos.

Volcano plots (Fig. 12) display genes based on their statistical significance (p- value) and fold change at each of the different time points. The p-values are plotted with an inverse log so the larger the p-value (higher on the chart) the more statistically 85

significant the difference. The fold change is plotted so a negative value is a down- regulation and a positive indicates an up-regulation of genes in Gam1 embryos relative to controls. An increase in the number of differentially expressed genes occurs between the EG (Fig. 12 A) and LG (Fig. 12 B) time points and again between the LG and the EN

(Fig. 12 C) time points. This indicates that a greater number of genes are affected by loss of SUMOylation activity as development proceeds caused either by direct or secondary effects due to down regulation of transcription factors at the earliest time point leading to changes in target genes at the later time points. There is also an increase in the magnitude of changes at the later time points (Fig. 12 D) consistent with an increased or compounding effect throughout development.

Three biological replicates were analyzed on different microarray chips allowing corrections to be made for variations that may occur between egg clutches to be eliminated. Expression values obtained from each experiment were compared to the other two biological replicates for control and Gam1 injected embryos at each time point (Fig. 13). Scatter plots with calculated R-values show that there is a strong correlation between each of the replicates placing confidence in the Gam1 injection and subsequent microarray analysis.

Figure 12. Volcano plots and comparative histogram of genes measured on microarray. (A-C) Genes measured by the microarray chip were plotted, y-axis: –log10(pvalues) and x-axis: log2(fold changes) to compare expression values. A significance values of p<0.05 (black line) indicates the cut off for differential expression. (A, pg.88) early gastrula, (B, pg.88) late gastrula, (C, pg.89) early neurula. (D) Page 89: Histogram of all the measured genes at the three times points showing the number of genes (y- axis) for each measured fold change (x-axis).

Differentially expressed genes, p<0.05, were compiled into various lists for further analysis to identify underlying biological causes for their change and links to phenotypes. At the time of analysis, the X. laevis genome was not sequenced and as a result, many probes on the chip do not have assigned gene names or known functions.

Due to the amount of data generated in this study, only those probes with annotated gene names were used for further analysis. This leaves a number of probes which showed differential expression that are not considered in further analysis. As the function and annotation of more X. laevis genes becomes known, it will be beneficial to revisit the data to update gathered information.

Table A.1 lists the differentially expressed genes from the early gastrula (stage

10, 9 hpf) time point. A total of 94 genes, measured by 94 different probes, were misregulated (53 down-regulated and 41 up-regulated). Additionally, Table A.2 and

Table A.3 list the differentially expressed genes from the late gastrula (stage 12, 13 hpf) and early neurula (stage 14, 16 hpf) time point, respectively. A total of 447 different genes, measured by 474 probes, were misregulated (263 down-regulated and 211 up- regulated) at LG and 742 different genes, measured by 781 probes, were misregulated

(394 down-regulated and 387 up-regulated) at the EN. The difference in numbers between the genes and probes represents a duplication of some probes on the microarray chip. In some cases, multiple probes are made that measure the expression of one gene in order to increase the likelihood of measuring changes since hybridization of targets is often dependant on probe design (162).

Figure 13. Comparison of biological replicates from Gam1 and H2O injected embryos. Expression values for each biological replicate (1, 2, 3) were compared to each other for both H2O (top panel) and Gam1 (bottom panel) injected embryos for each time point. (A, pg. 92) early gastrula, (B, pg. 92) late gastrula, and (C, pg. 93) early neurula. Correlation values (R2) for each comparison are shown above the corresponding scatter plot.

Quantitative real time PCR (qRT-PCR) verification of microarray data

Microarrays provide a large amount of data from one experiment; however, validation by qRT-PCR is standard practice to verify that the microarray data collected can be corroborated by an alternative method. This allows for judgment of the quality of the microarray data collected. Table 2 provides a list of the genes analyzed by the qRT-PCR along with the forward and reverse primers used for each gene. Genes were selected based upon various criteria: magnitude of the differential expression (DMRTA1, ribosomal protein L8, cyclin G1), comparison to an earlier microarray analysis of early X. laevis development (160) (exportin 1, Wnt8b, keratin), and components of signaling pathways that pattern the early embryo (Xbra, forkhead box C1, Wnt8b, goosecoid, chordin) (20). Total RNA from the same samples prepared for the microarray experiments was used as the template for qRT-PCR, and each was run in duplicate.

Samples were taken from both water-injected embryos and Gam1-injected embryos at three time points, early gastrula (EG), late gastrula (LG), and early neurula (EN). Fold changes between the water injected and the Gam1 injected embryos were calculated using the ΔΔCT method of analysis (163) using GAPDH expression to normalize the data.

The fold changes calculated from the qRT-PCR analysis were compared to those calculated from the microarray analysis. Graphical representations of those comparisons are shown in Figure 14 A where fold change is expressed on the log2 scale.

Therefore, any fold change greater than 1.0 indicates an up-regulation, where less than

1.0 indicates a down regulation. Previous studies have shown a strong correlation (80- 94

87%) between microarray data and qRT-PCR verification in direction of regulation (i.e., up-regulated versus down-regulated) (160, 164). In our study there is a 93% directional agreement indicating that the quality of our microarray data is high. Additionally, the microarray fold changes for each gene at each time point were compared to the qRT-

PCR fold changes with a Student’s t-test and no statistical significance (p<0.05) between measurements was found (Fig. 14 B). Discrepancies in directionality between the microarray and qRT-PCR values are characterized by p-values, shown in parentheses, to indicate statistical significance.

Three genes measured (Chrd, Gsc, and rpl8) show little to no differential expression when measured in either the microarray and qRT-PCR experiments.

Similarly, Krt shows minimal down regulation in the microarray or qRT-PCR from LG and

EN. However, the EG qRT-PCR data shows an up-regulation for this gene in disagreement with the microarray (p=0.97). The magnitude and directionality of expression levels for Xbra varied a small degree for the EG (p=0.55) and LG (p=0.69) time points between the microarray and qRT-PCR measurements. Foxc1 gene expression is down-regulated at both the EG and LG time points but shows a discrepancy between the microarray and qRT-PCR data at EN (p=0.45). There is an overall magnitude agreement between qRT-PCR and microarray confirming the extreme down-regulation of Dmrta1 and up-regulation of Ccng1 and Xpo. While Wnt8b expression shows a down regulation for all three time points, the magnitude of down regulation varies between the two measurements.

Figure 14. Comparison of microarray (dark bars) and qRT-PCR (light bars) data. (A) Page 97: Measurements of select genes from the microarray were verified using qRT-PCR. Asterisks indicate samples in which RNA levels were too low to measure by qRT- PCR. Each pair of bars corresponds to one time point ordered from early gastrula to late gastrula to early neurula. (B) Page 98: p-values calculated using Student’s t-test to compare microarray fold changes to qRT-PCR fold changes. A p-value less than 0.05 corresponds to a statistically significant difference between the

calculated fold changes.

* *

B Gene EG LG EN

Dmrta1 ---- 0.11 0.41 rpl8 0.24 0.34 0.99 Xpo 0.34 0.55 0.45 Xbra 0.55 0.69 0.79 Foxc1 0.24 0.88 0.45

98 Wnt8b ---- 0.5 0.39 Krt 0.97 0.36 0.71 Gsc 0.84 0.45 0.84 Chrd 0.83 0.29 0.48 Ccng1 0.49 0.73 0.81

Analysis of microarray data

Differentially regulated genes for each time point were assembled into gene lists to further analyze using data mining software including MetaCore®, BiNGO, DAVID, and the Gene Ontology database. MetaCore® is a software suite compiled for analysis of different types of –omics data including microarrays (165). Specifically, MetaCore® contains powerful algorithms to search experimentally validated data for pathways, functional characterization, protein-protein interactions, and transcriptional regulation.

Differentially regulated genes were uploaded and analyzed through various MetaCore® network building algorithms (Transcriptional Regulation and Enrichment) and pathway maps to form hypotheses about the biological mechanisms affected by the knockdown of SUMOylation.

Transcription factor network building

The majority of changes in gene expression are anticipated to be the result of

SUMO-dependant regulation of transcription factor activity as they are a major target of this modification (7, 166) with initial studies indicating SUMOylation had an overall repressive effect on gene transcription (109, 167, 168). However, as additional targets of SUMOylation are discovered, it is clear that repression is not the sole outcome. For this reason, the network building algorithm ‘Transcription Factor Regulation’ was used 99

to identify common transcription factors that control many of the differentially (both up- and down-regulated) expressed genes. The Transcription Factor Regulation algorithm adds a transcription factor, to the provided gene list, as a seed node and builds a network of interactions around the given factor. Gene lists, from each time point, were analyzed separately and the top 30 transcription factors found to control the most genes from each time point were returned. The connections made within each network are based on direct control of the target gene by the seed transcription factor and, inversely, target gene control of the seed transcription factor. Additionally, the networks include secondary interactions involving target gene interactions with each other. Table 3 lists the top transcription factors returned for the early gastrula time point and the GeneGo developmental processes associated with the 94 differentially expressed genes. Table 4 and Table 5 contain the same analysis and top transcription factors for the late gastrula (447 genes) and early neurula (742 genes) time points, respectively. The lists of transcription factors for each time point are similar and include those like Sp1, HNF4-alpha, c-myc, and p53, that regulate many different genes.

It seems that the effects of SUMOylation are far ranging and, consequently, the most abundant transcription factors were identified by this study. In order to determine if these transcription factors are specific to genes controlled by SUMOylation and thus differentially expressed, the same analysis was carried out using genes not differentially regulated (p>0.05) in the microarray data. Table 6-8 shows the results of the analysis of

11,702 genes from the early gastrula (Table 6), 11,322 genes from late gastrula (Table

7), and 11,019 genes from early neurula (Table 8) time points along with the GeneGo 100

processes associated with these genes. The results for unaffected genes (those not changed by the depletion in SUMOylation activity) and the differentially expressed genes (those changed) are very similar. At the early gastrula time point (Table 3,6) 18 out of 30 transcription factors were common to both sets of genes with the top 5 factors being identical. For the late gastrula stage (Table 4,7) 21 out of 30 factors are common and again the top 5 are identical. At the early neurula time point (Table 5,8) 20 out of 30 factors are common with the top 5 factors remaining identical between the lists. Of the top 5 identical transcription factors, four are common across all three time points (SP1, HNF4-alpha, c-myc, p53). These transcription factors are ubiquitously expressed in most organisms and this analysis indicates a SUMO-dependant control mechanism which allows each factor to specifically regulate a large subset of genes.

This provides one explanation for how ubiquitously expressed factors can have developmentally specific targets.

While the transcription factors between each gene list are similar, the Gene

Ontology processes associated with each list are distinct. Those genes whose expression is not regulated by SUMOylation are enriched for general processes such as cellular process, metabolic process, biosynthetic process, translational elongation, and biological processes. In contrast, differentially regulated genes are associated with processes such as embryo development, regulation of morphogenesis, cell fate specification, organismal development, eye development, and negative regulation of cell communication.

101

TABLE 3.

TOP 30 TRANSCRIPTION FACTORS WHICH REGULATE THE LARGEST NUMBER OF

DIFFERENTIALLY EXPRESSED GENES AT EARLY GASTRULA ALONG WITH THEIR

ASSOCIATED GENE ONTOLOGY PROCESSES. 1

Transcription Gene Ontology Processes Number of Factor Genes SP1 camera-type eye development (31.2%), eye 15 development (31.2%), sensory organ development (31.2%), embryo development (37.5%), positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (37.5%) HNF4-alpha positive regulation of dendrite morphogenesis 14 (13.3%), negative regulation of signaling pathway (26.7%), negative regulation of cellular process (53.3%), negative regulation of microtubule polymerization or depolymerization (13.3%), negative regulation of cell communication (26.7%) c-Myc cell cycle process (42.9%), negative regulation of 13 transcription, DNA-dependent (35.7%), negative regulation of RNA metabolic process (35.7%), cell cycle (42.9%), detection of mechanical stimulus involved in sensory perception of sound (14.3%) p53 negative regulation of transcription from RNA 9 polymerase II promoter (40.0%), negative regulation of transcription, DNA-dependent (40.0%), negative regulation of RNA metabolic process (40.0%), negative regulation of transforming growth factor beta receptor signaling pathway (20.0%), cellular monovalent inorganic cation homeostasis (20.0%) Oct-3/4 cell fate specification (37.5%), positive regulation of 7 neurogenesis (37.5%), regulation of multicellular organismal development (62.5%), positive regulation of dendrite morphogenesis (25.0%), positive regulation of cell development (37.5%)

1 There are 94 differentially regulated genes at this time point.

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TABLE 3 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes STAT3 generation of neurons (71.4%), neurogenesis 6 (71.4%), negative regulation of transcription from RNA polymerase II promoter (57.1%), negative regulation of transcription, DNA-dependent (57.1%), negative regulation of RNA metabolic process (57.1%) CREB1 regulation of apoptosis (71.4%), regulation of 6 programmed cell death (71.4%), regulation of cell death (71.4%), negative regulation of apoptosis (57.1%), negative regulation of programmed cell death (57.1%) YY1 negative regulation of transcription from RNA 5 polymerase II promoter (66.7%), negative regulation of transcription, DNA-dependent (66.7%), negative regulation of RNA metabolic process (66.7%), negative regulation of transcription (66.7%), negative regulation of gene expression (66.7%) HNF1-alpha carbohydrate homeostasis (40.0%), glucose 4 homeostasis (40.0%), negative regulation of chemokine production (20.0%), response to glucose stimulus (40.0%), response to hexose stimulus (40.0%) Sry positive regulation of transcription from RNA 4 polymerase II promoter (80.0%), positive regulation of transcription, DNA-dependent (80.0%), positive regulation of RNA metabolic process (80.0%), cell fate commitment (60.0%), regulation of transcription, DNA-dependent (100.0%) FKHR regulation of molecular function (100.0%), 4 regulation of MAPKKK cascade (60.0%), negative regulation of MAPKKK cascade (40.0%), negative regulation of biological process (100.0%), regulation of apoptosis (80.0%)

103

TABLE 3 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes SRF positive regulation of cell projection organization 4 (60.0%), positive regulation of filopodium assembly (40.0%), regulation of filopodium assembly (40.0%), regulation of protein complex assembly (60.0%), positive regulation of dendrite morphogenesis (40.0%) NANOG somatic stem cell maintenance (40.0%), stem cell 4 maintenance (40.0%), stem cell development (40.0%), negative regulation of transcription from RNA polymerase II promoter (60.0%), stem cell differentiation (40.0%) E2F1 response to growth factor stimulus (40.0%), nerve 4 growth factor production (20.0%), DNA replication involved in S phase (20.0%), neurotrophin production (20.0%), zygotic determination of anterior/posterior axis, embryo (20.0%) ETS1 regulation of multicellular organismal development 4 (80.0%), negative regulation of cellular process (100.0%), negative regulation of biological process (100.0%), regulation of BMP signaling pathway (40.0%), regulation of developmental process (80.0%) EGR1 learning or memory (60.0%), cognition (60.0%), cell 4 differentiation (100.0%), cellular developmental process (100.0%), behavior (60.0%) HIF1A elastin metabolic process (20.0%), epithelial tube 4 morphogenesis (40.0%), regulation of transcription from RNA polymerase II promoter (60.0%), positive regulation of macromolecule biosynthetic process (60.0%), connective tissue replacement involved in inflammatory response wound healing (20.0%) AP-2 cellular lipid catabolic process (50.0%), neuropore 3 closure (25.0%), anterior neuropore closure (25.0%), diacylglycerol catabolic process (25.0%), lipid catabolic process (50.0%)

104

TABLE 3 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes ATF-6 alpha retina development in camera-type eye (50.0%), 3 positive regulation of gene-specific transcription involved in unfolded protein response (25.0%), positive regulation of transcription, DNA-dependent (75.0%), positive regulation of RNA metabolic process (75.0%), positive regulation of transcription (75.0%) RelA (p65 negative regulation of microtubule polymerization or 3 NF-kB depolymerization (50.0%), regulation of microtubule subunit) polymerization or depolymerization (50.0%), regulation of microtubule cytoskeleton organization (50.0%), regulation of microtubule-based process (50.0%), negative regulation of cytoskeleton organization (50.0%) C/EBPalpha response to chemical stimulus (100.0%), response to 3 vitamin B2 (25.0%), glucocorticoid mediated signaling pathway (25.0%), diacylglycerol catabolic process (25.0%), regulation of developmental process (75.0%) ER81 response to growth factor stimulus (50.0%), cell 3 differentiation (100.0%), cellular developmental process (100.0%), positive regulation of transcription (75.0%), response to mechanical stimulus (50.0%) HSF1 snRNP protein import into nucleus (25.0%), rRNA 3 modification (25.0%), embryonic process involved in female pregnancy (25.0%), regulation of microtubule depolymerization (25.0%), negative regulation of microtubule depolymerization (25.0%) ESR1 ovarian follicle development (50.0%), female gonad 3 (nuclear) development (50.0%), development of primary female sexual characteristics (50.0%), ovulation cycle process (50.0%), female sex differentiation (50.0%)

105

TABLE 3 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes USF2 cellular lipid catabolic process (50.0%), lipid 3 metabolic process (75.0%), positive regulation of transcription from RNA polymerase II promoter by carbon catabolites (25.0%), positive regulation of transcription by carbon catabolites (25.0%), regulation of transcription from RNA polymerase II promoter by carbon catabolites (25.0%) AP-1 cellular response to reactive oxygen species (50.0%), 3 cellular response to oxidative stress (50.0%), response to reactive oxygen species (50.0%), nerve growth factor production (25.0%), neurotrophin production (25.0%) Androgen steroid hormone mediated signaling pathway 3 receptor (50.0%), chordate embryonic development (75.0%), embryo development ending in birth or egg hatching (75.0%), male somatic sex determination (25.0%), activation of prostate induction by androgen receptor signaling pathway (25.0%) MYOD myoblast cell fate determination (25.0%), positive 3 regulation of skeletal muscle tissue regeneration (25.0%), regulation of skeletal muscle tissue regeneration (25.0%), muscle organ development (50.0%), myoblast cell fate commitment (25.0%) FOXO3A initiation of primordial ovarian follicle growth 3 (25.0%), cellular response to chemical stimulus (75.0%), cellular response to stress (75.0%), cellular response to insulin stimulus (50.0%), cellular response to peptide hormone stimulus (50.0%) AP-2A regulation of ossification (50.0%), nerve growth 3 factor production (25.0%), detection of mechanical stimulus involved in equilibrioception (25.0%), neurotrophin production (25.0%), zygotic determination of anterior/posterior axis, embryo (25.0%)

106

TABLE 4.

TOP 30 TRANSCRIPTION FACTORS WHICH REGULATE THE LARGEST NUMBER OF

DIFFERENTIALLY EXPRESSED GENES AT LATE GASTRULA ALONG WITH THEIR ASSOCIATED

GENE ONTOLOGY PROCESSES. 2

Transcription Gene Ontology Processes Number of Factor Genes HNF4-alpha nucleic acid metabolic process (48.5%), nucleobase, 104 nucleoside, nucleotide and nucleic acid metabolic process (52.5%), nitrogen compound metabolic process (54.5%), cellular metabolic process (72.3%), cellular nitrogen compound metabolic process (53.5%) SP1 organ development (54.5%), system development 101 (57.6%), multicellular organismal development (61.6%), anatomical structure development (59.6%), developmental process (61.6%) c-Myc cellular macromolecule biosynthetic process 92 (50.6%), macromolecule biosynthetic process (50.6%), regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (52.8%), negative regulation of transcription (25.8%), negative regulation of transcription, DNA-dependent (23.6%) ESR1 regulation of transcription from RNA polymerase II 75 (nuclear) promoter (42.5%), positive regulation of transcription, DNA-dependent (37.0%), positive regulation of RNA metabolic process (37.0%), positive regulation of transcription (38.4%), positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (39.7%)

2 There are 447 differentially regulated genes at this time point.

107

TABLE 4 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes p53 regulation of transcription from RNA polymerase II 67 promoter (39.1%), positive regulation of gene expression (37.5%), positive regulation of transcription (35.9%), positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (37.5%), positive regulation of transcription, DNA-dependent (32.8%) E2F1 nucleic acid metabolic process (69.0%), positive 60 regulation of macromolecule metabolic process (50.0%), cellular macromolecule metabolic process (82.8%), positive regulation of cellular metabolic process (50.0%), negative regulation of transcription (37.9%) NF-Y organ development (61.4%), regulation of 59 transcription from RNA polymerase II promoter (45.6%), multicellular organismal development (70.2%), positive regulation of transcription, DNA-dependent (36.8%), system development (63.2%) Androgen regulation of transcription from RNA polymerase II 58 receptor promoter (48.2%), positive regulation of transcription, DNA-dependent (42.9%), positive regulation of gene expression (46.4%), positive regulation of RNA metabolic process (42.9%), positive regulation of transcription (44.6%) EGR1 regulation of transcription from RNA polymerase II 54 promoter (51.0%), positive regulation of transcription, DNA-dependent (43.1%), positive regulation of RNA metabolic process (43.1%), positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (47.1%), positive regulation of macromolecule metabolic process (52.9%)

108

TABLE 4 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes c-Jun regulation of transcription from RNA polymerase II 53 promoter (49.0%), positive regulation of transcription, DNA-dependent (43.1%), positive regulation of RNA metabolic process (43.1%), positive regulation of transcription from RNA polymerase II promoter (39.2%), positive regulation of gene expression (45.1%) ETS1 positive regulation of nucleobase, nucleoside, 53 nucleotide and nucleic acid metabolic process (52.9%), positive regulation of macromolecule metabolic process (58.8%), positive regulation of nitrogen compound metabolic process (52.9%), positive regulation of gene expression (51.0%), positive regulation of macromolecule biosynthetic process (52.9%) GCR-alpha positive regulation of transcription, DNA-dependent 53 (39.2%), positive regulation of RNA metabolic process (39.2%), positive regulation of transcription (41.2%), regulation of transcription from RNA polymerase II promoter (43.1%), positive regulation of gene expression (41.2%) YY1 regulation of transcription from RNA polymerase II 52 promoter (50.0%), negative regulation of transcription, DNA-dependent (38.0%), positive regulation of metabolic process (54.0%), negative regulation of RNA metabolic process (38.0%), positive regulation of macromolecule metabolic process (52.0%) Oct-3/4 regulation of transcription, DNA-dependent (69.4%), 51 regulation of RNA metabolic process (69.4%), regulation of transcription from RNA polymerase II promoter (55.1%), positive regulation of transcription, DNA-dependent (49.0%), positive regulation of RNA metabolic process (49.0%)

109

TABLE 4 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes RelA (p65 regulation of transcription from RNA polymerase II 51 NF-kB promoter (49.0%), positive regulation of subunit) transcription, DNA-dependent (42.9%), positive regulation of RNA metabolic process (42.9%), negative regulation of transcription, DNA-dependent (38.8%), negative regulation of RNA metabolic process (38.8%) Oct-1 regulation of transcription from RNA polymerase II 50 promoter (56.2%), positive regulation of transcription, DNA-dependent (50.0%), positive regulation of RNA metabolic process (50.0%), positive regulation of transcription from RNA polymerase II promoter (45.8%), positive regulation of transcription (50.0%) SRF regulation of transcription from RNA polymerase II 50 promoter (58.3%), positive regulation of transcription, DNA-dependent (50.0%), positive regulation of RNA metabolic process (50.0%), regulation of transcription, DNA-dependent (68.8%), regulation of RNA metabolic process (68.8%) HIF1A regulation of transcription from RNA polymerase II 50 promoter (52.1%), positive regulation of transcription, DNA-dependent (45.8%), positive regulation of RNA metabolic process (45.8%), positive regulation of transcription from RNA polymerase II promoter (41.7%), positive regulation of macromolecule biosynthetic process (50.0%) MYOD regulation of transcription from RNA polymerase II 49 promoter (57.4%), positive regulation of transcription, DNA-dependent (51.1%), positive regulation of RNA metabolic process (51.1%), positive regulation of transcription from RNA polymerase II promoter (46.8%), regulation of transcription, DNA-dependent (68.1%)

110

TABLE 4 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes C/EBPbeta regulation of transcription from RNA polymerase II 49 promoter (51.1%), positive regulation of transcription, DNA-dependent (44.7%), positive regulation of RNA metabolic process (44.7%), positive regulation of transcription from RNA polymerase II promoter (40.4%), negative regulation of gene expression (42.6%) Bcl-6 negative regulation of transcription (50.0%), 48 negative regulation of gene expression (50.0%), negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (50.0%), negative regulation of cellular macromolecule biosynthetic process (50.0%), negative regulation of nitrogen compound metabolic process (50.0%) TCF7L2 negative regulation of transcription (50.0%), 48 (TCF4) regulation of transcription, DNA-dependent (69.6%), regulation of RNA metabolic process (69.6%), negative regulation of gene expression (50.0%), negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (50.0%) HSF1 regulation of transcription from RNA polymerase II 48 promoter (58.7%), regulation of transcription, DNA-dependent (71.7%), regulation of RNA metabolic process (71.7%), positive regulation of transcription, DNA-dependent (50.0%), positive regulation of RNA metabolic process (50.0%) HNF6 regulation of transcription from RNA polymerase II 48 promoter (54.3%), negative regulation of transcription (45.7%), regulation of transcription, DNA-dependent (65.2%), regulation of RNA metabolic process (65.2%), positive regulation of transcription, DNA-dependent (45.7%)

111

TABLE 4 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes E2F4 nucleic acid metabolic process (75.6%), regulation of 47 transcription from RNA polymerase II promoter (48.9%), nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (75.6%), positive regulation of transcription, DNA-dependent (40.0%), positive regulation of RNA metabolic process (40.0%) SMAD3 positive regulation of transcription, DNA-dependent 47 (56.8%), positive regulation of RNA metabolic process (56.8%), regulation of transcription from RNA polymerase II promoter (61.4%), positive regulation of transcription (56.8%), regulation of transcription, DNA-dependent (72.7%) SOX4 regulation of transcription from RNA polymerase II 47 promoter (55.6%), regulation of transcription, DNA-dependent (66.7%), regulation of RNA metabolic process (66.7%), negative regulation of transcription, DNA-dependent (42.2%), negative regulation of RNA metabolic process (42.2%) STAT1 regulation of transcription from RNA polymerase II 46 promoter (47.7%), positive regulation of transcription, DNA-dependent (40.9%), positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (45.5%), positive regulation of RNA metabolic process (40.9%), positive regulation of nitrogen compound metabolic process (45.5%) c-Myb regulation of transcription from RNA polymerase II 46 promoter (52.3%), positive regulation of transcription, DNA-dependent (45.5%), positive regulation of RNA metabolic process (45.5%), regulation of transcription, DNA-dependent (63.6%), regulation of macromolecule metabolic process (79.5%) SREBP1 regulation of transcription from RNA polymerase II 46 (nuclear) promoter (48.8%), positive regulation of transcription, DNA-dependent (41.9%), positive regulation of RNA metabolic process (41.9%)

112

TABLE 5.

TOP 30 TRANSCRIPTION FACTORS WHICH REGULATE THE LARGEST NUMBER OF

DIFFERENTIALLY EXPRESSED GENES AT EARLY NEURULA ALONG WITH THEIR

ASSOCIATED GENE ONTOLOGY PROCESSES. 3

Transcription Gene Ontology Processes Number Factor of Genes SP1 cellular process (95.0%), primary metabolic process 119 (70.8%), developmental process (50.0%), metabolic process (76.7%), cellular metabolic process (69.2%) HNF4-alpha cellular process (85.7%), cellular metabolic process 114 (64.3%), primary metabolic process (62.5%), metabolic process (68.8%), cellular macromolecule metabolic process (46.4%) c-Myc cellular process (94.4%), cellular metabolic process 106 (76.6%), primary metabolic process (76.6%), metabolic process (80.4%), cellular macromolecule metabolic process (60.7%) p53 cellular process (94.9%), regulation of biological 77 process (83.3%), regulation of cellular process (80.8%), cellular macromolecule metabolic process (65.4%), biological regulation (84.6%) ESR1 cellular process (95.7%), cellular metabolic process 69 (nuclear) (73.9%), biological regulation (81.2%), anatomical structure development (47.8%), primary metabolic process (72.5%) C/EBPbeta response to stress (49.0%), cellular response to 50 stimulus (39.2%), cellular process (96.1%), regulation of apoptosis (33.3%), positive regulation of transcription, DNA-dependent (27.5%) NF-kB apoptosis (28.3%), programmed cell death (28.3%), 45 response to reactive oxygen species (15.2%), positive regulation of transcription, DNA-dependent (26.1%), positive regulation of RNA metabolic process (26.1%)

3 There are 742 differentially regulated genes at this time point.

113

TABLE 5 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes STAT1 positive regulation of biological process (53.8%), 38 positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (33.3%), positive regulation of macromolecule metabolic process (38.5%), positive regulation of cellular process (51.3%), positive regulation of nitrogen compound metabolic process (33.3%) Androgen cellular process (100.0%), cell death (31.6%), death 37 receptor (31.6%), developmental process (60.5%), anatomical structure development (55.3%) CREB1 cellular response to chemical stimulus (23.7%), 37 embryonic organ development (15.8%), primary metabolic process (71.1%), regulation of apoptosis (26.3%), cellular process (89.5%) HSF1 regulation of macromolecule metabolic process 36 (70.3%), regulation of gene expression (64.9%), cellular macromolecule metabolic process (78.4%), embryo development (37.8%), gene expression (62.2%) MYOD positive regulation of transcription, DNA-dependent 36 (45.9%), positive regulation of RNA metabolic process (45.9%), regulation of macromolecule metabolic process (78.4%), positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (48.6%), positive regulation of nitrogen compound metabolic process (48.6%) SRF regulation of transcription, DNA-dependent (62.2%), 36 regulation of transcription from RNA polymerase II promoter (48.6%), regulation of RNA metabolic process (62.2%), regulation of transcription (64.9%), positive regulation of transcription, DNA-dependent (37.8%) EGR1 developmental process (67.6%), multicellular 36 organismal development (59.5%), cellular macromolecule metabolic process (70.3%), positive regulation of macromolecule metabolic process (37.8%), cell differentiation (45.9%)

114

TABLE 5 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes E2F1 cellular metabolic process (91.9%), cellular 36 macromolecule metabolic process (81.1%), primary metabolic process (89.2%), macromolecule metabolic process (81.1%), metabolic process (91.9%) AP-1 response to chemical stimulus (54.3%), cellular 35 response to chemical stimulus (31.4%), embryo development (31.4%), cellular response to stimulus (37.1%), regulation of monocyte differentiation (8.6%) RelA (p65 cell differentiation (51.4%), cellular developmental 34 NF-kB process (51.4%), developmental process (65.7%), subunit) anatomical structure development (60.0%), positive regulation of DNA replication (14.3%) C/EBPalpha regulation of macromolecule metabolic process 32 (72.7%), regulation of metabolic process (78.8%), regulation of transcription, DNA-dependent (54.5%), regulation of RNA metabolic process (54.5%), positive regulation of transcription, DNA-dependent (36.4%) p21 regulation of transcription (78.8%), regulation of 32 transcription, DNA-dependent (69.7%), regulation of RNA metabolic process (69.7%), regulation of cellular macromolecule biosynthetic process (78.8%), regulation of macromolecule biosynthetic process (78.8%) ATF-2 macromolecule metabolic process (84.4%), response 31 to hydrogen peroxide (21.9%), positive regulation of apoptosis (34.4%), positive regulation of programmed cell death (34.4%), cellular response to stress (37.5%) YY1 cellular metabolic process (93.3%), cellular 29 biosynthetic process (70.0%), cellular macromolecule biosynthetic process (63.3%), biosynthetic process (70.0%), macromolecule biosynthetic process (63.3%)

115

TABLE 5 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes HIF1A response to oxygen levels (23.3%), response to 29 hypoxia (20.0%), regulation of transforming growth factor-beta production (10.0%), positive regulation of myeloid cell differentiation (13.3%), response to stress (46.7%) p63 cellular response to stress (36.7%), metabolic 29 process (93.3%), macromolecule metabolic process (80.0%), cellular macromolecule metabolic process (73.3%), response to DNA damage stimulus (26.7%) SP3 developmental process (65.5%), cellular process 28 (100.0%), anatomical structure development (58.6%), multicellular organismal development (58.6%), interspecies interaction between organisms (24.1%) NF-Y cellular response to stimulus (44.8%), response to 28 chemical stimulus (55.2%), response to organic substance (41.4%), response to stress (48.3%), protein oligomerization (20.7%) AP-4 positive regulation of transcription, DNA-dependent 28 (41.4%), positive regulation of RNA metabolic process (41.4%), positive regulation of transcription from RNA polymerase II promoter (37.9%), positive regulation of transcription (41.4%), positive regulation by host of viral transcription (13.8%) c-Jun response to oxidative stress (29.6%), cellular 27 metabolic process (92.6%), response to hydrogen peroxide (22.2%), response to reactive oxygen species (22.2%), metabolic process (92.6%) GATA-1 positive regulation of biological process (65.4%), 26 positive regulation of cellular process (61.5%), regulation of metabolic process (73.1%), regulation of macromolecule metabolic process (65.4%), positive regulation of macromolecule metabolic process (42.3%)

116

TABLE 5 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes STAT3 regulation of transcription, DNA-dependent (59.3%), 26 regulation of RNA metabolic process (59.3%), regulation of transcription from RNA polymerase II promoter (44.4%), cellular macromolecule metabolic process (81.5%), regulation of transcription (63.0%) Oct-3/4 embryo development (60.0%), embryonic 24 morphogenesis (48.0%), organ development (72.0%), anatomical structure morphogenesis (64.0%), tissue development (56.0%)

117

TABLE 6.

TOP 30 TRANSCRIPTION FACTORS WHICH REGULATE THE LARGEST NUMBER OF NON-

DIFFERENTIALLY EXPRESSED GENES AT EARLY GASTRULA ALONG WITH THEIR

ASSOCIATED GENE ONTOLOGY PROCESSES. 4

Transcription Gene Ontology Processes Number Factor of Genes HNF4-alpha cellular metabolic process (62.6%), metabolic 1372 process (68.7%), cellular process (81.2%), primary metabolic process (59.8%), cellular macromolecule metabolic process (47.0%) SP1 cellular process (90.6%), cellular metabolic process 1082 (68.1%), metabolic process (75.0%), primary metabolic process (67.2%), developmental process (46.2%) c-Myc cellular metabolic process (69.4%), cellular process 1033 (88.3%), primary metabolic process (67.9%), metabolic process (73.9%), cellular macromolecule metabolic process (52.9%) p53 cellular process (90.7%), negative regulation of 486 cellular process (35.9%), negative regulation of biological process (36.5%), positive regulation of cellular process (36.7%), regulation of cell cycle (17.0%) ESR1 cellular process (82.5%), positive regulation of 479 (nuclear) biological process (33.5%), cellular metabolic process (60.0%), primary metabolic process (59.6%), response to organic substance (21.9%) CREB1 cellular process (89.5%), response to organic 467 substance (28.4%), negative regulation of cellular process (34.4%), negative regulation of biological process (35.9%), cellular metabolic process (65.6%)

4 There are 11,702 non-differentially regulated genes at this time point.

118

TABLE 6 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes AP-1 response to organic substance (32.7%), cellular 378 process (90.4%), response to chemical stimulus (42.8%), response to endogenous stimulus (23.7%), response to stimulus (54.5%) NF-Y cellular response to stimulus (28.6%), cell cycle 310 (23.1%), cellular process (87.0%), developmental process (46.8%), primary metabolic process (66.6%) E2F1 DNA metabolic process (23.3%), cell cycle (26.8%), 342 DNA replication (14.5%), cellular macromolecule metabolic process (62.5%), cellular metabolic process (73.2%) NF-kB response to organic substance (34.5%), regulation 314 of programmed cell death (29.1%), regulation of cell death (29.1%), regulation of apoptosis (28.4%), positive regulation of biological process (44.7%) AP-2 response to organic substance (31.5%), response to 306 chemical stimulus (42.0%), primary metabolic process (70.8%), cellular process (89.2%), cellular metabolic process (69.2%) YY1 translational elongation (15.7%), cellular metabolic 303 process (76.3%), cellular process (91.7%), metabolic process (79.7%), cellular biosynthetic process (50.3%) EGR1 anatomical structure development (50.0%), 300 developmental process (54.7%), cellular process (91.9%), response to organic substance (33.4%), multicellular organismal development (50.0%) GATA-1 positive regulation of biological process (45.5%), 283 positive regulation of cellular process (43.0%), developmental process (52.7%), negative regulation of cellular process (38.4%), negative regulation of biological process (40.1%) Elk-1 cellular protein metabolic process (37.9%), cellular 214 metabolic process (69.2%), cellular macromolecule metabolic process (57.9%), primary metabolic process (68.7%), protein metabolic process (40.2%)

119

TABLE 6 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes ETS1 macromolecule metabolic process (68.4%), positive 250 regulation of macromolecule metabolic process (33.6%), positive regulation of cellular metabolic process (34.0%), positive regulation of metabolic process (34.8%), primary metabolic process (76.1%) Androgen cellular process (90.6%), positive regulation of 258 receptor macromolecule metabolic process (26.4%), positive regulation of biological process (39.4%), regulation of cell cycle (17.3%), positive regulation of cellular process (36.6%) HIF1A positive regulation of cellular process (47.2%), 236 positive regulation of biological process (48.1%), cellular process (93.1%), positive regulation of metabolic process (34.3%), positive regulation of cellular metabolic process (33.5%) C/EBPbeta response to organic substance (40.9%), response to 235 chemical stimulus (48.5%), positive regulation of biological process (48.9%), positive regulation of cellular process (46.0%), developmental process (55.7%) Oct-3/4 negative regulation of gene expression (30.0%), 236 regulation of transcription (52.7%), regulation of cellular macromolecule biosynthetic process (54.9%), regulation of gene expression (55.7%), regulation of transcription, DNA-dependent (44.3%) c-Jun response to organic substance (36.1%), 233 developmental process (57.0%), positive regulation of biological process (48.3%), positive regulation of cellular process (45.7%), cellular process (93.0%) E2F4 cell cycle (33.6%), DNA metabolic process (26.5%), 229 DNA replication (18.1%), regulation of cell cycle (26.1%), cellular process (95.1%) RelA (p65 positive regulation of biological process (51.8%), 224 NF-kB positive regulation of cellular process (48.2%), subunit) response to organic substance (36.0%), response to chemical stimulus (45.0%), negative regulation of biological process (42.8%)

120

TABLE 6 (CONTINUED)

Transcription Gene Ontology Processes Number of Factor Genes SP3 developmental process (60.5%), anatomical 206 structure development (55.6%), multicellular organismal development (55.6%), organ development (43.9%), response to organic substance (36.6%) GCR-alpha cellular process (93.2%), positive regulation of 205 biological process (46.3%), negative regulation of cellular process (40.0%), response to organic substance (32.2%), negative regulation of biological process (41.0%) HNF6 cellular metabolic process (73.7%), metabolic 205 process (79.0%), positive regulation of macromolecule metabolic process (29.8%), positive regulation of cellular metabolic process (30.2%), primary metabolic process (70.7%) Oct-1 positive regulation of biological process (48.7%), 202 developmental process (56.9%), negative regulation of cellular process (43.1%), positive regulation of cellular process (45.7%), negative regulation of biological process (44.2%) AP-2A cellular process (94.5%), developmental process 200 (57.7%), negative regulation of cellular process (42.8%), positive regulation of cellular process (44.8%), positive regulation of biological process (46.8%) AHR positive regulation of cellular process (46.6%), 192 positive regulation of biological process (47.6%), positive regulation of gene expression (27.5%), positive regulation of metabolic process (34.4%), positive regulation of nitrogen compound metabolic process (28.0%) HNF1-alpha positive regulation of gene expression (26.7%), 192 positive regulation of transcription from RNA polymerase II promoter (22.0%), positive regulation of transcription (25.7%), cellular metabolic process (73.3%), positive regulation of transcription, DNA-dependent (23.0%)

121

TABLE 7.

TOP 30 TRANSCRIPTION FACTORS WHICH REGULATE THE LARGEST NUMBER OF NON-

DIFFERENTIALLY EXPRESSED GENES AT LATE GASTRULA ALONG WITH THEIR ASSOCIATED

GENE ONTOLOGY PROCESSES. 5

Transcription Gene Ontology Processes Number Factor of Genes HNF4-alpha cellular metabolic process (62.3%), metabolic 1335 process (68.5%), cellular process (81.4%), primary metabolic process (59.5%), cellular macromolecule metabolic process (46.7%) SP1 cellular process (90.8%), cellular metabolic process 1053 (68.3%), metabolic process (75.2%), primary metabolic process (67.2%), developmental process (45.9%) c-Myc cellular process (88.4%), cellular metabolic process 1002 (69.0%), primary metabolic process (67.5%), metabolic process (73.5%), cellular macromolecule metabolic process (52.6%) p53 cellular process (90.8%), positive regulation of 461 cellular process (38.8%), positive regulation of biological process (40.1%), negative regulation of cellular process (35.3%), regulation of cell cycle (17.4%) ESR1 cellular process (82.8%), cellular metabolic process 463 (nuclear) (61.1%), primary metabolic process (60.7%), positive regulation of biological process (33.3%), metabolic process (66.5%) CREB1 cellular process (89.6%), response to organic 452 substance (27.9%), negative regulation of cellular process (34.4%), negative regulation of biological process (36.1%), cellular metabolic process (65.4%)

5 There are 11,322 non-differentially regulated genes at this time point.

122

TABLE 7 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes AP-1 response to organic substance (32.5%), cellular 370 process (90.4%), response to chemical stimulus (42.9%), response to stimulus (54.9%), response to endogenous stimulus (23.5%) E2F1 DNA metabolic process (22.9%), cellular process 335 (92.2%), DNA replication (14.5%), cell cycle (26.5%), cellular metabolic process (72.9%) NF-Y cell cycle (23.7%), cellular response to stimulus 301 (28.1%), cellular process (87.3%), regulation of cell cycle (17.4%), cell cycle process (18.7%) NF-kB response to organic substance (35.2%), regulation 302 of programmed cell death (29.6%), regulation of cell death (29.6%), regulation of apoptosis (28.9%), positive regulation of biological process (45.5%) AP-2 cellular process (89.7%), primary metabolic process 300 (70.0%), cellular metabolic process (69.3%), positive regulation of biological process (40.7%), metabolic process (75.3%) EGR1 cellular process (92.8%), developmental process 293 (55.2%), anatomical structure development (50.3%), response to organic substance (33.4%), multicellular organismal development (50.3%) YY1 translational elongation (15.6%), cellular metabolic 290 process (76.0%), cellular process (91.7%), translation (17.4%), cellular biosynthetic process (50.0%) GATA-1 positive regulation of biological process (44.1%), 276 positive regulation of cellular process (41.5%), developmental process (50.7%), cellular process (89.0%), negative regulation of cellular process (37.1%) Elk-1 cellular metabolic process (70.1%), cellular protein 211 metabolic process (38.4%), cellular macromolecule metabolic process (58.8%), primary metabolic process (69.7%), cellular process (88.2%)

123

TABLE 7 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes ETS1 macromolecule metabolic process (69.0%), primary 248 metabolic process (76.7%), positive regulation of cellular process (44.5%), positive regulation of macromolecule metabolic process (32.2%), positive regulation of metabolic process (33.5%) Androgen cellular process (90.5%), positive regulation of 255 receptor cellular process (38.9%), positive regulation of biological process (40.9%), developmental process (48.0%), positive regulation of macromolecule metabolic process (26.6%) HIF1A positive regulation of cellular process (46.8%), 233 cellular process (93.5%), positive regulation of biological process (48.1%), developmental process (55.8%), positive regulation of metabolic process (33.8%) C/EBPbeta response to organic substance (39.1%), response to 230 chemical stimulus (47.4%), positive regulation of biological process (47.8%), positive regulation of cellular process (44.8%), cellular process (91.7%) E2F4 cell cycle (34.7%), DNA replication (18.2%), DNA 228 metabolic process (26.2%), regulation of cell cycle (25.8%), cellular process (95.6%) Oct-3/4 regulation of cellular macromolecule biosynthetic 227 process (53.1%), regulation of transcription (50.9%), negative regulation of gene expression (28.1%), regulation of transcription, DNA-dependent (43.0%), regulation of RNA metabolic process (43.4%) c-Jun developmental process (57.7%), positive regulation 222 of biological process (48.2%), cellular process (93.2%), positive regulation of cellular process (45.5%), anatomical structure development (50.9%) RelA (p65 positive regulation of biological process (51.9%), 216 NF-kB positive regulation of cellular process (48.6%), subunit) response to organic substance (35.5%), negative regulation of biological process (43.5%), regulation of cell death (29.4%)

124

TABLE 7 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes SP3 developmental process (60.4%), anatomical 203 structure development (55.4%), multicellular organismal development (55.4%), organ development (43.6%), response to organic substance (36.6%) SRF developmental process (57.1%), anatomical 188 structure development (51.6%), anatomical structure morphogenesis (34.8%), positive regulation of cellular process (44.6%), positive regulation of macromolecule metabolic process (32.1%) AP-2A cellular process (94.4%), developmental process 197 (57.6%), negative regulation of cellular process (43.9%), negative regulation of biological process (44.9%), positive regulation of cellular process (44.4%) HNF6 cellular metabolic process (73.1%), metabolic 197 process (78.2%), positive regulation of macromolecule metabolic process (29.4%), positive regulation of cellular metabolic process (29.9%), positive regulation of metabolic process (29.9%) Oct-1 positive regulation of biological process (49.5%), 195 positive regulation of cellular process (46.3%), developmental process (56.4%), negative regulation of cellular process (41.5%), primary metabolic process (74.5%) GCR-alpha cellular process (93.8%), positive regulation of 195 biological process (45.6%), negative regulation of cellular process (40.5%), negative regulation of biological process (41.5%), response to organic substance (32.3%) AHR positive regulation of cellular process (46.8%), 189 positive regulation of biological process (47.8%), positive regulation of metabolic process (34.4%), positive regulation of gene expression (26.9%), positive regulation of macromolecule metabolic process (32.3%)

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TABLE 8.

TOP 30 TRANSCRIPTION FACTORS WHICH REGULATE THE LARGEST NUMBER OF NON-

DIFFERENTIALLY EXPRESSED GENES AT EARLY NEURULA ALONG WITH THEIR

ASSOCIATED GENE ONTOLOGY PROCESSES. 6

Transcription Gene Ontology Processes Number Factor of Genes HNF4-alpha cellular metabolic process (62.8%), metabolic 1313 process (69.0%), cellular process (81.3%), primary metabolic process (59.9%), cellular macromolecule metabolic process (47.3%) SP1 cellular process (90.7%), cellular metabolic process 1028 (68.8%), metabolic process (75.6%), primary metabolic process (67.7%), developmental process (46.3%) c-Myc cellular metabolic process (69.1%), cellular process 987 (87.9%), primary metabolic process (67.5%), metabolic process (73.7%), cellular macromolecule metabolic process (52.7%) p53 cellular process (90.8%), negative regulation of 460 cellular process (35.2%), positive regulation of cellular process (37.8%), regulation of cell cycle (17.7%), positive regulation of biological process (38.9%) ESR1 cellular process (82.3%), cellular metabolic process 455 (nuclear) (60.9%), positive regulation of biological process (33.8%), primary metabolic process (60.7%), response to organic substance (22.1%) CREB1 cellular process (89.5%), response to organic 451 substance (28.1%), negative regulation of cellular process (34.5%), negative regulation of biological process (36.1%), cellular metabolic process (65.5%)

6 There are 11,019 non-differentially regulated genes at this time point.

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TABLE 8 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes AP-1 response to organic substance (33.2%), response to 365 chemical stimulus (43.8%), cellular process (90.3%), response to stimulus (55.7%), response to endogenous stimulus (24.1%) E2F1 DNA metabolic process (22.5%), cellular process 332 (91.8%), cellular metabolic process (72.9%), cellular macromolecule metabolic process (62.0%), macromolecule metabolic process (64.7%) NF-Y cell cycle (23.9%), cellular process (87.7%), 303 regulation of cell cycle (17.6%), developmental process (47.2%), cell cycle process (18.6%) NF-kB response to organic substance (34.3%), regulation 298 of programmed cell death (28.6%), positive regulation of biological process (45.1%), regulation of cell death (28.6%), response to chemical stimulus (44.1%) YY1 translational elongation (16.2%), cellular metabolic 294 process (77.0%), cellular process (91.4%), cellular biosynthetic process (50.9%), translation (17.5%) AP-2 response to organic substance (31.2%), primary 293 metabolic process (71.2%), response to chemical stimulus (42.1%), cellular metabolic process (70.2%), cellular process (89.0%) EGR1 anatomical structure development (50.5%), cellular 289 process (92.3%), developmental process (54.4%), response to organic substance (33.7%), organ development (38.9%) GATA-1 positive regulation of biological process (44.1%), 266 positive regulation of cellular process (41.8%), developmental process (51.7%), negative regulation of cellular process (38.0%), negative regulation of biological process (39.5%) Elk-1 cellular protein metabolic process (38.8%), cellular 206 macromolecule metabolic process (59.7%), cellular metabolic process (69.9%), macromolecule metabolic process (61.7%), protein metabolic process (41.3%)

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TABLE 8 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes ETS1 macromolecule metabolic process (68.6%), positive 242 regulation of macromolecule metabolic process (33.9%), primary metabolic process (76.6%), positive regulation of metabolic process (35.1%), positive regulation of cellular metabolic process (34.3%) Androgen cellular process (90.2%), positive regulation of 249 receptor macromolecule metabolic process (26.5%), positive regulation of biological process (39.6%), positive regulation of cellular process (37.1%), cellular metabolic process (66.1%) Oct-3/4 negative regulation of gene expression (29.6%), 232 regulation of transcription (52.4%), regulation of gene expression (55.4%), negative regulation of transcription (27.9%), regulation of cellular macromolecule biosynthetic process (54.1%) c-Jun developmental process (59.3%), positive regulation 224 of biological process (50.2%), organ development (43.4%), positive regulation of cellular process (47.1%), anatomical structure development (52.5%) E2F4 cell cycle (34.1%), DNA metabolic process (25.9%), 223 regulation of cell cycle (25.9%), DNA replication (17.3%), cellular process (95.0%) HIF1A positive regulation of cellular process (48.4%), 222 positive regulation of biological process (48.9%), positive regulation of metabolic process (35.6%), positive regulation of macromolecule metabolic process (33.8%), cellular process (93.6%) C/EBPbeta response to organic substance (41.0%), response to 217 chemical stimulus (49.3%), positive regulation of biological process (49.3%), positive regulation of cellular process (46.5%), developmental process (56.7%) RelA (p65 positive regulation of biological process (51.4%), 214 NF-kB positive regulation of cellular process (48.1%), subunit) response to organic substance (36.3%), negative regulation of biological process (44.3%), negative regulation of cellular process (41.0%)

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TABLE 8 (CONTINUED)

Transcription Gene Ontology Processes Number Factor of Genes SP3 anatomical structure development (55.6%), organ 199 development (45.5%), developmental process (60.1%), response to organic substance (37.4%), multicellular organismal development (55.6%) GCR-alpha cellular process (92.9%), response to organic 198 substance (33.3%), positive regulation of biological process (45.5%), negative regulation of cellular process (39.9%), negative regulation of biological process (41.4%) HNF6 cellular metabolic process (73.7%), positive 198 regulation of macromolecule metabolic process (30.8%), positive regulation of cellular metabolic process (31.3%), metabolic process (79.3%), positive regulation of metabolic process (31.3%) AP-2A cellular process (94.9%), developmental process 194 (57.9%), negative regulation of cellular process (43.6%), negative regulation of biological process (44.6%), cellular metabolic process (74.9%) Oct-1 developmental process (57.4%), negative regulation 192 of cellular process (43.6%), negative regulation of biological process (44.7%), positive regulation of biological process (47.3%), response to organic substance (34.0%) HNF1-alpha positive regulation of gene expression (27.4%), 187 positive regulation of transcription from RNA polymerase II promoter (22.6%), positive regulation of transcription (26.3%), positive regulation of transcription, DNA-dependent (23.7%), positive regulation of RNA metabolic process (23.7%) SRF positive regulation of cellular process (47.5%), 185 developmental process (58.0%), positive regulation of macromolecule metabolic process (34.8%), positive regulation of metabolic process (35.9%), regulation of transcription from RNA polymerase II promoter (29.8%)

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Therefore, genes necessary for organism viability, such as those needed for metabolism, would be controlled in a SUMO-independent manner. Conversely, those genes which require a more regulated expression pattern would be controlled by SUMO modified transcription factors allowing for differential expression during development.

This observation suggests that SUMO modification generates distinct pools of a given transcription factor with different specificities. Furthermore, SUMOylated forms of these transcription factors appear to be biased towards more highly regulated processes.

Studies in Drosophila revealed a differential role for transcription factor

SUMOylation in proliferating and non-proliferating cells during development (92).

Specifically, mutations in both subunits of the E1 activating enzyme did not affect larval growth or survival but did cause defects in imaginal disk development. The larval stage of Drosophila is composed mainly of non-diving cells, while imaginal disks, which form the future appendages, are rapidly dividing structures. Therefore, SUMOylation activity is most important to those processes involving development and not necessary for larval viability.

The necessity of SUMOylation for embryo viability is a topic that has been studied utilizing multiple invertebrate and vertebrate systems. Knockout studies of various components of the SUMOylation machinery in C. elegans, Drosophila, and zebrafish show that it is necessary for the proper development and survival of early embryos (12, 153, 169, 170). Studies in mice, however, have proven to be more controversial. In the first study, an upstream component of the SUMOylation 130

machinery, Ubc9, was removed leading to embryonic lethality just following the blastocyst stage due to disruptions in nuclear integrity and chromosome organization

(13). Additionally, Sumo1 heterozygosity in mice resulted in cleft lip and palate with both heterozygotes and homozygotes having an increase in embryonic lethality (14).

Concurrent with these results, a study in 2011 revealed that SUMO -/- mice have a higher rate of embryonic lethality and heterozygotes display atrial and ventricle septal defects and a high rate of post-natal death (15). These results are contrary to two additional reports in which SUMO-1 knockout mice have no discernible phenotypes or decrease in viability (16, 17). In each study, experiments indicated that other isoforms, SUMO-2/3, may compensate for the lack of SUMO-1. While the mechanisms behind these inconsistencies are not fully understood, there is evidence that the genetic background of the mouse strain used has an impact on the cardiac phenotypes and embryonic survival (171). The three studies which observed an increase in lethality upon disruption of SUMOylation all utilized the C57BL6 mouse strain while the opposing studies used a combination of 129/SvEv and ICR mice, thereby complicating direct comparison of the results.

These discrepancies also highlight the need for more consistent model for testing disruptions in SUMOylation and the benefit of experiments utilizing microinjection of Gam1 mRNA. Gam1 mRNA injection decreases the levels of SAE1 and does decrease embryo viability at a higher concentration, while lower concentrations effect proper embryonic development which revealed those genes whose expression levels are most sensitive to this post translational modification. This approach allows 131

the degree of SUMOylation knockdown to be controlled by adjusting the amount of injected mRNA, thus producing viable embryos with specific developmental defects.

Therefore, this experimental strategy has provided a necessary method of studying this post-translational modification in early embryonic development.

The notion that SUMOylation alters the specificity of a transcription factor has support from recent studies. SUMO modification can act as a repressor or activator of transcription factors switching the factor between these two states depending on the context. The transcription factor Sp3 can act as both a repressor and an activator depending on the particular target gene and the cells in which it is expressed (156).

SUMOylation of Sp3 is proposed to recruit a complex of repressor proteins including histone deacetylase activity to the promoters of target genes through SIMs (172).

Mutation of the basic amino acids on the SUMO of the SUMO-Sp3 fusion protein, which interact with the SIM of the repressor protein, decreased the repressive activity of Sp3.

Additionally, unmodified Sp3 is associated with actively expressed genes shown by studies which utilized a non-SUMOylatable form of Sp3 or co-expression of wild-type

Sp3 with SUMO-specific proteases.

SUMO conjugation to regulatory domains of transcription factors, such as c-Myb, can change their activity. The negative regulatory domain of c-Myb contains a

SUMOylation site which controls its repressive function (173). SUMO modification of this domain blocks the recruitment of the p300 co-activator while de-sumoylation converts the factor to a potent activator of transcription.

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Another way that SUMOylation controls the activity of transcription factors is through nuclear localization. For the transcription factor NFAT1, SUMOylation of one lysine residue promotes the SUMOylation of an additional lysine, in NFAT1, which facilitates the interaction with the nuclear matrix (174). Mutations blocking both

SUMOylation sites disrupt the nuclear localization and transcriptional activation activity of NFAT1; whereas, mutation of the second SUMOylation site only blocks the attachment to the nuclear matrix. This dual SUMOylation provides a mechanism to fine tune the activity of this transcription factor. The transcription factor analysis of both the differentially and not-differentially expressed genes supports a dual role for many transcription factors with SUMOylation acting as a potential molecular switch in many cases.

A total of 50 different transcription factors were identified through the

Transcription Factor Regulation algorithm as being associated with the differentially expressed gene lists (at one or more time point). These factors were analyzed for potential SUMOylation sites using published protein sequences and the SUMO prediction site algorithm SUMOsp2.0 (98). A literature search was also carried out to identify published reports providing supporting evidence for SUMOylation of transcription factors and to verify predicted SUMOylation data (Table 9). A question mark indicates an ambiguous case in which the Xenopus protein sequence was not available for analysis. Of the 50 transcription factors, 37 (74%) contain potential

SUMOylation sites, only 4 (8%) lack a SUMO motif, and 9 (18%) lack sufficient sequence information to make a prediction. 133

TABLE 9.

COMPILED LIST OF TRANSCRIPTION FACTORS, IDENTIFIED TO REGULATE

DIFFERENTIALLY EXPRESSED GENES FROM ALL THREE TIME POINTS, ANALYZED FOR

POTENTIAL CONSENSUS AND NON-CONSENSUS SUMOYLATION SITES.

SUMO site Transcription Factor EG LG EN Reference No C/EBPbeta‡ * * (176) Yes c-Jun‡ * * (177, 178) ? NF-Y * * -- Yes STAT1‡ * * (179) Yes AP-1‡ * * (177) Yes C/EBPalpha‡ * * (176) Yes CREB1‡ * * (180) Yes STAT3 * * -- Yes Androgen receptor‡ * * * (85, 181) Yes c-Myc * * * -- Yes E2F1‡ * * * (182) Yes EGR1‡ * * * (183) Yes ESR1 (nuclear) * * * -- Yes HIF1A‡ * * * (184) Yes HNF4-alpha‡ * * * (185) Yes HSF1‡ * * * (87) Yes MYOD * * * -- ? Oct-3/4‡ * * * (186) p53‡ (88, 178, Yes * * * 187) Yes RelA (p65 NF-kB subunit)‡ * * * (188) Yes SP1‡ * * * (74, 75) Yes SRF‡ * * * (189) Yes YY1‡ * * * (190) Yes ETS1‡ * * (191) ? AP-4 * -- Yes ATF-2 * --

134

TABLE 9 (CONTINUED)

SUMO site Transcription Factor EG LG EN Reference Yes GATA-1‡ * (87) Yes NF-kB * -- Yes p21 * -- Yes p63‡ * (192) SP3‡ (144, 146, Yes * 156) Yes Bcl-6 * -- Yes c-Myb‡ * (193) Yes E2F4 * -- ? GCR-alpha‡ * (194) ? HNF6 * -- Yes Oct-1 * -- No SMAD3‡ * (195) No SOX4 * -- No SREBP1 (nuclear) * -- Yes TCF7L2 (TCF4)‡ * (196) Yes HNF1-alpha * -- Yes Sry * -- ? FKHR * -- ? NANOG * -- ? AP-2‡ * (197) ? ATF-6 alpha * -- Yes ER81‡ * (198) Yes USF2 * -- Yes FOXO3A * -- Yes AP‑2A * --

A question mark indicates that no X. laevis sequence for that transcription factor was present in the database. An asterisk indicates that the transcription factor is one of the top thirty which regulate the largest number of differentially expressed genes at that time point. ‡ is present next to transcription factors which were verified as SUMOylation targets through a literature search (references provided).

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Additionally, 28 of the 50 are experimentally verified to be SUMOylated, as indicated by the provided references. The high number of transcription factors which can potentially be SUMOylated and are associated with our differentially expressed data indicates a powerful and widespread role for SUMOylation in controlling a large number of genes. It also provides validation that the microarray is indeed measuring changes in gene expression due to misregulation of transcription factors caused by the knockdown of SUMOylation.

GeneGo functional analysis of differentially expressed genes

The lists of the differentially expressed genes were also analyzed with respect to function in order to understand if the genes controlled by SUMOylation could be tied to specific cellular processes. As mentioned above, the differentially expressed genes connected to each transcription factor were associated with processes important for development. In order to understand this observation further, gene lists were organized according to which Gene Ontology category they belonged. The Gene

Ontology is a comprehensive bioinformatics resource that organizes known gene products into specific categories including cellular components, biological processes, and molecular function (175). Our analysis focused on those terms associated with the category ‘biological process’ which is defined as ‘series of events accomplished by one or more ordered assemblies of molecular functions’.Each gene was placed into one of ten different categories, biological regulation, cellular component organization or 136

biogenesis, death, developmental process, localization, metabolic process, multicellular organismal process, response to stimulus, signaling, or other. Genes from each time point were found to be enriched for different biological processes and the number of enriched biological processes changed between time points (Fig. 15). At the early gastrula time point, the greatest number of genes (31%) was attributed to biological regulation and second to metabolic process (18%). The late gastrula time point changes to developmental process (34%) being the most enriched and at the early neurula, metabolic process (37%) becomes the most enriched.

The breakdown of enrichment terms for each time point is interesting when the biological changes that are occurring at each stage are taken into account. At start of gastrulation, many developmental processes are just beginning, prior to that point the embryo is primarily a ball of rapidly dividing cells. At this stage, genes necessary for embryo viability (biological regulation and metabolic process) are highly expressed while developmental regulators are present at lower levels. During gastrulation, many patterning events are initiated and the germ layers are migrating into place. The top enrichment term for the late gastrula time point reflects the many developmental processes that are occurring and that are apparently influenced by SUMOylation. At the early neurula stage in development, organs are beginning to form and the embryo has depleted the store of saved yolk proteins. Therefore, various metabolic processes, up to this point unnecessary, must begin.

137

Figure 15. Functional enrichment analysis. Differentially expressed genes from each time point were separated into ten functional groups based on GeneGo categorization of their annotated protein function. The percentage displayed is the portion of the total number of genes from each developmental time point that are contained within each functional group. The category other represents genes which did not fall into the previous nine groups.

138

139

This could be the explanation for the enrichment of genes involved in metabolic processes at this time point. The expression of those genes would need to change in order to meet the needs of the changing embryo.

Co-expression analysis

For all three time points, heat maps were generated to determine how the expression profiles of differentially regulated genes are related (Fig. 16 A-C). Heat maps are a tool used to understand the relationship between genes based on the degree of regulation. The brackets at the top designate the relationship among the different biological replicates. In this case, each of the water-injected controls group together and the Gam1-injected embryos group together, providing further evidence that the data is consistent across all three replicates. The brackets on the left hand side group genes based on the level of up-regulation or down-regulation, clustering together those genes that are similarly expressed. There are two distinct groups, down-regulation

(upper portion) or up-regulation (lower portion), which can be sub-divided into smaller clusters. Genes whose expression has been changed to a similar degree due to the decrease in SUMOylation are connected by shorter brackets. These heat maps provide a detailed analysis of the expression patterns of the differentially expressed genes at each distinct time point. Future analysis of this co-expression may identify common factors that regulate similarly expressed genes and thus understand how SUMOylation controls gene expression at these stages in development. 140

Figure 16. Heat maps of differentially expressed genes for three developmental time points. Expression values for differentially expressed genes (from Gam1 and H2O injected embryos) were normalized to values between -3.0 (down regulated-green) and 3.0 (up regulated-red) for three biological replicates. Brackets (to the left) indicate the similarity of expression values for each gene, the closer the brackets the more similar the expression profile. Biological replicates are clustered by brackets at the top of each map showing experiment reproducibility. Official gene symbols are listed at the right. (A, pg. 142) early gastrula, (B, pg. 143-144) late gastrula, (C, pg. 145) early neurula.

141

142

143

144

145

A separate, yet similar, analysis of the differentially expressed genes was undertaken to determine how the expression levels of the genes were related. The expression of the differentially expressed genes were compiled across the three time points and compared using the Clustal algorithm program (199). The differentially regulated genes were compiled into eight syn-expression clusters based on the similarity of expression across all three time points with respect to directionality (up- or down-regulation) and magnitude of change (Fig. 17). The distance between each point represents the degree of similarity between the expression profiles of each gene, with shorter distances equating to more similarly expressed genes and longer lines to less similar. This clustering allows for the genes most similarly expressed to be analyzed for relationships in regulation and function.

The genes lists for individual cluster groups were uploaded into the MetaCore® software suite for analysis. The most enriched transcription factors for each gene list were determined through the transcriptional regulation algorithm as mentioned previously. Each cluster returned a similar set of top transcription factors (Table 10). In addition to the transcription factor lists for each cluster being similar to each other, they are similar to the individual lists returned for each time point analysis for both the differentially and not differentially expressed genes. This observation supports the idea that the most highly connected transcription factors are those that are widely used to control a variety of genes. Consequently, this analysis does not explain the expression similarities within the clusters, indicating that the co-expression cannot be traced to a single transcription factor or set of transcription factors for each cluster. 146

Figure 17. Syn-expression cluster analysis of differentially expressed genes. Eight clusters were identified using Qcut software and each cluster is mapped with a different color. The distance between each data point is a measurement of the expression profile similarity, the shorter the distance the more similar the expression profile.

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TABLE 10.

TOP TRANSCRIPTION FACTORS CONTROLLING THE LARGEST NUMBER OF GENES FROM EACH OF THE EIGHT SYN-EXPRESSION

CLUSTER GROUPS WITH THE NUMBER IN PARENTHESES INDICATING THE TOTAL NUMBER OF GENES PRESENT IN EACH GROUP.

Cluster 1 (180) Cluster 2 (130) Cluster 3 (107) Cluster 4 (120) Cluster 5 (180) Cluster 6 (122) Cluster 7 (139) Cluster 8 (136) CREB1 (61) HNF4-alpha HNF4-alpha SP1 (17) HNF4-alpha HNF4-alpha SP1 (24) HNF4-alpha (24) (17) (33) (31) (30) c-Myc (41) SP1 (18) c-Myc (17) c-Myc (17) SP1 (30) SP1 (23) HNF4-alpha c-Myc (18) (22) ESR1 c-Myc (18) SP1 (15) ESR1 c-Myc (26) c-Myc (21) c-Myc (22) SP1 (18) (nuclear) (28) (nuclear) (14)

148 SP1 (27) ESR1 p53 (11) p53 (12) p53 (18) p53 (16) NF-kB (14) CREB1 (10)

(nuclear) (14) Oct-3/4 (25) SRF (7) C/EBPbeta (9) HNF4-alpha Androgen EGR1 (15) p53 (13) ESR1 (12) receptor (15) (nuclear) (8) GCR-alpha p53 (7) ESR1 Androgen E2F1 (12) p73 (12) AP-1 (10) p53 (7) (24) (nuclear) (8) receptor (11) YY1 (24) E2F1 (6) Androgen AP-1 (10) ESR1 p63 (12) C/EBPbeta (9) RelA (p65 receptor (8) (nuclear) (12) NF-kB subunit) (6) Androgen NANOG (6) AP-1 (7) NF-Y (10) CREB1 (11) NF-Y (11) ESR1 Oct-3/4 (6) receptor (22) (nuclear) (9) p53 (21) STAT3 (6) GCR-alpha (7) C/EBPbeta (9) ETS1 (10) ESR1 HIF1A (8) SREBP1 (nuclear) (11) (nuclear) (5)

TABLE 10 (CONTINUED)

Cluster 1 (180) Cluster 2 (130) Cluster 3 (107) Cluster 4 (120) Cluster 5 (180) Cluster 6 (122) Cluster 7 (139) Cluster 8 (136) NANOG (21) STAT1 (5) HIF1A (7) p63(9) AP-2A (9) RelA (p65 CREB1 (8) ERR1 (5) NF-kB subunit) (11) HIF1A (20) NF-Y (5) CREB1 (7) HSF1 (8) C/EBPbeta (8) E2F1 (11) STAT1 (7) AP-2 (5) TCFL2 (TCF4) c-Jun (5) Oct-3/4 (7) STAT4 (8) E2F4 (8) TFIIIA (10) YY1 (7) Androgen (19) receptor (5) c-jun (19) PAX6 (5) E2F1 (6) YY1 (8) SP3 (6) Bcl-6 (10) RelA (p65 GCR-alpha (5) NF-kB subunit) (7) SMAD3 (18) HSF1 (5) MYOD (6) NF-kB (8) NF-Y (6) YY1 (10) AP-2 (7) c-Jun (5) C/EBPbeta ZNF206 (4) AP-2 (5) p300 (8) HIF1A (6) FKHR (10) AP-2A (5) NF-kB (4) 149 (18)

MYOG (17) HNF1-alpha GATA-3 (5) EGR1 (8) c-Jun (6) Androgen GATA-1 (5) HSF1 (3) (4) receptor (10) SRF (17) EGR1 (4) ETS1 (5) GATA-1 (8) AP-2 (6) HOXA9 (10) E2F1 (5) NF-Y (3) SOX2 (16) PU.1 (4) ATF-4 (4) GCR-alpha (7) SRF (6) c-Myb (10) AP-4 (5) CDX2 (3) GATA-3 (16) Androgen RelA (p65 E2F1 (7) GCR-alpha (6) XBP1 (10) XBP1 (5) XBP1 (3) receptor (4) NF-kB subunit) (4) RARbeta (16) AML1 Oct-1 (4) TCF7L2 Bcl-6 (6) NRF2 (9) NF-Y (4) C/EBPbeta (3) (RUNX1) (4) (TCF4) (7) RelA (p65 c-Fos (4) c-Myb (7) p21 (6) PPARGC1 Androgen SF1 (3) NF-kB (PGC1-alpha) receptor (4) subunit) (16) (9)

TABLE 10 (CONTINUED)

Cluster 1 (180) Cluster 1 (180) Cluster 1 (180) Cluster 1 (180) Cluster 1 (180) Cluster 1 (180) Cluster 1 (180) Cluster 1 (180) GLI-1 (15) ETS1 (4) HNF6 (4) SMAD3 (7) Elk-1 (6) IRF1 (9) SP3 (4) SP3 (3) STAT1 (15) SP3 (4) E2F4 (4) STAT3 (7) AP-1 (6) Pdx-1 (IPF1) NRF2 (4) EGR1 (3) (9) EGR1 (15) AP-2 (4) PU.1 (4) RelA (p65 STAT1 (5) SOX4 (9) NF-kB1 (p50) Elk-1 (3) NF-kB (4) subunit) (7) E2F1 (15) CREB1 (4) c-Jun (4) c-Jun (6) C/EBPalpha p300 (9) EGR1 (4) YY1 (3) (5) NK31 (15) WT1 (4) NF-Y (3) GATA-2 (6) RelA (p65 SRF (9) c-Rel (NF-kB E2F1 (3)

150

NF-kB subunit) (4) subunit) (5) p63 (15) SREBP1 AP-2A (3) PU.1 (6) YY1 (5) c-Jun (9) GCR-alpha (3) NANOG (3) (nuclear) (4) Pitx2 (15) TCF7L2 AP-4 (3) CREB1 (6) E2F3 (5) COUP-TFI (9) TBP (3) p63 (3) (TCF4) (3) SMAD4 (15) SLUG (3) NF-kB (3) E2F4 (6) Oct-3/4 (5) ETS1 (9) AHR (3) Lef-1(2) ETS1 (15) ATF-2 (3) TCF7L2 AHR (6) RARalpha (5) Ikaros (9) SREBP2 TCF7L2 (TCF4) (3) (nuclear) (3) (TCF4) (2)

The data suggests that SUMOylation creates different pools of individual transcription factors and/or the activity of these factors is influenced by SUMOylation of secondary, gene specific factors.

The gene lists for each cluster were also subjected to analysis with the Gene

Ontology (GeneGo) processes enrichment algorithm. All eight clusters were enriched for specific processes using the GeneGo analysis (Table 11). An equivalent analysis using the BiNGO (200) program was done in order to verify the findings from MetaCore® which identified six clusters as specifically enriched. Both analyses were in agreement for the majority of the clusters indicating specific processes contain a subset of similarly expressed genes which are controlled by SUMOylation. Clusters 1 and 2 were enriched for GeneGo processes specific to embryo development such as patterning and organ morphogenesis. Clusters 3 and 4 also contained genes identified as developmentally important, however, cluster 3 was also enriched for metabolic processes (nucleic acid and nitrogen metabolism) and cluster 4 genes were associated with cellular organization. Genes from clusters 5, 6, and 7 were primarily metabolism related with some response to stress and DNA repair (cluster 6). Cluster 8 genes were identified as regulators of membrane organization and vesicle formation.

Identification of the association of co-expressed genes with distinct processes indicates that SUMOylation provides a mechanism to control gene expression which may differ depending on the gene function. The clustering of differentially expressed genes based on similar regulation and function will help to understand how SUMO modification affects different biological processes. 151

TABLE 11.

PROCESS ENRICHMENT ANALYSIS FOR EACH OF THE EIGHT SYN-EXPRESSION CLUSTER GROUPS PERFORMED WITH METACORETM

AND BINGO. A PROCESS NOT LISTED MEANS THAT THE GENES WERE NOT SIGNIFICANTLY ENRICHED.

MetaCore® Analysis BiNGO Analysis Cluster 1 Embryo development, regionalization, pattern specification process, organ Patterning, signaling, immune response, negative morphogenesis, anatomical structure morphogenesis regulation of morphogenesis, regulation of DNA transcription, organ morphogenesis, molecular transport, response to stimulus, metabolic processing

152 Cluster 2 Embryo development, pattern specific process, digestive tract Embryonic development, embryonic development ending

morphogenesis, digestive system development in birth or egg hatching, chordate embryonic development, pattern specific process, regionalization, Cluster 3 Nucleic acid metabolic process, nitrogen compound metabolic process, RNA metabolic process, embryo development, nucleoside primary metabolic process, cellular metabolic process, anterior/posterior metabolic process, cell fate determination, somatogenesis pattern formation, organ morphogenesis, regionalization Cluster 4 Cellular component organization, positive regulation of dendritic spine development, positive regulation of smooth muscle cell chemotaxis, cellular component organization or biogenesis Cluster 5 Cellular metabolic process, primary metabolic process, cellular macromolecular metabolic process, biosynthetic process, mitosis, nuclear division, cellular process Cluster 6 Cellular response to stimulus, response to DNA damage stimulus, cellular Cellular process, metabolic process, nucleotide response to stress, double strand break repair, regulation of phosphorus biosynthesis, RNA processing, DNA repair, signaling metabolic process response to stress, development, lymphocyte/T cell

TABLE 11 (CONTINUED)

MetaCore® Analysis BiNGO Analysis Cluster 7 Cellular metabolic process, catabolic process, hydrogen peroxide catabolic Cellular catabolic process, catabolic process, cellular process, cellular response to oxidative stress, cellular protein catabolic metabolic process, metabolic process, cellular process process Cluster 8 Vesicle-mediated transport, actin cortical patch assembly, cellular process, Vesicle-mediated transport, membrane organization catabolic process, cellular membrane organization, membrane invagination, endocytosis, Golgi vesicle transport

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Pathway analysis of differentially expressed genes

A list of differentially expressed genes from each time point was uploaded into

MetaCore® and analyzed for their presence in known biochemical pathways.

MetaCore® contains over 70,000 pathways built from experimentally established biochemical interactions of proteins, DNA, RNA, and metabolites. Pathway maps, graphic interpretations of these interactions, were manually examined to determine how the misregulated genes overlayed onto important developmental pathways.

Analysis focused on those pathways with outcomes related to the observed phenotypes of blastopore closure failure, spina bifida, shortened axis, and cardiovascular deformations. Four pathways, explained in detail below, contain multiple differentially expressed genes: (i) non-canonical Wnt signaling, (ii) YY1 transcription factor regulation of neural tube closure and heart development, (iii) Twist/Snail control of epithelial-to- mesenchymal transition, and (iv) Ets-1 regulation of key genes during gastrulation and heart development. For each pathway SUMOylation controls regulation of top transcription factors and disruptions in gene regulation can be related to the observed phenotypes.

Non-canonical Wnt signaling

Wnt signaling encompasses a large family of proteins that relay signals across the cell membrane through the binding of an extra cellular Wnt ligand to the membrane

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bound frizzled (Fzd) receptor (201, 202). Research in Xenopus has identified 19 distinct

Wnt genes and 10 different genes encoding frizzled (Fzd) receptors (203, 204). Wnt signaling can be divided into two broad groups, canonical and non-canonical, based on the signaling outcome. Canonical signaling is dependent on β-catenin and regulates transcription of target genes, while non-canonical is a β-catenin independent signaling cascade that regulates cell polarity and calcium levels (205).

Both pathways begin with the binding of a Wnt ligand to a Fzd receptor and subsequent recruitment of the disheveled (Dvl) protein to the plasma membrane where it is phosphorylated. In the canonical signaling pathway, activation of disheveled leads to inhibition of the β-catenin destruction protein complex which allows β-catenin to accumulate in the cytoplasm and be transported into the nucleus (Fig. 18 A). Once in the nucleus, it complexes with transcription factors such as TCF/LEF and activates target genes. Alternatively, activation of the non-canonical signaling pathway leads to association of Dvl and Dishevelled-associated activator of morphogenesis 1 (DAAM1) with Fzd at the plasma membrane (Fig. 18 B). This association activates the G-protein,

Rho, through recruitment of guanine nucleotide exchange factors (GEF), leading to activation of Rho-associated kinases (ROK) that results in the reorganization of the cytoskeleton and changes in cell polarity. Thus, the primary outcome of canonical Wnt signaling is transcriptional regulation, while non-canonical Wnt signaling is responsible for cytoskeleton rearrangements.

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Figure 18. SUMO regulation of Wnt pathway leading to defects in cytoskeleton remodeling. Wnt signaling can be separated into two different pathways. (A) Canonical Wnt signaling involves the binding of a Wnt ligand to the membrane bound frizzled (Fzd) receptor. This binding recruits disheveled (Dvl) to the membrane where it is phosphorylated. Activated Dvl inhibits the β-catenin protein destruction complex causing β-catenin to accumulate in the cytoplasm and subsequently be transported into the nucleus where it activates transcription of target genes. (B) Non-canonical signaling also leads to the activation of Dvl which then recruits Dishevelled-associated activator of morphogenesis 1 (DAAM1) to the membrane. DAAM1 recruits a guanine nucleotide exchange factor which activates the G-protein Rho leading to

156 activation of Rho-associated kinases (ROK) and consequently cytoskeleton remodeling. p53, which is a target of SUMOylation, controls the expression of Wnt ligands and Fzd receptors. Notch signaling, the binding of ligand to the Notch protein and interaction with intracellular cofactors, controls the expression of the G-protein Rho1. Several Notch co-factors are targets of SUMO modification. Protein interactions are indicated by colored arrows and the developmental stage(s) of misregulation in Gam1-injected embryos are indicted by corresponding green or red numbers indicating up- or down-regulation, respectively. p-values are listed when the values are larger than the differential expression cut off of 0.05.

A B

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Specific Wnt proteins are classified as canonical or non-canonical based on their ability to induce secondary axes or dorsalize Xenopus embryos. Wnt1, Wnt2B, Wnt3A, and Wnt8A (206–209) are known activators of the canonical signaling pathway, while

Wnt4, Wnt5a, and Wnt11 (210–212) have little or no dorsalizing effect and do not activate known Wnt target genes such as Xnr-3 and siamois.

Analysis of the microarray data identified several Wnt and Fzd genes that are misregulated in SUMOylation deficient embryos (Table 12). Four Fzd genes (Fzd2, Fzd7,

Fzd8, and Fzd10) are down regulated at one or more time points. Previous experiments have shown each of these Fzd genes is responsible for controlling the non-canonical

Wnt signaling pathway in development or cell movements (213–217).

TABLE 12.

GENES FROM THE WNT AND FRIZZLED (FZD) FAMILY DOWN REGULATED (PERCENTAGE)

AT ONE OR MORE OF THE TIME POINTS IN DEVELOPMENT.

Gene EG LG EN Wnt8b -- 46% 55%

Wnt11 -- 43% 50% (p=0.08) Fzd2 47% 27% --

Fzd7 -- 16% 16% Fzd8 -- 19% --

Fzd10a 40% -- 35% Fzd10b -- -- 38%

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Fzd2 and Fzd7 seem to have redundant roles in convergence and extension and mice with mutations in both genes show cleft palate and ventricular septal defects

(218). The frequencies of defects increase when these mutations are combined with mutations in additional genes in the Wnt pathway, including Dvl3, Wnt3a, Wnt5a, or

Wnt11. Additionally, Fzd7 controls chondrocyte polarity in developing bones and down-regulation disrupts the size and integrity of long bones (217). Expression of a mutant form of the Fzd8 protein in Xenopus disrupts normal convergence and extension patterns during gastrulation through disruption of the non-canonical signaling pathway

(216). Over-expression of the Fzd10 protein in sarcoma cells promotes lamellipodia formation and cell growth due to activation of non-canonical signaling (219).

Two Wnt genes (Wnt8b and Wnt11) are also down regulated in late gastrula and early neurula. Wnt11 is a ligand for the Fzd7 receptor and both over-expression and knockdown studies point to a necessary balance of both proteins in Xenopus convergence and extension and their function in non-canonical Wnt signaling (213).

Researchers determined that a constitutively active form of the Rho GTPase, Cdc42, could rescue axis elongation effects caused by injection of a truncated form of Fzd7 which does not contain the intracellular signaling domain. Additionally, a dominant negative form of Cdc42 rescues the inhibition of axis elongation which occurs with

Wnt11 and Fzd7 overexpression. Therefore, both Wnt11 and Fzd7 are required for proper non-canonical signaling through the Rho GTPase, Cdc42, and both a decrease and an increase in signaling activity can lead to gastrulation defects.

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Wnt8b has not been specifically implicated in non-canonical signaling; however it is often placed in the same group as Wnt11 due to other similarities such as their presence as maternal mRNA during oogenesis (220, 221). In addition to their presumed role in non-canonical signaling during gastrulation, maternal mRNA for both Wnts activates the canonical signaling pathway. In the case of Wnt8b, expression before the

MBT can induce a secondary axis but causes no axis duplication when expression is induced post-MBT (221). Wnt11 has a dorsalizing effect, indicating canonical signaling, when overexpressed in stage 6 oocytes prior to fertilization but not when overexpressed in one cell embryos (222). This indicates a role, in the earliest stages of development, for the maternal mRNA in canonical signaling which is separate from non-canonical signaling. The similarities between Wnt8b and Wnt11 in early development and the fact that they are both down regulated at the same stage suggests a yet undetermined role for Wnt8b in gastrulation and the non-canonical Wnt signaling.

As mentioned previously, another important protein in non-canonical Wnt signaling is the G-protein Rho. In SUMO deficient embryos Rho1 (rnd1) is markedly up- regulated, 87% in late gastrula and 140% in early neurula. Rho1 activation is necessary for the integrity and remodeling of the cytoskeleton during Xenopus gastrulation as it promotes cell rearrangements by mediating interactions with cadherin (223, 224). A decrease in Rho1 leads to decreased cell migration and severe gastrulation defects. In addition, an increase in levels of Rho1 leads to loss of cell adhesion due to a decrease in cadherin interaction thus promoting a more mesenchymal cell phenotype. While the loss of these cadherin interactions is necessary for gastrulation to occur, some cell-cell 160

contacts must remain in order to allow directed cell migration. The extreme up- regulation of Rho1 may abolish all of the cadherin interactions and therefore destroy the cell-cell contacts which are necessary for directed cell migration needed for gastrulation.

A genome wide study in mouse embryonic stem cells revealed p53 as a major regulator of Wnt signaling genes through ChIP-chip and gene expression microarray assays (225). As all of the Wnt pathway genes are down regulated in the SUMO deficient embryos, it is reasonable to think that they share some common regulators. p53 controls a large subset of genes, not simply the Wnts, and, through the transcription factor regulation analysis, it was also identified as a transcription factor highly connected to both differentially and not-differentially expressed genes indicating that there most likely are two active pools of this factor. While, p53 is a well established target of SUMOylation, the outcome of the modification on its activity is a debated issue providing further evidence that this modification affects a subpopulation of the transcription factor (226). In vitro DNA binding assays indicate that SUMOylation of p53 prevents site specific promoter binding thus repressing p53 activity (88). In contrast, two studies utilizing in vivo luciferase reporter systems determined that SUMO modified p53 enhances its transcriptional activity (187, 227). An additional in vivo cellular study showed that p53 SUMOylation had no effect on the transcriptional activity or nuclear localization of the factor (228) further complicating the role of this modification on p53 activity. Due to these widespread effects, it is possible that SUMOylation of p53 is

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acting as a transcriptional activator of non-canonical Wnt pathway genes during

Xenopus gastrulation and acting in another capacity for additional Wnt pathway genes.

Expression of the Xenopus Rho1 (rnd1) gene is regulated by the Notch signaling pathway (223). This pathway involves the binding of a ligand to the Notch transmembrane protein causing the proteolytic cleavage of the intracellular domain.

Once detached from the membrane, the intracellular domain enters the nucleus and, through the recruitment of co-factors, alters transcription of target genes (229). Studies in C. elegans show that SUMOylation inhibits Notch signaling during vulval development although the direct target and model was not described (230). Separate studies of two

Notch signaling cofactors, MAML1 and KyoT2, show that they are targets of

SUMOylation and modification of this cofactor inhibits the transcriptional activity of the

Notch factor (231, 232). Since SUMOylation inhibits the Notch signaling pathway, a decrease in SUMOylation would lead to an increase in signaling and a subsequent increase in the expression of Notch target genes, accounting for the observed increase in Rho1 in Gam1-injected embryos.

The defects seen in SUMOylation deficient embryos during gastrulation can be, at least in part, attributed to disruptions in cytoskeleton remodeling (34, 35) in addition to disruptions in cell migration and adhesion. Non-canonical Wnt signaling is one pathway that controls the regulation of cytoskeleton remodeling in Xenopus embryos through the activation of Rho associated kinases (233, 234). Disruptions in either of the two types of Wnt signaling, canonical versus non-canonical, produce different phenotypes in Xenopus embryos. Blocking canonical Wnt signaling leads to a decrease 162

in β-catenin and dorsalizes the embryo while an increase in β-catenin creates a secondary axis. Alternatively, disruptions in non-canonical Wnt signaling cause patterning defects and disruptions in gastrulation. The specific role of SUMOylation in controlling non-canonical Wnt signaling as opposed to canonical signaling is suggested from the observed phenotypes. SUMOylation does not induce a secondary axis or dorsalization of the embryos which are hallmarks of disruption in the canonical Wnt signaling. Instead, embryos display defects in gastrulation and axis formation.

Additionally, target genes used to measure canonical Wnt signaling such as Xnr-3 and sia are not misregulated in SUMO deficient embryos as would be expected if canonical Wnt signaling was disrupted.

YY1 regulation of neural tube closure and heart development

A major finding from this work is that SUMO controls genes that span a large variety of biological processes. This is also evident from the number of different development defects that include failure of the blastopore to close, failure of the neural tube to closed, eye defects, edema, and heart defects in both chamber formation and looping. The defects not only span a large range of developmental processes but also a large range of developmental stages. The first can be seen during the earliest stages of gastrulation (stages 10-13) when the mesoderm invaginates forming the blastopore. In

Gam1-injected embryos, this cell migration does not occur and the blastopore does not close which propagates throughout neurulation (stages 14-24) as the neural folds do not

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fuse together leading to an open neural tube. These defects can both be attributed to a failure of cellular migration or adhesion. At later stages in development, more distinct defects in specific organs are observed including the heart and eyes which likely result from failure of cells to differentiate properly.

Assays to test the knockdown of E1 enzyme activity show a transient recovery of

SUMOylation activity at the mid-blastula stage followed by a decrease to undetectable levels until late neurula, however defects are seen well beyond this stage. These results indicate a role for SUMOylation at the earliest stages of embryonic patterning that may not be apparent until much later in development. Patterning of the Xenopus heart begins at early gastrula (stage 10) when the pre-cardiac cells migrate inward as a part of the presumptive mesoderm (235, 236). At the beginning of neurulation (stage 14), two heart fields located adjacent to the neural folds contain cells fated to be cardiac even though they are not yet expressing cardiac markers. By stage 28, past late neurula, a heart tube structure is formed and contraction begins soon after at stage 33 (237).

Concurrent with the onset of cardiac muscle contraction is looping, a process which will eventually form the three chambers of the amphibian heart. The two earliest stages of heart development, cell fate determination and heart field formation, occur at a time when SUMOylation is still inhibited in Gam1 expressing embryos. This may account for heart defects observed in later stages (beyond neurula) when SUMOylation activity has recovered and suggests a role for regulation by SUMO at the earliest stages in patterning the vertebrate heart.

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The transcription factor Yin Yang 1 (YY1) regulates a large number of the differentially expressed genes as determined through the MetaCore® transcription factor regulation analysis. YY1 is a transcription factor in the GLI-Kruppel family of factors that acts as both an activator and a repressor, depending on the cell type and developmental stage (238, 239). YY1 proteins are highly conserved across several vertebrate species and regulate a wide variety of developmentally important genes.

Mice embryos homozygous for a mutant form of YY1 were able to implant in the uterus but died before gastrulation; heterozygous embryos survived, but showed growth retardation and neurulation defects (240). In Xenopus, YY1 regulates the expression of neural marker genes and induces neural tissue along with patterning the anterior- posterior axis (241, 242). In developing zebrafish embryos, YY1, through interaction with β-arrestin1, controls the cdx4-hox pathway which is important in hematopoiesis

(243). YY1, along with its interacting SMAD partners, regulates the expression of the

Nkx2.5 gene and is vital to many aspects of proper chick heart development (244). YY1 has other functions during heart development including repression of the cardiac α- actin gene and both repression and activation of cardiac Mlc2 gene (245).

YY1 is implicated in heart development and neural tube closure with knockdowns of the protein displaying similar phenotypes to SUMO deficient embryos

(244–246). YY1 can act as both a repressor or an activator of transcription depending on the cell type or stage in development (238, 247). Additionally, YY1 is SUMOylated on lysine 288, with PIAS4 acting as the E3 ligase, which alters the effect YY1 has on the transcription of target genes (190). Studies using in vitro luciferase reporter assays 165

showed that YY1, when co-expressed with PIAS4, inhibited the transcription of c-myc and EZH2 and increased the transcription of cdc6. This increase in transcription of cdc6 was confirmed using in vivo RT-PCR measurements upon over expression of YY1 and

PIAS4 (190). This data provides evidence for the SUMOylation of YY1 acting as a regulatory mechanism to convert the factor between an activator and a repressor.

The microarray data supports this hypothesis as the differentially expressed genes controlled by YY1 were both up-regulated and down-regulated when

SUMOylation was inhibited. Table 13 lists differentially expressed genes regulated by

YY1. Specifically, cdc6 was down-regulated at the early neurula time point which agrees with the previous data that SUMOylated YY1 acts as an activator of cdc6 transcription.

Experiments have determined that YY1 binds to the promoter of the hoxb4 and msx2 gene and increases their transcription while binding to the rad51 promoter decreases transcription (248–250). The microarray data indicates a down regulation of hoxb4 and msx2 and an up regulation of rad51 providing evidence for the inactivation of YY1 due to the knockdown of SUMOylation. YY1 also binds to the promoter of psmd8, which is up regulated at early neurula, but experiments did not indicate whether YY1 acts as an activator or repressor of this gene (251). Additionally, the misregulation of many YY1 controlled genes can be linked to developmental defects seen in the SUMO deficient embryos, as previously mentioned.

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TABLE 13.

DIFFERENTIALLY EXPRESSED GENES (UP-REGULATED OR DOWN-REGULATED)

CONTROLLED BY THE TRANSCRIPTION FACTOR YY1 AT EACH OF THE THREE TIME

POINTS.

Up-regulated Down-regulated EG LG EN EG LG EN prdx5 rps12 tnfrsf10b atp5a1 msx1 msx1 rad51 rps9 ptbp1 aldh2 mre11a psmd8 rpl3 sema3a hmgcs1 rad51 sfrs5 cnbp fdps ndufs7 ier3ip1 msx2 tp53 psen1 cnbp cdc6 hspa5 prdx6 icmt rrm1 pcna gjb2 ndufa8 fdps msx2 ndufs8 hoxb4 parp1

Several genes controlled by YY1 which are down regulated in SUMOylation deficient embryos are also necessary for proper embryonic development. The msh homeobox 1(msx1), which is down regulated 60% in late gastrula and 40% in early neurula, is a transcription factor that controls early genes involved in craniofacial development and mutations of this gene are seen in patients with cleft lip and palate

(252, 253). In mice, the polypyrimidine tract binding protein 1 (ptbp1), an RNA-binding protein, is vital to gastrulation. Mice lacking ptbp1 showed growth retardation in pre- gastrulation stages of development and failed to differentiate the germ layers post gastrulation (254). Ptbp1 is down regulated 20% at late gastrula in SUMOylation- 167

deficient embryos which may contribute to the gastrulation defects observed.

Presenilin-1 (PS1) is a protein that has been linked to familial Alzheimer’s disease due to its role in processing amyloid precursor protein and is up-regulated (5%) in late neurula

SUMOylation-deficient embryos. Additionally, mice lacking the PS1 protein show defects in neuronal, somite, and skeletal system development along with disruptions in angiogenesis (255). This small but statistically significant increase may disrupt proper heart and circulatory system development in developing X. laevis.

The genes described above represent a small fraction of those genes regulated by YY1. However, they do highlight that the knockdown in SUMOylation and subsequent disruption in YY1 transcriptional activity affects a variety of aspects of development which contribute to the observed gastrulation and heart development defects seen in Gam1-injected embryos.

Twist/Snail control of epithelial to mesenchymal transition

Epithelial to mesenchymal transition (EMT) is a hallmark of early vertebrate development and patterning of the embryo (256, 257). In this process, stationary epithelial cells lose their connections to basal membranes, become polarized, and migrate in order to form different germ layers and tissues. During EMT, epithelial markers such as ZO1-3 and E-cadherin are down regulated and mesenchymal markers including vimentin and N-cadherin are up-regulated creating cells which are more motile. In Xenopus, EMT changes allow for the presumptive mesoderm to migrate

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inward during gastrulation, and disruptions in EMT signaling cascades lead to developmental defects in gastrulation and later patterning of the embryo (35).

There are numerous pathways and factors that are involved in EMT which change throughout development. One important pathway involves control of target

EMT genes by the transcription factors Snail1 and Twist (Fig. 19) (258).

Figure 19. SUMO regulation of Twist/Snail which controls the epithelial to mesenchymal transition. The transcription factors Twist and Snail bind to the promoter of the Zeb-2 gene which in turn regulates genes necessary for the epithelial to mesenchymal transition, up-regulating those which promote a mesenchymal phenotype (Vim, Laminin) and down regulating those which promote an epithelial phenotype (ZO-2). High mobility group A2 (HMGA2), a target of SUMOylation, binds to the promoter of both Twist and Snail. Binding of HMGA2 to the Twist promoter is direct while binding to the Snail promoter requires SMAD cofactor association. 169

These two factors have been implicated as major regulators of the EMT reprogramming but they seem to be under different regulatory control.Studies have determined that both Snail1 and Twist are regulated by the high mobility group A2

(HMGA2) factor (259). HMGA2 is a DNA binding protein which contains three AT-hook domains that facilitate binding to the minor groove of DNA (260). HMGA2 can then recruit other factors to the promoters or enhancers of target genes important for EMT.

In the case of Snail1 regulation, HMGA2 binding to the promoter element is enhanced by forming a complex with phosphorylated SMADs, specifically 3 and 4, which are activated by TGFβ signaling (261). Conversely, HMGA2 is able to bind directly to the

Twist promoter in the absence of TGFβ signaling indicating a different mechanism for control (259).

Analysis of the microarray data reveal several disruptions in this pathway that would lead to increased epithelial and decreased mesenchymal characteristics producing the observed gastrulation defects and failure of the blastopore to close.

Specifically, Twist is down regulated at all three sampled time points to varying degrees;

10% at EG, 50% at LG, and 37% at EN. In contrast, no misregulation of Snail1 is observed at any time point. Twist and Snail1 form a complex to control the EMT specific transcription factor, Zeb-2, which is down regulated 20% at the EN stage. Zeb-2 is responsible for activation of genes that promote a mesenchymal phenotype and repression of genes promoting epithelial phenotypes, thereby regulating the transition from epithelial to mesenchymal (262). Zonula occludens-2 (ZO-2), an integral component of tight junctions and normally down regulated in mesenchymal cells which 170

allows for increased cell movement, is up-regulated 15% in both LG and EN stages.

Vimentin, a protein necessary for cytoskeleton remodeling and normally up regulated in mesenchymal cells, is down regulated approximately 20% by early neurula. While not statistically significant, there is a slight down regulation of laminin β, necessary for extracellular matrix remodeling, by the early neurula stage. These changes in gene regulation promote an epithelial-like cell phenotype preventing cell migration and leading to the observed gastrulation defects.

The initial down regulation, but not complete loss, of Twist can explain the subsequent misregulation of downstream genes necessary to complete epithelial to mesenchymal change and also how some EMT genes remained unchanged (E-cadherin,

N-cadherin, fibronectin, and occludin). The constant levels of Snail1 could also account for the unperturbed levels of these EMT remodeling targets. Additionally, the disruption of only one segment of the pathway explains how the embryos remain viable well beyond gastrulation into free swimming tadpole stage.

As mentioned previously, HMGA2 controls the expression of both Snail1 and

Twist during EMT; however, it is through different mechanisms (259, 261). This data indicates that SUMOylation affects the expression of Twist but not the expression of

Snail1. HMGA2 is a target of SUMOylation at two adjacent lysine residues (position 66 and 67) located between the second and third AT-hook DNA binding domain (263).

These lysine residues are conserved across many vertebrate species including zebrafish, mouse, rat, and humans indicating a functional importance. HMGA2 binds directly to the promoter of Twist through the three AT-hook domains and activates its 171

transcription. It is possible that SUMOylation promotes the binding of HMGA2 to specific target sequences such as those in the Twist promoter. An equally logical scenario is that SUMOylation of HMGA2 facilitates the binding of additional transcription factors necessary for the specific activation of the Twist gene but not other

HMGA2 target genes. In either case, loss of SUMOylation would lead to a decrease in the transcription of Twist, as seen in the microarray data. This would lead to the downstream affects in gene misregulation and subsequent defects in gastrulation, axis elongation, and patterning.

In contrast, the binding of HMGA2 to the Snail1 promoter is increased though interactions with the SMAD proteins and that result from TGFβ signaling (261). The microarray data indicates that this is a SUMO-independent mechanism as it is not affected by the knockdown of SUMOylation activity. It is well established that only a small portion of a total pool of protein is SUMOylated at one time, and this data supports the hypothesis that there are different forms of transcription factors creating specificity for different genes (7, 184).

Ets-1 regulation of key genes during gastrulation and heart development

Ets-1 is a member of the Ets transcription factor family of proteins characterized by a highly conserved 80 amino acid C-terminal DNA binding domain along with an N- terminal transactivation domain important for protein-protein interactions (264). Ets proteins have been shown to control a wide variety of genes important for cell cycle

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control, heart development, neural tube closure, and cell differentiation. Several post- translational modifications such as phosphorylation, ubiquitination, and SUMOylation are responsible for the regulation of Ets-1 during development (154, 191).

Ets-1 is directly modified by SUMOylation at two lysine residues (K15 and K227) which are located in the large N-terminal transactivation domain. Attachment of SUMO at these residues represses the transcriptional activity of Ets-1 most likely through changes in protein-protein or protein-DNA interactions as SUMO modification does not change the stability or subcellular localization of Ets-1 (191).

The transcriptional activity of Ets-1 can also be indirectly influenced by

SUMOylation through regulation of the Ras/Raf-MEK-MAPK phosphorylation pathway

(Fig. 20). This pathway is an important signaling cascade necessary for proper cell differentiation, proliferation, and survival (265, 266). Extra cellular signaling of certain growth factors (e.g. BMP, EGF) initiate this cascade through the activation of the GTPase

Ras, through formation of the GTP-bound state, which promotes binding to its target

Raf. The Ras/Raf complex is a kinase that phosphorylates the MAPK/ERK kinase (MEK) leading to its activation. This activation allows MEK to in turn phosphorylate Mitogen- activated protein kinase (MAPK) which translocates to the nucleus and regulates target transcription factors such as Ets-1 though phosphorylation.

Ets-1 is phosphorylated at threonine 38, located in the transactivation domain, by MAPK following Ras activation and this phosphorylation increases the transcriptional activity of Ets-1. SUMOylation has been shown to control this pathway through two distinct and opposing mechanisms. Ras-1 is a target for SUMO modification and 173

knockdown of SUMO-1 in Drosophila cells was shown to decrease MEK activation and

MAPK phosphorylation due to disruptions in Ras/Raf signaling (170). Loss of MAPK phosphorylation would lead to a decrease in Ets-1 phosphorylation and subsequent decrease in transcriptional activity. In contrast, MEK SUMOylation blocks MAPK activation by disrupting interactions of MEK and MAPK (267). Therefore, knockdown of

SUMOylation should lead to an increase in MAPK phosphorylation and subsequent increase in Ets-1 phosphorylation creating an increase in its transcriptional activity which would be further amplified by the absence of the repressive effects of Ets-1

SUMOylation. Ets-1 is important for the regulation of many developmentally specific genes, and Ets-1 knockouts display cardiovascular defects in many model organisms, including Drosophila, chick, and mouse embryos, due to lack of cardiac neural crest cell migration (268, 269). Ets-1 regulates a subset of genes including polo-like kinase 3

(PLK3), S-phase kinase associated protein (Skp1), cell division cycle (CDC) 16 and 23 (part of the anaphase promoting complex), and CDC25a; all of which are misregulated in

SUMO deficient embryos as shown by the microarray data (Table 14) (264). These genes are necessary for the activation of the pocket protein family which includes retinoblastoma binding protein (Rbp), p107, and p130 (Fig. 20). The anaphase promoting complex (APC) proteins, CDC16 and CDC23, along with Skp1, are ubiquitin ligases which promote the ubiquitination of CDC25a thus leading to its degradation

(270).

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Figure 20. SUMO regulation of Ets-1 and control of gastrulation and heart development. Activity of the transcription factor Ets-1 can be controlled by SUMOylation through two distinct mechanisms. Ets-1 is a direct target of SUMO modification which represses its transcriptional activity. Ets-1 activity can also be controlled indirectly by SUMOylation

175 through the Ras/Raf-MEK-MAPK pathway. Both Ras and MEK (MAPK/ERK kinase) are targets of SUMOylation that potentially can have opposing effects on the activity of Ets- 1. SUMO modification of Ras increases MEK activation while SUMOylation of MEK decreases its activity. MEK in turn is the kinase that phosphorylates MAPK (Mitogen- activated protein kinase) which is then translocated to the nucleus and controls Ets-1 activity. Ets-1 controls the expression of platelet derived growth factor alpha (PDGFα) and its receptor (gastrulation) along with genes (PLK3, skp1, CDC16, CDC23, CDC25a) which activate pocket family proteins (Rbp, p107,p130) and alter their binding to transcription factors E2F1 and E2F4 (heart development).

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TABLE 14.

DIFFERENTIALLY EXPRESSED GENES REGULATED EITHER DIRECTLY OR INDIRECTLY BY

ETS-1.

Up-regulated EG LG EN -- Egr1 (2.28) Skp1 (1.19) Rrm1 (1.19) CDC23 (1.18)

Down-regulated EG LG EN

PLK3 (0.74) PDGFα (0.32) PDGFαR (0.64) CDC16 (0.94) PDGFαR (0.68) CDC16 (0.80) Dusp1(0.76) CDC25a (0.76) Dusp1 (0.81) IGF2 (0.31)

Directionality of differential expression is indicated as either up-regulation or down-regulation and the developmental stage in which it is misregulated. The fold change for each gene is indicated in parentheses.

Additional control of CDC25a comes from phosphorylation by PLK3 which increases the former’s stability due to an interaction with Cdk1/cyclin B complex (271).

An increase in the levels of CDC16 and Skp1, both responsible for the ubiquitination of

CDC25a, and a decrease in the levels of PLK3, which stabilizes CDC25a, would lead to a decrease in the amount of this protein. This decrease would be exacerbated by the decreased levels of Cdc25a mRNA at the early neurula time point. CDC25a de- 177

phosphorylates cyclin dependant kinase 4 (CDK4) and resulting in its activation (272).

Therefore, a decrease in the levels of CDC25a would lead to a decrease in activated

CDK4 protein even though a change in the mRNA level is not observed on the microarray. CDK4 is the kinase responsible for phosphorylating the pocket proteins Rbp, p107, and p130, that triggers interaction of these proteins with their binding partners, two of which are the transcription factors E2F1 and E2F4 (273, 274). The abolishment of these interactions contributes to downstream changes that can account for several of the observed phenotypes.

Vertebrate heart development is a complex and well orchestrated process that requires the involvement of multiple signaling pathways and coordinated regulation.

Pathway analysis of the differentially expressed genes identified those involved in the regulation of the pocket protein family (Rbp, p107, p130) and their binding partners, transcription factors E2F1 and E2F4. Both the Rbp family of proteins and the E2F1/E2F4 transcription factors are necessary for proper cardiac development (275–277).

Mutations in p107 and loss of Rbp lead to increased mouse embryonic lethality and heart defects due to improper migration and proliferation of precursor heart cells (275).

E2F4 is necessary for cell-cycle control in mouse cardiomyocytes (276) and E2F1 along with Rbp regulates the cell entry into S phase in myocardial cells (277). The E2F transcription factors are required for the patterning of the anterior-posterior axis by controlling region specific gene expression (278). E2F1 and E2F4 also control a number of genes, including IGF2, Dusp1, and Egr1, whose misregulation can contribute to the observed phenotypes. Insulin like growth factor II (IGF2), which is down regulated at 178

early neurula, is necessary for proper growth, cell differentiation, and angiogenesis

(279). In lung cancer cells, dual specific phosphatase 1 (Dusp1) promotes metastasis and angiogenesis and cells lacking this enzyme show decreased invasion and angiogenic potential (280). Dusp1 is down-regulated at late gastrula and early neurula in SUMO deficient embryos and may contribute to the decreased cell migration and heart defects seen in these embryos. Early growth response protein 1 (Egr1) is up-regulated at late gastrula and controls cell differentiation in a variety of cell types (281–283).

Ribonucleotide reductase M1 (Rrm1) is also up-regulated at late gastrula but has not been specifically identified as a regulator of any processes involving cell migration or heart development at this time.

In addition to heart development, Ets-1 binds to the promoter of genes necessary for proper gastrulation specifically platelet derived growth factor α (PDGFα) and its receptor (PDGFαR) (284, 285). Mice with mutations in the PDGFαR display severe skeletal abnormalities, cleft face, and spina bifida (286, 287). These defects have been attributed to increase apoptosis or migration defects in the neural crest cells.

Gastrulation defects in developing Xenopus embryos are also observed when PDGFα signaling is disrupted (40, 41, 288). PDGFα is expressed in the blatocoel roof of the developing embryos and its receptor is expressed in the migrating mesoderm. This complementary expression of ligand and receptor orients and directs the movement of mesodermal cells inward during gastrulation. Interfering with PDGFα signaling randomizes internal movement of these cells and prevents proper convergence and

179

extension leading to improper patterning and some of the phenotypes (incomplete blastopore closure, shortened axis) observed in the Gam1-injected embryos.

Analysis of the microarray data shows a down regulation of both PDGFα (LG-

70%) and PDGFαR (LG-30%, EN-45%). As described above, a decrease in SUMOylation could lead to a decrease in the transcriptional activity of Ets-1 thereby explaining the decrease in transcription of the PDGFα and PDGFαR genes (Fig. 20). Studies in vascular smooth muscle cells show that cooperation between Ets-1 and the transcription factor

Sp1 is necessary for transcription of PDGFα (284). As Sp1 is also a target of

SUMOylation (74, 75), it is possible that SUMO modification of either or both factors could impact the expression of PDGFα.

Expression levels from the microarray data for both the H2O and Gam1-injected embryos were compared to previously published mRNA expression values for all differentially regulated genes in the Ets-1 pathway (Fig. 21). In the majority of cases, the general trends are in agreement between the H2O-injected embryos and previously measured X. laevis data; however, the measured expression levels are lower than initially reported (289). The exception is Skp1 as prior measurements show a slight up- regulation as development proceeds while the recent data indicates a slight down regulation. Additionally, Egr1 expression levels are higher than previously measured.

Discrepancies in the data may be attributed to the difference in microarray chips,

Agilent versus Affymetrix, or the normalization methods. The previous report used linear extrapolation from spike-in measurements while the data above utilized the quantile normalization method. 180

Notwithstanding these disagreements, the differentially expressed genes can be grouped together based on how SUMOylation knockdown has affected their expression.

PDGFα, PDGFαR, and IGF2 are all down regulated in Gam1 injected embryos due to the gene not being activated at the proper stage. PLK3, CDC25a, and Dusp1 are also down regulated, however, these changes in expression are due to deactivation of the gene occurring too early in development. CDC16 should be reactivated slightly at early neurula and remains at a steady state in Gam1-injected embryos causing a down- regulation to be measured.

Up-regulation observed for CDC23, Skp1, and Egr1 appears to be due to each gene being reactivated when it should be repressed. Rrm1 is also up-regulated but this change is due to a failure of the gene to be deactivated to the extent seen in H2O- injected embryos. The various forms of regulation observed indicate that SUMOylation of transcription factors controlling these genes can have different affects depending on the target gene. This analysis further supports the hypothesis that SUMOylation of one transcription factor can create very diverse outcomes, such as activation versus repression, depending on what is required.

181

Figure 21. Gene expression levels (log10) from microarray data compared to previously measured mRNA levels for genes regulated by Ets-1. H2O (blue) and Gam1-injected (black) expression is plotted for EG (st. 10), LG (st. 12), and EN (st. 14) stage embryos. mRNA levels from X. topicalis (green) and X. laevis (red) are plotted for stage 2-18 embryos. An asterisk denotes the time points where genes are differentially regulated, pg. 183-184.

182

183

184

Cell migration analysis using Keller explant sandwiches

The observed phenotypes of SUMOylation deficient embryos and the pathway analysis have pointed to a role for this post-translational modification in early cell migration and patterning which has also been shown in previous studies (153, 184).

As cell movement during gastrulation is primarily internal and cannot be easily observed, Keller explant assays are used to visualize those movements by dissecting out the migrating tissue and observing development (290, 291). Convergence and extension in Xenopus embryos requires very specific and coordinated cell movements (292). At the beginning of gastrulation, prospective mesodermal and endodermal cells migrate inward as the blastopore forms. Subsequently, prospective ectodermal cells remain external and cover the embryo through a process called epiboly. As internalized cells migrate along the blastocoel roof, they also undergo mediolateral or radial intercalation in which multiple layers of cells become one layer thereby flattening and elongating the tissue resulting in convergence and extension of the anterior-posterior embryonic axis.

The purpose of Keller explants is to dissect the portions of the embryo which contain prospective mesodermal and endodermal cells to allow for direct visualization of cell movements responsible for convergence and extension (34). Explants are made by sandwiching together a portion of two embryos, dorsal to the blastopore lip, which contain migrating cells. This sandwiching causes the mesodermal and endodermal cells to elongate in a plane rather than involuting beneath ectodermal cells which enables quantification of the degree of convergence and extension. 185

The phenotypes of a shortened axis and failure of the blastopore and neural tube to close, observed in the SUMO deficient embryos, indicate that SUMOylation somehow controls convergence and extension movements. In order to test that directly, Keller explants were prepared from embryos injected with either H2O or Gam1 mRNA and measurements were taken to establish the degree of convergence and extension that had occurred in each. Embryos injected with H2O showed normal convergence and extension while those injected with 2.5 ng Gam1 failed to elongate properly (Fig. 22 A). Additionally, when the amount of Gam1 mRNA was doubled to 5.0 ng, explants not only failed to converge and extend but cells of the explants failed to adhere to each other. This indicated not only a role of SUMOylation in controlling cell migration but also in controlling cell adhesion.

The degree of convergence and extension of explants was quantified through measurement of the length (anterior to posterior) and the convergence (at the boundary of the non-involuting and involuting marginal zones (Fig. 22 B,C). Two-tailed t-tests show that both convergence (p=0.001) and extension (p=0.00005) are significantly decreased in those embryos injected with Gam1 compared to H2O.

Disruption of convergence and extension in SUMO deficient embryos explains the observed phenotypes and also validates the use of the microarray data in identifying those pathways controlled by SUMOylation.

186

Figure 22. Gam1 disrupts convergence and extension in Keller sandwich explants. (A) Explants made from embryos injected with H2O (top) show normal convergence and extension, while those from embryos injected with 2.5 ng Gam1 mRNA (middle) display a decrease in both convergence and extension. Explants from embryos injected with 5 ng of Gam1 mRNA (bottom) fail to adhere. Explants were measured in order to quantify the degree of convergence (B) and extension (C). Explants were measured along the indicated red line for each quantification. Error bars shown are the standard deviation for control (n=17) and 2.5 ng Gam1 (n=18) explants.

187

B C

188

The data presented in this thesis highlight the extensive and complicated roles of

SUMOylation in early Xenopus laevis development and several major conclusions can be drawn from the experiments described above. The knockdown of SUMOylation activity through Gam1 expression is an effective means of studying the necessity of

SUMOylation in vertebrate early development as it provides viable embryos with distinct and reproducible phenotypes.

This approach has highlighted the fact that SUMOylation regulates a vast number of genes and in turn a large number of processes in the early stages of development. This regulation is most likely due to SUMO modification of transcription factors which can create two pools of factors which have different target genes and can also act as a molecular switch to convert factors between activators and repressors of gene expression. Enrichment analysis has identified several pathways whose disruption may be responsible for the observed phenotypes and further analysis of these pathways may provide insight into birth defects such as spina bifida and congenital heart defects.

189

APPENDIX A

Negative numbers for the log2 differential expression and numbers less than 1 for the fold change indicate down regulation in embryos injected with Gam1 mRNA.

Positive numbers for the log2 differential expression and numbers greater than 1 for the fold change indicate up regulation.

Table A.1: Page 191. Genes differentially expressed (p<0.05) between Gam1 mRNA and

H2O injected embryos at early gastrula.

Table A.2: Page 198. Genes differentially expressed (p<0.05) between Gam1 mRNA and

H2O injected embryos at late gastrula.

Table A.3: Page 234. Genes differentially expressed (p<0.05) between Gam1 mRNA and

H2O injected embryos at early neurula.

190

TABLE A.2

GENES DIFFERENTIALLY EXPRESSED (P<0.05) BETWEEN GAM1 MRNA AND H2O INJECTED EMBRYOS AT EARLY GASTRULA.

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol

191 Xl2.956.1.S1_a_at 0.020049387 0.075485617 1.053715661 abl-interactor 2 abi2

Xl2.29261.1.S2_at 0.017137783 -0.446812526 0.733662003 ADP-ribosylation factor-like 4A arl4a Xl2.3975.1.S1_at 0.014686125 0.034344396 1.024091339 alkB, alkylation repair homolog 6 alkbh6 ARP3 actin-related protein 3 Xl2.23453.1.S1_at 0.009922489 -0.112690746 0.924861509 homolog actr3 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha Xl2.29153.1.S1_at 0.003414482 -0.357753152 0.780378993 subunit, isoform 1 atp5a1 Xl2.45543.1.S1_at 0.016521929 -0.083081394 0.944039157 cell division cycle 16 homolog cdc16 Xl2.47502.1.S1_at 0.024915427 -0.41564215 0.749685732 chloride intracellular channel 3 clic3 chromosome 15 open reading Xl2.2458.1.S1_at 0.008277989 0.177332382 1.13079106 frame 26 c15orf26 Xl2.532.1.S1_at 0.038538477 0.782525296 1.720139174 coiled-coil domain containing 18 ccdc18 Xl2.31947.1.S1_at 0.048402897 -0.318623338 0.801834647 cullin 4A cul4a

TABLE A.1 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.15931.1.S1_at 0.001092749 -0.060431221 0.958977438 DMRT-like family A2 dmrta2 Xl2.31935.1.S2_at 0.025131738 -0.575633542 0.670991529 dual specificity phosphatase 6 dusp6 Xl2.47694.1.S1_x_at 0.011246633 0.462412949 1.377844377 E3 ubiquitin ligase Smurf2 LOC398372 Xl2.46713.1.S1_at 0.025085237 0.493401901 1.40776049 early endosome antigen 1 eea1 Xl2.24906.1.S1_at 0.040469343 0.219157118 1.1640533 enigma (LIM domain protein) enigma Xl2.277.1.S1_at 0.006479956 -0.702093959 0.6146794 even-skipped homeobox 1 evx1 family with sequence similarity Xl2.48955.1.S1_at 0.002107049 0.011819388 1.008226227 184, member A fam184a 192 family with sequence similarity Xl2Affx.24.1.S1_s_at 0.035694339 0.096391972 1.069096419 49, member A fam49a Fibrous sheath-interacting Xl2.56044.2.A1_at 0.012859902 -0.154293653 0.898572208 protein 1 fsip1 Xl2.53777.1.S1_at 0.004453465 -1.413153379 0.37549006 forkhead box C1 foxc1 Xl2.66.1.S2_at 6.55E-05 -0.025129149 0.982732621 forkhead box D1 foxd1 Xl2.1040.1.S1_at 0.045172344 -0.734247722 0.601131397 frizzled homolog 10 fzd10-a Xl2.18511.1.S1_at 0.043631062 0.1086324 1.078205669 glycerol kinase 5 (putative) gk5 golgi-associated, gamma adaptin ear containing, ARF binding Xl2.47630.1.S1_at 0.033597042 -0.243340572 0.844786935 protein 1 gga1 GTPase-activating protein and VPS9 domain-containing protein Xl2.9260.1.A1_at 0.042611187 -0.119673644 0.920395832 1 gapvd1

TABLE A.1 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.1205.1.S1_a_at 0.045017083 -0.412355299 0.751395667 H1 histone family, member 0 h1f0 high-mobility group nucleosomal Xl2.22529.1.S1_a_at 0.028541638 -0.054853618 0.962692117 binding domain 2 hmgn2 Xl2.36404.1.S1_at 0.008496957 0.066736249 1.047344636 histone cluster 2, H3d hist2h3d Xl2.17674.1.S1_a_at 0.007803871 0.521780418 1.435725972 hypothetical LOC495139 LOC495139 hypothetical protein Xl2.6985.1.S1_x_at 0.049837735 0.211424781 1.157831074 LOC100049125 LOC100049125 Hypothetical protein 193 Xl2.50948.1.S1_at 0.030146438 0.295971878 1.227711752 LOC100137673 LOC100137673

Xl2.15452.1.S1_at 0.018700435 0.241647168 1.182341806 hypothetical protein LOC432214 LOC432214 Xl2.33980.1.S1_at 0.012752989 -0.174758805 0.885915618 hypothetical protein LOC443643 LOC443643 Xl2.32906.1.S1_at 0.015678109 0.437251108 1.354021932 hypothetical protein LOC733182 LOC733182 Xl2.25101.1.A1_at 0.012858795 0.409618564 1.328334567 hypothetical protein LOC733326 LOC733326 Xl2.32455.1.S1_at 0.025811885 -0.223565322 0.856446289 hypothetical protein LOC733360 LOC733360 hypothetical protein MGC115288 MGC115288 /// Xl2.27469.2.S1_s_at 0.016217623 -0.270025787 0.829304723 /// serine/threonine kinase 17a stk17a Xl2.16117.1.S1_at 0.04045585 0.094684677 1.067831992 hypothetical protein MGC115313 MGC115313 Xl2.18687.2.A1_x_at 0.030457439 0.416416853 1.334608737 Hypothetical protein MGC81356 MGC81356 Xl2.13292.1.S1_at 0.020968168 0.155960251 1.114162953 hypothetical protein MGC81394 MGC81394 Xl2.54413.1.S1_at 0.03484506 0.378396153 1.299895955 lipase, hormone-sensitive Lipe Xl2.25260.1.A1_s_at 0.028136387 -0.234522534 0.849966253 lysophosphatidic acid receptor 4 lpar4

TABLE A.1 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.3702.1.A1_at 0.002110241 -0.32360748 0.799069293 male-specific lethal 2 homolog msl2 MID1 interacting protein 1 (gastrulation specific G12 Xl2.47645.1.S1_at 0.008798208 0.072745417 1.051716173 homolog) mid1ip1 minichromosome maintenance Xl2.7149.1.S1_at 0.039447545 -0.504607214 0.704852253 complex component 6 mcm6.2 mitochondrial ribosomal protein Xl2.50120.1.S1_at 0.003964668 0.231587761 1.174126426 L11 mrpl11 194 MKI67 (FHA domain) interacting

Xl2.13504.1.S1_at 0.004617126 -0.088911494 0.940231881 nucleolar phosphoprotein mki67ip MOB1, Mps One Binder kinase Xl2.5955.1.S1_at 0.010020578 0.06542261 1.046391415 activator-like 3 mobkl3 Xl2.53972.1.S1_at 2.62E-05 -0.215329688 0.8613493 N-glycanase 1 ngly1 NOP58 ribonucleoprotein Xl2.47829.1.S1_at 0.00073053 0.142375534 1.103721002 homolog nop58 nuclear Y/CCAAT-box binding factor C subunit NF-YC /// nuclear Xl2.6752.1.S1_a_at 0.038507214 -0.325160011 0.798209851 transcription factor Y, gamma nfyc /// nfyc Xl2.4315.1.S1_at 0.041873808 -0.15307258 0.899333067 nucleoporin 160kDa nup160 Xl2.19055.1.S1_at 0.028286967 0.086828656 1.062033045 omega-amidase NIT2-B MGC82469 Xl2.847.1.S2_at 0.006499412 -0.067838567 0.954066302 Otogelin Otog Xl2.47227.1.S1_at 0.027858621 0.087354196 1.06241999 OTU domain containing 6B otud6b

TABLE A.1 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.16235.1.S1_at 0.028746551 0.170334338 1.125319242 peroxiredoxin 5 prdx5 phosphatidic acid phosphatase Xl2.48740.1.S1_x_at 0.03616056 -0.530438461 0.692344286 type 2B ppap2b Xl2.19981.1.S1_at 0.04218148 -0.428558897 0.743003599 polo-like kinase 3 plk3 Xl2.55576.1.S1_at 0.017604144 0.14275053 1.104007926 prefoldin subunit 5 pfdn5 proprotein convertase Xl2.48635.1.S2_a_at 0.005710345 -0.155211948 0.898000437 subtilisin/kexin type 6 pcsk6 Protein prenyltransferase alpha 195 subunit repeat-containing protein

Xl2.18328.1.S1_x_at 0.045183035 -0.109048134 0.927199611 1-B ptar1-b Xl2.24523.1.S1_at 0.02097959 -0.207975305 0.865751385 prothymosin, alpha ptma-b Xl2.12030.1.S1_at 0.028276243 -0.121552091 0.91919822 RAN binding protein 3 ranbp3 Xl2.1987.4.S1_a_at 0.047988516 -0.248331915 0.841869246 reticulon 4 b rtn4b Xl2.23633.1.S1_at 0.019485826 0.209932933 1.156634414 Rho GTPase activating protein 12 arhgap12 ribosomal Xl2.55093.1.A1_x_at 0.041881034 0.12970501 1.094069973 Ribosomal protein S1a protein protein S1a ribosome binding protein 1 Xl2.19941.1.S1_at 0.030481588 0.259864096 1.197365906 homolog 180kDa (dog) rrbp1 Xl2.48056.3.S1_a_at 0.02273193 0.646210761 1.565052182 Scc2-1B scc2-1 Xl2.31476.1.S1_at 0.005662092 -0.080338413 0.945835756 septin 12 sep12 Xl2.27469.2.S1_at 0.021910989 -0.291824318 0.816868458 serine/threonine kinase 17a stk17a

TABLE A.1 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol serine/threonine kinase receptor Xl2.52043.1.S1_at 0.019493364 -0.146356842 0.903529217 associated protein Strap serum/glucocorticoid regulated Xl2.7842.1.S1_at 0.037036753 -0.439012878 0.737639144 kinase 1 sgk1 SIN3 homolog A, transcription Xl2.7792.1.S1_at 0.020067561 -0.080938042 0.945442719 regulator sin3a Xl2.46600.1.S1_at 0.00505918 -0.234255734 0.850123453 Sin3A-associated protein, 30kDa sap30 solute carrier family 37 (glucose- 196 6-phosphate transporter),

Xl2.24203.1.S1_at 0.025318453 0.14293676 1.104150446 member 4 slc37a4 sprouty homolog 1, antagonist of Xl2.10087.3.S1_at 0.011375463 -0.424746271 0.744969739 FGF signaling spry1-b SRY (sex determining region Y)- Xl2.188.1.S2_at 0.009036786 -0.807214983 0.571484002 box 2 sox2 StAR-related lipid transfer Xl2.18908.2.S1_at 0.030447531 -0.108360438 0.927641689 (START) domain containing 4 stard4 Xl2.4631.1.S1_a_at 0.010946428 -0.091280565 0.93868918 stem-loop binding protein Slbp striatin, calmodulin binding Xl2.25909.1.S1_at 0.001515455 -0.11545995 0.923087971 protein Strn structural maintenance of Xl2.928.1.S1_at 0.03044341 0.483860015 1.398480381 chromosomes 2 smc2

TABLE A.1 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol TAF3 RNA polymerase II, TATA box binding protein (TBP)- Xl2.33423.1.S1_at 0.022894407 0.415294658 1.333571019 associated factor, 140kDa taf3 tankyrase, TRF1-interacting ankyrin-related ADP-ribose Xl2.33022.1.A1_s_at 0.013887918 0.287750436 1.220735326 polymerase 2 tnks2 Xl2.10706.2.S1_at 0.02585658 -0.158401281 0.89601744 Tctex1 domain containing 1 tctex1d1 TRAF family member-associated 197 Xl2.53193.1.S1_at 0.033758884 0.447729302 1.363891899 NFKB activator Tank

Xl2.49796.1.S1_at 0.022651376 0.198338026 1.147375825 transcription factor Dp-1 b tfdp1b Xl2.53918.1.S1_at 0.039066944 0.080403111 1.057313429 translation initiation factor SUI1 sui1 twist homolog 1 /// twist Xl2.879.1.S1_s_at 0.013462256 -0.15265128 0.899595731 homolog 2 twist1 /// twist2 UPF2 regulator of nonsense Xl2.45425.1.S1_at 0.025178113 0.190640806 1.141270524 transcripts homolog upf2 vascular endothelial zinc finger 1 Xl2.53359.1.S1_at 0.011840582 -0.269511137 0.829600612 b vezf1b voltage-dependent anion Xl2.6273.1.S1_at 0.023286138 -0.045511791 0.968946026 channel 1 vdac1 XK, Kell blood group complex Xl2.16705.1.S1_at 0.048589673 -0.682842206 0.622936837 subunit-related family, member 5 xkr5 Xl2.15800.1.S1_at 0.025424397 -0.211776191 0.863473501 Yip1 domain family, member 6 yipf6 Xl2.23505.1.S1_at 0.025031025 -0.349331976 0.784947475 zincfinger protein clone LcGF48.2 lcgf48.2-a

TABLE A.2

GENES DIFFERENTIALLY EXPRESSED (P<0.05) BETWEEN GAM1 MRNA AND H2O INJECTED EMBRYOS AT LATE GASTRULA.

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol 3-hydroxy-3-methylglutaryl- Xl2.13148.1.S1_s_at 0.034841948 0.721130997 1.648473845 Coenzyme A synthase 1 hmgcs1 3-hydroxy-3-methylglutaryl- Xl2.13148.1.S1_x_at 0.03868647 0.544813537 1.458831784 Coenzyme A synthase 1 hmgcs1

198 3-hydroxyisobutyrate

Xl2.22389.1.S1_at 0.034834182 0.208280651 1.155310508 dehydrogenase Hibadh Xl2.49747.1.S1_at 0.001782831 5.217340787 37.20283828 5'-nucleotidase, cytosolic III nt5c3 abhydrolase domain containing Xl2.11489.1.S1_at 0.031373319 -0.379188819 0.76886978 3 abhd3 Xl2.29221.1.S1_a_at 0.007061602 0.233811349 1.175937471 actin, gamma 1 actg1 Xl2.6255.1.S1_at 0.026325 -0.180938277 0.882129105 additional sex combs like 1 asxl1 Xl2.47277.2.S1_x_at 8.36525E-05 1.067030168 2.095116064 adenosine deaminase CECR1 cecr1 ADP-ribosylation-like factor 6 Xl2.16810.1.S1_at 0.017835801 0.286646128 1.219801274 interacting protein 6 arl6ip6 Xl2.32071.1.S1_at 0.005930942 -0.626674203 0.647667743 alkaline ceramidase 3 acer3 Xl2.3522.1.S1_at 0.009136369 -0.261754205 0.834073134 alkB, alkylation repair homolog 5 alkbh5 ankyrin repeat and SOCS box- Xl2.16856.1.S1_at 0.048467602 0.584865097 1.49989873 containing 13 asb13

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.10696.1.S1_at 0.000622402 -0.286348629 0.81997474 annexin A4 anxa4 Xl2.25847.1.A1_at 0.014575094 -1.515602439 0.349750387 anterior gradient 2 agr2 Xl2.25391.1.S1_at 0.009671984 1.285441055 2.437565617 apoptosis enhancing nuclease Aen apoptosis-inducing factor, Xl2Affx.8.1.S1_s_at 0.04390107 0.059139689 1.0418443 mitochondrion-associated, 2 aifm2 Xl2.892.1.S1_a_at 0.021950124 1.094977784 2.136097923 arginase type II arg2-b Xl2.1242.1.S1_at 0.012481188 -0.467035667 0.723449557 arginase, liver arg1 Xl2.34147.1.A1_a_at 0.03836787 0.3901494 1.31052911 aspartoacylase (aminocyclase) 3 acy3

199 Xl2.34147.2.A1_x_at 0.038792745 0.370428496 1.292736731 aspartoacylase (aminocyclase) 3 acy3 ATP synthase, H+ transporting, mitochondrial F1 complex, Xl2.25560.1.S1_at 0.012554296 0.206425757 1.153826063 gamma polypeptide 1 atp5c1 Xl2.610.1.A1_at 0.008679587 -0.175438132 0.885498561 Axin-related protein Xarp BAH domain and coiled-coil Xl2.12374.1.A1_at 0.026873623 -0.593763847 0.662611962 containing 1 bahcc1 Xl2.53752.1.S2_at 0.036799524 -0.678361314 0.624874636 BAI1-associated protein 2-like 1 baiap2l1 Xl2.43086.1.S1_at 0.02591924 -0.520978423 0.696899042 B-cell CLL/lymphoma 7A bcl7a BMP and activin membrane- Xl2.4715.1.S1_at 0.026026185 -0.457717409 0.728137386 bound inhibitor Bambi

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol BMP and activin membrane- bound inhibitor /// MGC81574 bambi /// Xl2.25777.1.S1_s_at 0.011055791 -0.367477245 0.775136748 protein MGC81574 Xl2.2964.1.S1_at 0.02170837 0.502104913 1.416278426 Boc homolog Boc bone morphogenetic protein 2 /// bone morphogenetic protein Xl2.1140.1.S1_s_at 0.021411457 -0.317755826 0.802316946 2 B bmp2 /// bmp2-b Xl2.1141.1.S1_x_at 0.049816276 -0.098118696 0.93425048 bone morphogenetic protein 4 bmp4 200 Xl2.53712.1.S1_at 0.016236809 -1.039574197 0.486471032 Bowline2 protein bowline2

breast cancer metastasis- Xl2.21343.1.S1_at 0.03860019 0.342081656 1.267584265 suppressor 1-like brms1l cAMP responsive element Xl2.16347.1.S1_at 0.039890958 0.576735123 1.49147017 binding protein 3 creb3 carboxypeptidase N, Xl2.14830.1.S1_at 0.023998124 -0.895801559 0.537448507 polypeptide 1 cpn1 CCAAT/enhancer binding Xl2.3789.2.S1_a_at 0.00939713 -0.929456249 0.525056198 protein (C/EBP), alpha Cebpa CCAAT-enhancer binding protein Xl2.25857.1.S1_at 0.031098673 -0.790442963 0.578166545 delta LOC398729 CCHC-type zinc finger, nucleic Xl2.985.1.S1_a_at 0.039518503 -0.184891675 0.879715127 acid binding protein Cnbp Xl2.8146.1.S1_at 0.003472423 -0.334211049 0.793217797 CDC-like kinase 2 clk2

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol CDK5 regulatory subunit Xl2.13079.1.S1_at 0.037955802 0.199374237 1.14820022 associated protein 1-like 1 cdkal1 Xl2.14197.1.S1_at 0.021140084 -0.431102838 0.741694596 cell division cycle associated 7 cdca7 chemokine (C-X-C motif) ligand Xl2.29309.1.S1_at 0.005076695 -0.963682376 0.512746493 12 (stromal cell-derived factor 1) cxcl12 Xl2.56738.1.S1_at 0.019619133 -0.003873149 0.997318938 Chimerin (chimaerin) 1 chn1 chromosome 1 open reading Xl2.3649.1.S1_at 0.005135849 -0.20973851 0.864693944 frame 144 c1orf144 201 chromosome 10 open reading

Xl2.52870.1.S1_at 0.019129534 -0.86506422 0.549021967 frame 140 c10orf140 chromosome 16 open reading Xl2.9881.1.S1_at 0.006325728 0.463752076 1.379123902 frame 57 c16orf57 chromosome 17 open reading Xl2.3785.1.S1_at 0.041744921 -0.211953508 0.863367381 frame 75 c17orf75 chromosome 2 open reading Xl2.51233.1.S1_at 0.008198684 -0.088526714 0.940482684 frame 42 c2orf42 chromosome 21 open reading Xl2.4468.1.S1_at 0.033457832 -0.269963839 0.829340333 frame 59 c21orf59 chromosome 22 open reading Xl2.6011.2.A1_a_at 0.039864315 1.101004225 2.145039515 frame 28 c22orf28 chromosome 3 open reading Xl2.4117.1.S1_at 0.04195263 -0.807806256 0.571249833 frame 54 c3orf54-b

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol chromosome 3 open reading Xl2.19250.1.S1_at 0.043740569 1.254367029 2.385624582 frame 63, gene 2 c3orf63.2 chromosome 6 open reading Xl2.7519.1.S1_at 0.030044355 0.276893948 1.211583594 frame 182 c6orf182 Xl2.50132.1.S1_at 0.00912956 -0.6202079 0.650577169 claudin 12 cldn12 Xl2.53796.1.S2_at 0.041540268 -0.213371236 0.862519371 claudin 4 cldn4 coiled-coil domain containing Xl2.15231.1.S1_at 0.045174515 -0.237575852 0.848169287 104 ccdc104 202 coiled-coil domain containing

Xl2.10892.1.S1_at 0.00052119 0.31750538 1.246173877 109A ccdc109a Xl2.5123.1.S2_at 0.00060485 4.361137979 20.55101834 complement component 9 c9 Xl2.5123.1.S1_at 0.006657718 4.59320767 24.13755547 complement component 9 c9 Xl2.5028.1.S1_at 0.021957509 -0.694960038 0.617726429 complement factor I Cfi Xl2.511.2.S1_a_at 0.025774263 -0.391748209 0.76220543 cone-rod homeobox Crx core promoter element binding Xl2.48835.1.S1_s_at 0.032265351 -0.466715791 0.723609979 protein /// Kruppel-like factor 6 copeb /// klf6 core-binding factor, beta Xl2.25702.1.S1_at 0.048133576 -0.515419423 0.699589517 subunit Cbfb core-binding factor, runt domain, alpha subunit 2; Xl2.4960.1.S1_at 0.042863078 -0.930872861 0.524540888 translocated to, 2 cbfa2t2

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol coronin, actin binding protein, Xl2.908.1.S1_at 0.010518207 0.123077549 1.089055554 1C coro1c Xl2.4103.1.S1_at 0.031469815 0.398547669 1.318180258 creatine kinase, brain Ckb Xl2.4103.1.S1_x_at 0.035445139 0.28558713 1.218906218 creatine kinase, brain Ckb creatine kinase, mitochondrial Xl2.2151.1.S1_at 0.000531791 -0.983915445 0.505605673 1B ckmt1b-a CUE domain-containing protein Xl2.48669.1.S1_s_at 0.024389787 0.186571188 1.138055715 2-B cuedc2-b 203 CWC25 spliceosome-associated

Xl2.4727.1.S1_a_at 0.038798519 -0.247826273 0.84216436 protein cwc25 Xl2.55726.1.A1_at 0.000849102 3.096090512 8.550984385 cyclin G1 ccng1 Xl2.55582.1.A1_at 0.005568367 2.9302808 7.62258747 cyclin G1 ccng1 Xl2.4340.1.S1_at 0.0477468 0.223584174 1.167630802 cyclin K Ccnk Xl2.50818.1.S1_at 0.017845999 -0.613028029 0.653822972 cyclin O Ccno cytochrome P450, family 26, Xl2.55931.1.S1_at 0.01206736 -1.974010552 0.254544438 subfamily C, polypeptide 1 cyp26c1 Xl2.14357.1.S1_at 0.044956971 -0.63623597 0.643389381 cytokine receptor-like factor 3 crlf3 dapper, antagonist of beta- Xl2.7602.1.S2_at 0.036960023 -0.36187932 0.778150266 catenin dact1-b dehydrogenase/reductase (SDR Xl2.13360.1.S1_at 0.030477401 -0.221483686 0.857682931 family) member 3 dhrs3 Xl2.54916.1.A1_s_at 0.009788473 -0.336760369 0.791817376 delta-like 1 dll1

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.14759.1.S1_at 0.045950981 -0.442699027 0.73575685 delta-like 1 dll1 derriere (tgf-beta family Xl2.457.1.S1_at 0.036323195 0.527085936 1.441015582 member) Der Xl2.47149.1.S1_at 0.000358111 -0.527491427 0.693760003 DMRT-like family A1 dmrta1 DnaJ (Hsp40) homolog, Xl2.5180.1.S1_at 0.044079735 0.279499036 1.213773339 subfamily B, member 11 dnajb11 Xl2.19069.1.S1_at 0.041055378 0.301193872 1.232163643 drebrin-like Dbnl Xl2.1243.1.S1_x_at 0.021909437 -0.376516769 0.770295142 dual specificity phosphatase 1 dusp1 204 Xl2.1243.1.S1_at 0.026826472 -0.410975947 0.752114415 dual specificity phosphatase 1 dusp1

early growth response 1 /// Early growth response protein 1- Xl2.637.1.S1_s_at 0.008298345 1.190056827 2.281617301 B egr1 /// egr1-b Xl2.25747.1.S1_at 0.023732876 -0.151388398 0.900383548 elongation factor 1 homolog elof1 emopamil binding protein Xl2.2628.1.S1_at 0.029590683 0.199866203 1.148591828 (sterol isomerase) Ebp endoplasmic reticulum Xl2.19291.1.S1_at 0.018989027 0.234832454 1.176770066 metallopeptidase 1 ermp1 Xl2.11684.1.S1_at 0.039429345 0.150199365 1.109722813 enhancer of mRNA decapping 4 edc4 Xl2.1028.2.S1_a_at 0.027109145 -0.246373734 0.843012698 EPH receptor B1 ephb1 Xl2.10209.1.S1_a_at 0.009078701 2.180022286 4.531605542 estrogen-related receptor alpha Esrra eukaryotic translation initiation Xl2.51779.1.S1_at 0.035461366 0.145622326 1.106207728 factor 3, subunit E eif3e-b

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.35568.1.S1_at 0.009420413 0.189476965 1.140350218 exosome component 1 exosc1 family with sequence similarity Xl2.50643.1.S1_at 0.039856158 1.092208902 2.132002163 158, member A fam158a family with sequence similarity Xl2.13086.2.S1_at 0.016533529 1.685948203 3.217517963 46, member A fam46a family with sequence similarity Xl2.48344.1.S1_at 0.003888255 1.459203238 2.749564705 69, member A fam69a farnesyl diphosphate synthase 205 (farnesyl pyrophosphate

synthetase, dimethylallyltranstransferase, Xl2.50432.1.S2_a_at 0.037007642 0.313690469 1.242882979 geranyltranstransferase) Fdps Xl2.18973.1.A1_at 0.042788978 0.234384142 1.176404446 F-box protein 27 fbxo27 Xl2.56920.1.S1_at 0.020416042 0.309097459 1.238932389 F-box protein 7 fbxo7 Xl2.16197.1.S1_at 0.010354455 -1.98461593 0.25268012 FEZ family zinc finger 2 fezf2 fibroblast growth factor 8 Xl2.32349.1.A1_s_at 0.02293519 -0.216292417 0.860774702 (androgen-induced) fgf8 fibroblast growth factor 8 Xl2.20011.1.S1_at 0.031877305 -0.295443751 0.814821667 (androgen-induced) fgf8 fibroblast growth factor Xl2.1008.1.S1_at 0.021167984 -0.514913535 0.699834875 receptor 4 fgfr4

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol fibroblast growth factor Xl2.17381.1.S1_at 0.03652879 -0.29281635 0.816306952 receptor substrate 2 frs2 fibroblast growth factor Xl2.48321.1.S2_at 0.028472023 -0.607871489 0.656164074 receptor-like 1 fgfrl1 Xl2.403.1.S1_at 0.011505974 -0.728462421 0.603546813 forkhead box B1 foxb1 Xl2.642.1.S1_at 0.039584474 -0.187285316 0.878256762 forkhead box D4-like 1, gene 1 foxd4l1.1-a Xl2.182.1.S1_at 0.01775872 -1.207498447 0.433018796 forkhead box F1 foxf1 Xl2.1396.1.S1_at 0.0220439 -1.182246813 0.440664685 forkhead box J1 foxj1 206 Xl2.48978.1.S1_at 0.005654829 -0.135396113 0.91041983 forkhead box N2 foxn2

Xl2.50790.1.S1_at 0.006843513 -1.734117926 0.300592741 forkhead box N4 foxn4 Xl2.2074.1.S2_at 0.013886039 1.184621171 2.273037005 forkhead box O3 foxo3 Xl2.508.1.S1_at 0.022322516 -0.552382714 0.681893003 forkhead box protein C2-B foxc2b Xl2.508.1.S1_x_at 0.02757539 -0.647587438 0.638346904 forkhead box protein C2-B foxc2b Xl2.34501.1.S1_at 0.022997155 -0.70839057 0.612002491 forkhead box protein J1-A foxj1a Xl2.48526.1.S1_at 0.04871037 -0.352447336 0.783254286 fragile histidine triad gene Fhit Xl2.560.1.S1_at 0.000119997 -0.449615555 0.732237947 frizzled homolog 2 fzd2 Xl2.633.1.S2_at 0.025789434 -0.244560011 0.84407318 frizzled homolog 7 fzd7 Xl2.412.1.S1_at 0.030005198 -0.30092128 0.811733872 frizzled-8 xfz8 Xl2.48195.1.S1_at 0.016783172 -0.646638193 0.638767053 FSCN1 protein fscn1 Xl2.16274.1.S1_at 0.036805994 -0.39683812 0.759521063 G patch domain containing 3 gpatch3

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol G protein-coupled receptor, Xl2.3029.2.S1_at 0.046197567 -0.951838116 0.516973374 family C, group 5, member B gprc5b gap junction protein, beta 2, Xl2.8924.1.S1_s_at 0.022144497 -0.397246186 0.759306263 26kDa gjb2 Xl2.28002.1.A1_at 0.026791279 -1.209121879 0.432531803 GATA binding factor-3 XGATA-3 Xl2.792.1.S2_at 0.008600563 -0.747563704 0.59560852 GATA binding protein 2 gata2 Xl2.792.1.S1_a_at 0.036083427 -0.649540104 0.637483495 GATA binding protein 2 gata2 Xl2.793.1.S1_at 0.014428542 -1.557869589 0.339652272 GATA binding protein 3 gata3 207 Xl2.183.1.S1_at 0.012108146 -0.253614459 0.838792311 GATA binding protein 6 gata6-b Xl2.23009.1.S1_at 0.037322166 -0.575141374 0.671220473 GATA binding protein 6 gata6-a Xl2.860.1.S1_at 0.046119929 1.128724187 2.186652833 general transcription factor 3A gtf3a general transcription factor IIA, Xl2.29248.1.S1_at 0.002548666 1.200469039 2.298143744 1, 19/37kDa gtf2a1 Xl2.481.1.S1_at 0.042922757 -2.212980367 0.215688271 GLI family zinc finger 2 gli2 Xl2.1041.1.S1_at 0.02230079 -0.899827185 0.535950927 GLI family zinc finger 3 gli3 glioma tumor suppressor Xl2.3545.1.S1_at 0.045489655 -0.200120099 0.870478096 candidate region gene 2 gltscr2 glucan (1,4-alpha-), branching Xl2.29829.1.S1_at 0.045538581 0.258487741 1.196224143 enzyme 1 gbe1 glucosamine (N-acetyl)-6- Xl2.46117.1.S1_at 0.036645968 -0.60834103 0.655950553 sulfatase Gns

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.52838.1.S1_at 0.028560747 0.389663101 1.310087435 glucosidase, alpha; neutral AB Ganab glutamate-ammonia ligase Xl2.47079.1.S2_at 0.016573034 -0.233025854 0.850848482 (glutamine synthase) Glul Xl2.46936.1.S1_at 0.038907673 -0.269377954 0.8296772 Golgi phosphoprotein 3-like A golph3l-a Xl2.48194.1.S1_at 0.026156518 -0.4326382 0.74090568 golgin A1 golga1 Xl2.28684.1.S1_at 0.034672181 0.492013245 1.406406111 golgin A2 golga2 Xl2.801.1.A1_at 0.004756116 -0.282262282 0.822300561 goosecoid protein goosecoid Xl2.12148.1.S1_at 0.029795232 0.50953332 1.423589621 GRAM domain containing 1C gramd1c 208 Xl2.34033.1.S1_s_at 0.021386718 -0.621838413 0.649842312 growth arrest-specific 6 gas6 Xl2.21546.1.S1_at 0.033312971 0.474397217 1.389337609 GTP-binding protein like 1 xG28K guanine nucleotide binding Xl2.46974.1.S1_at 0.02960324 -0.322610967 0.799621425 protein (G protein), gamma 5 gng5 Xl2.25977.3.S1_a_at 0.042321995 -0.417672828 0.748631248 hairy and enhancer of split 4 hes4-a hairy and enhancer of split 7, Xl2.24379.1.S1_at 0.004313779 -0.276615645 0.825525311 gene 2 hes7.2 hairy and enhancer of split 7, Xl2.24379.1.S1_x_at 0.034046122 -0.247500626 0.842354476 gene 2 hes7.2 HAUS augmin-like complex, Xl2.37779.1.S1_at 0.007931151 0.153832138 1.112520668 subunit 7 haus7 Xl2.3985.1.S1_at 0.007174995 0.228812417 1.171869904 HBS1-like hbs1l

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol heat shock 70kDa protein 5 (glucose-regulated protein, Xl2.21814.1.S1_at 0.033637266 0.807455515 1.750122024 78kDa) hspa5 Xl2.34774.1.S1_at 0.018154786 0.250766984 1.189839505 Heat shock protein gp96 TRA1 Xl2.50380.1.S1_at 0.013461221 -0.347587506 0.785897187 heat shock transcription factor 2 hsf2 Xl2.4985.1.S1_a_at 0.012747775 -0.321995114 0.799962838 helicase, lymphoid-specific Hells Xl2.131.1.A1_at 0.013426687 -1.075461013 0.474519404 HESX homeobox 1 hesx1 heterogeneous nuclear 209 Xl2.40721.2.S1_a_at 0.033592559 -0.15284576 0.899474471 ribonucleoprotein D-like-B hnrpdl-b

Xl2.42618.1.S1_at 0.022521368 -0.072655639 0.950886044 high-mobility group box 2 hmgb2 Xl2.25274.1.A1_at 0.016963389 0.300547658 1.231611854 HLA-B associated transcript 4 bat4 HMG box mitochondrial Xl2.8873.1.S1_at 0.014403729 0.204421355 1.152224113 transcription factor mttfa-A Xl2.9542.1.S1_at 0.002157029 2.255268168 4.774230324 HMG-box transcription factor 1 hbp1 Xl2.54392.1.S1_at 0.018985121 -0.88370814 0.541972614 homeobox B4 hoxb4 Xl2.3370.1.S1_at 0.003446443 -0.509369904 0.7025292 homeobox D1 hoxd1 Xl2.1003.1.A1_at 0.02783062 -1.366970097 0.387704639 homeobox protein (XANF-2) anf2 hyaluronan synthase related Xl2.23252.1.S1_at 0.008819989 -0.175481846 0.885471731 sequence protein has-rs hydroxyacid oxidase (glycolate Xl2.3534.1.S1_at 0.015269472 -0.513425127 0.700557257 oxidase) 1 hao1

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol hydroxyprostaglandin Xl2.39977.1.S1_at 0.029432002 -0.519510968 0.697608262 dehydrogenase 15-(NAD) Hpgd Xl2.9137.1.S1_at 0.013404046 -0.01745447 0.987974376 hypothetical LOC494589 LOC494589 Xl2.13414.1.S1_at 0.02736956 2.121741236 4.352189081 hypothetical LOC494708 LOC494708 Xl2.25637.1.S1_at 0.026459254 0.126539265 1.091671857 hypothetical LOC495269 LOC495269 Xl2.49112.1.S1_at 0.002246985 0.392854646 1.312988833 hypothetical LOC495823 LOC495823 Xl2.33893.1.S1_at 0.048304837 0.443696734 1.360084922 hypothetical LOC495944 LOC495944 hypothetical LOC496089 /// LOC496089 /// 210 Xl2.14602.1.S1_s_at 0.01452251 0.222806644 1.167001685 peroxiredoxin 4 prdx4

hypothetical protein Xl2.56337.1.S1_at 0.000123006 -0.603838541 0.658000898 LOC100036848 LOC100036848 hypothetical protein Xl2.54900.1.S1_at 0.002522526 0.360913213 1.284238551 LOC100036945 LOC100036945 hypothetical protein Xl2.17518.1.A1_at 0.007222866 -0.516547706 0.699042606 LOC100037050 LOC100037050 Hypothetical protein Xl2.12197.1.A1_at 0.014079621 0.169965355 1.125031468 LOC100049759 LOC100049759 Hypothetical protein Xl2.54863.1.S1_x_at 0.001473956 -0.521481141 0.696656245 LOC100101311 LOC100101311 hypothetical protein Xl2.51712.3.S1_a_at 0.022266896 0.353149854 1.277346432 LOC100137672 LOC100137672

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol hypothetical protein Xl2.16774.1.S1_at 0.008712989 1.765621247 3.400203863 LOC100158389 LOC100158389 hypothetical protein Xl2.34769.1.S1_at 0.009631437 1.527968323 2.883794423 LOC100158389 LOC100158389 Xl2.25830.1.S1_at 0.037846466 -0.43234776 0.741054853 hypothetical protein LOC443610 LOC443610 Xl2.33550.1.S1_at 0.048527306 0.184675603 1.136561383 hypothetical protein LOC443647 LOC443647 Xl2.47371.1.S1_at 0.024175433 0.770669054 1.706060791 hypothetical protein LOC443672 LOC443672 Xl2.53985.1.S1_s_at 0.001604473 0.15198222 1.111095035 hypothetical protein LOC733279 LOC733279 211 Xl2.19274.1.S1_at 0.004110082 0.774929979 1.711107001 Hypothetical protein LOC733408 LOC733408

Xl2.21410.1.S1_at 0.040552 -0.237618087 0.848144458 Hypothetical protein LOC733436 LOC733436 hypothetical protein Xl2.48475.1.S1_at 0.004990815 0.49390274 1.408249286 MGC114707 MGC114707 hypothetical protein Xl2.7237.1.S1_at 0.029875642 0.351971774 1.276303798 MGC114747 MGC114747 Hypothetical protein Xl2.51115.1.S1_at 0.028227899 0.288131036 1.221057413 MGC115462 MGC115462 Hypothetical protein Xl2.262.1.S1_at 0.045134057 -0.253595379 0.838803404 MGC130625 MGC130625 hypothetical protein Xl2.19496.1.S1_at 0.046451859 0.579126415 1.493944357 MGC130950 MGC130950 hypothetical protein Xl2.55974.1.S1_at 0.041007894 -0.776241988 0.583885752 MGC130961 MGC130961

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol hypothetical protein Xl2.33942.1.S1_at 0.038892387 -0.767869581 0.587284073 MGC131046 MGC131046 hypothetical protein Xl2.50539.1.S1_at 0.041990091 -0.4283401 0.74311629 MGC131081 MGC131081 Hypothetical protein Xl2.12354.1.S1_at 0.015375363 -0.960010824 0.514053057 MGC131155 MGC131155 Xl2.8199.1.S1_at 0.007763043 -0.581014827 0.668493377 hypothetical protein MGC52980 MGC52980 Xl2.5427.1.S1_at 0.043634606 0.206809043 1.154132645 hypothetical protein MGC53066 MGC53066 212 Xl2.45569.1.S1_at 0.008421662 0.227900841 1.171129684 hypothetical protein MGC64382 MGC64382

Xl2.15877.1.S1_at 0.012842698 -0.343973053 0.787868602 hypothetical protein MGC64589 MGC64589 Xl2.34474.1.S1_at 0.02252817 0.243214184 1.183626731 hypothetical protein MGC68580 MGC68580 Xl2.17546.1.S1_at 0.034779285 -0.586140244 0.666122656 hypothetical protein MGC68615 MGC68615 Xl2.16456.1.S1_at 0.047150501 -0.421771072 0.746507639 hypothetical protein MGC68858 MGC68858 Xl2.2686.1.S1_at 0.0345729 -0.21171511 0.86351006 hypothetical protein MGC68893 MGC68893 hypothetical protein MGC68999 /// tight junction protein 2 (zona MGC68999 /// Xl2.8126.1.S1_at 0.039235012 0.206469878 1.15386135 occludens 2) tjp2 Xl2.18687.2.A1_at 0.047362341 -0.030839568 0.978850496 Hypothetical protein MGC81356 MGC81356 Xl2.45830.1.S1_at 0.004430948 0.210556597 1.157134524 hypothetical protein MGC81440 MGC81440 Xl2.15758.1.S1_at 0.018641846 -0.434638033 0.739879364 hypothetical protein MGC81482 MGC81482 Xl2.6173.1.S1_at 0.030343633 -0.447168214 0.733481145 hypothetical protein MGC81522 MGC81522

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.24912.1.A1_at 0.044199215 -0.355102103 0.78181431 Hypothetical protein MGC83076 MGC83076 Xl2.6330.1.S1_at 0.024783499 -0.574349981 0.671588773 hypothetical protein MGC84581 MGC84581 Xl2.13767.1.S1_at 0.048466095 -0.538548105 0.688463414 hypothetical protein MGC85058 MGC85058 Xl2.33610.1.S1_at 0.048810547 0.567361051 1.481810592 hypoxia up-regulated 1 hyou1 Xl2.8317.1.S1_at 0.031368565 -0.248589974 0.841718672 immediate early response 2 ier2-b immediate early response 3 Xl2.10898.1.S1_at 0.030255106 -0.06486466 0.956035 interacting protein 1 ier3ip1 Xl2.33398.1.S1_at 0.040876734 -0.287852848 0.819120243 immediate early response 5-like ier5l 213 Xl2.4654.1.S1_at 0.03614488 0.295272486 1.227116725 Importin alpha 5.2 protein LOC407836 Xl2.14733.1.A1_at 0.002812062 -0.990149992 0.503425433 insulinoma-associated 1 insm1 Xl2.24778.1.S1_at 0.033866691 -0.255178108 0.837883688 integral membrane protein 2A b itm2ab Xl2.11208.1.S1_at 0.048862237 -0.579258623 0.669307635 interferon regulatory factor 2 irf2 Xl2.69.1.S1_at 0.033788667 -0.425710145 0.744472186 iroquois homeobox 1 irx1 Xl2.155.1.S1_at 0.026771595 -0.458146719 0.727920743 iroquois homeobox 2 irx2 Xl2.4522.1.S1_at 0.008741311 -0.496037163 0.709051753 iroquois homeobox 3 irx3 isoprenylcysteine carboxyl Xl2.16.1.S2_at 0.019798157 -0.421425197 0.74668663 methyltransferase icmt Xl2.16315.1.S1_at 0.049635197 0.328763491 1.255936472 jumonji domain containing 6 jmjd6 Xl2.5482.1.S1_at 0.00380594 -0.472539077 0.720695091 keratin 19 krt19 Xl2.6950.1.S1_at 0.021649129 0.788123999 1.726827531 KIAA1370 kiaa1370

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol kynurenine 3-monooxygenase Xl2.52644.1.S1_at 0.001760215 1.414966705 2.666535814 (kynurenine 3-hydroxylase) kmo leucine carboxyl Xl2.50712.1.S1_at 0.040040248 0.320138365 1.248450278 methyltransferase 1 lcmt1 Xl2.10574.1.S1_at 0.013820361 2.480658691 5.581522437 ligase IV, DNA, ATP-dependent lig4 Xl2.45689.1.S1_at 0.025258683 -0.366302645 0.775768099 LIM domain and actin binding 1 lima1 Xl2.4461.1.S2_at 0.000668451 -0.295972755 0.814522945 LIM domain binding protein 1 xldb1 LysM, putative peptidoglycan- 214 Xl2.8367.1.S1_at 0.04918275 0.647568055 1.566525284 binding, domain containing 2 lysmd2 Xl2.244.1.S1_at 0.01467649 -0.258732283 0.835822046 lysophosphatidic acid receptor 1 lpar1 Xl2.53980.1.S1_at 0.024371519 -0.005109621 0.996464545 lysyl oxidase-like 3 loxl3 major facilitator superfamily Xl2.3500.1.A1_at 0.012898342 0.634496343 1.552395701 domain containing 7 mfsd7 Xl2.3702.1.A1_at 0.036735346 -0.649085159 0.637684554 male-specific lethal 2 homolog msl2 mannosyl (beta-1,4-)- glycoprotein beta-1,4-N- Xl2.47452.1.S1_at 0.000585249 -0.68362358 0.622599542 acetylglucosaminyltransferase mgat3 Xl2.10038.1.A1_at 0.024810565 -0.34487091 0.787378427 MARVEL domain containing 3 marveld3 Xl2.53912.1.S1_s_at 0.014752396 -0.471289251 0.721319709 mediator complex subunit 4 med4 Xl2.8738.1.S1_at 0.034301566 -0.492558506 0.710763497 mediator complex subunit 4 med4 Xl2.452.2.S1_at 0.017653831 -0.583045319 0.667553182 Meis homeobox 3 meis3-a

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol membrane-associated ring Xl2.8458.1.S1_at 0.037501018 0.469970968 1.385081595 finger (C3HC4) 8 mar8 Xl2.55.1.S1_at 0.016239106 -0.686456836 0.621378041 mesoderm posterior homolog A mespa methyl-CpG binding domain Xl2.11399.1.S1_at 0.02114801 -0.308685211 0.807377221 protein 1 mbd1 Xl2.4916.1.S1_at 0.029197663 -0.764129205 0.58880866 methyltransferase like 13 mettl13 Xl2.5253.1.S1_at 0.040494131 0.206621595 1.153982699 methyltransferase like 3 mettl3 Xl2.40829.1.A1_s_at 0.008670849 -0.228519882 0.853510093 mex-3 homolog C mex3c 215 AFFX-Xl-gapdh-

M_a_at 0.032247048 0.571209898 1.48576907 mg:bb02e05 mg:bb02e05 Xl2.23223.1.S1_a_at 0.034661084 0.629004626 1.546497633 mg:bb02e05 mg:bb02e05 AFFX-Xl-gapdh-5_a_at 0.038502353 0.579973121 1.494821399 mg:bb02e05 mg:bb02e05 AFFX-Xl-gapdh-3_a_at 0.048684996 0.620070804 1.536950609 mg:bb02e05 mg:bb02e05 Xl2.18233.1.S1_at 0.044401069 -0.143793781 0.905135834 MGC79134 protein MGC79134 Xl2.47581.1.S1_at 0.023242853 -0.612969002 0.653849723 MGC80400 protein MGC80400 Xl2.12327.1.S1_at 0.036574553 -1.012599534 0.495652347 MGC80468 protein MGC80468 Xl2.50370.1.S1_at 0.004948435 -0.337289883 0.791526807 MGC80563 protein MGC80563 Xl2.45603.1.S1_at 0.030626532 -0.339090503 0.790539523 MGC80941 protein MGC80941 Xl2.51207.1.S1_at 0.018191116 -0.456298912 0.728853663 MGC81672 protein MGC81672 Xl2.7809.1.S2_at 0.009322048 2.26435904 4.804409224 MGC81892 protein MGC81892 Xl2.7809.1.S1_at 0.030293635 2.026650811 4.074578459 MGC81892 protein MGC81892

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.7311.1.S1_at 0.032702859 -0.336271406 0.792085786 MGC82349 protein MGC82349 Xl2.48977.1.S1_at 0.00778811 1.534944318 2.897772465 MGC84470 protein MGC84470 microtubule-associated protein Xl2.14237.1.S1_at 0.02459301 1.490978051 2.810794637 1 light chain 3 alpha map1lc3a Xl2.15338.1.S1_at 0.037970325 -0.188698185 0.877397083 Midnolin midn minichromosome maintenance Xl2.7149.1.S1_at 0.044541809 -0.463946604 0.725000249 complex component 6 mcm6.2 mitochondrial ribosomal protein 216 Xl2.50343.1.S1_at 0.016555313 -0.221596872 0.857615644 L40 mrpl40

MKI67 (FHA domain) interacting Xl2.13504.1.S1_at 0.036559357 -0.304795329 0.809557058 nucleolar phosphoprotein mki67ip MRE11 meiotic recombination Xl2.488.1.S1_at 0.032533374 0.272209663 1.207656084 11 homolog A mre11a Xl2.45216.1.S1_at 0.01686064 -0.649013636 0.637716168 msh homeobox 1 msx1 Xl2.31078.1.S1_at 0.00269509 -0.792101466 0.577502275 msh homeobox 2 msx2 Xl2.51183.1.S1_at 0.022659843 3.333157828 10.07814231 myeloid zinc finger 1 mzf1 Xl2.146.1.S1_at 0.027440153 -0.452878735 0.730583595 myogenic factor 5 myf5 Xl2.29888.1.S1_at 0.017724965 -0.003459287 0.997605078 myotubularin-related protein 3 mtmr3 Na+/K+-transporting ATPase Xl2.6045.1.S2_at 0.039507331 -0.584291535 0.666976791 beta subunit atpb-3

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol NADH dehydrogenase (ubiquinone) 1 alpha Xl2.25913.1.S1_at 0.038241909 0.270141031 1.205925707 subcomplex, 10, 42kDa ndufa10a NADH dehydrogenase (ubiquinone) 1 alpha Xl2.6970.1.S1_at 0.01096492 1.609795987 3.052086788 subcomplex, 8, 19kDa ndufa8 Xl2.2279.1.S1_at 0.014310819 0.48129901 1.396000066 NDRG family member 2 ndrg2 N-ethylmaleimide-sensitive 217 factor attachment protein,

Xl2.47189.1.S1_at 0.046755464 0.195166402 1.144856202 gamma napg neuro-oncological ventral Xl2.13279.1.A1_at 0.035135703 -0.294667337 0.815260297 antigen 2 nova2 Xl2.46726.1.S1_at 0.008095485 -0.486342312 0.713832597 neuropilin 2 nrp2 Xl2.9528.2.S1_a_at 0.042936737 0.291413675 1.223838911 Nibrin nbn NIMA (never in mitosis gene a)- Xl2.14397.1.S2_at 0.039146836 0.800434024 1.741625003 related kinase 6 nek6 Xl2.9076.1.S1_at 0.019191916 -1.591831955 0.331749925 NK3 homeobox 1 nkx3-1 nodal homolog 5 /// nodal nodal5 /// homolog 5, gene 2 /// Xnr5-6 nodal5.2 /// xnr5- Xl2.55965.2.S1_s_at 0.049606153 2.264025338 4.80329807 protein /// Xnr5-9 protein 6 /// xnr5-9

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol nuclear factor of kappa light polypeptide gene enhancer in B- Xl2.48864.1.S1_at 0.021079848 0.638906176 1.557148112 cells 1 nfkb1 Xl2.7966.1.S1_at 0.00903605 0.609766515 1.52601222 nuclear receptor co-repressor 2 ncor2 Xl2.16506.1.S1_at 0.027718321 -0.333276927 0.793731558 nuclear VCP-like nvl Xl2.17520.1.S1_at 0.028372612 0.530976051 1.444906413 ORM1-like 1 ormdl1 ornithine aminotransferase Xl2.25660.1.S1_at 0.016039531 0.593745598 1.509159823 (gyrate atrophy) oat

18 orphan G protein-coupled

Xl2.22754.1.S1_at 0.024910238 -0.165658537 0.89152148 receptor Xflop LOC496402 Xl2.16133.1.A1_at 0.028359671 0.301120511 1.232100989 oxidation resistance 1 oxr1 Xl2.3219.2.S1_at 0.013251842 -1.537007411 0.344599518 Paralemmin palm Xl2.19495.1.S1_at 0.021021322 0.201990642 1.150284432 paraoxonase 2 pon2 Xl2.19495.1.S1_x_at 0.036646439 0.193250504 1.143336843 paraoxonase 2 pon2 Xl2.8529.1.S1_at 0.024708975 -0.116759835 0.922256633 partner of NOB1 homolog pno1 Xl2.1039.1.S1_at 0.005230315 -0.61442322 0.653190983 patched-2 xptch-2 Xl2.35430.2.S1_a_at 0.013136422 2.280585095 4.858749637 pheromone receptor-like xV2R1 phosphatase and actin regulator Xl2.23936.1.S1_at 0.023736283 -1.002044287 0.499292006 1 phactr1 phosphatidic acid phosphatase Xl2.48740.1.S1_s_at 0.027210161 -0.412331771 0.751407921 type 2B ppap2b

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol phosphatidic acid phosphatase Xl2.33366.1.S1_at 0.040672167 -0.57154134 0.672897497 type 2B ppap2bb phosphoinositide-3-kinase, regulatory subunit, polypeptide Xl2.1475.1.A1_at 0.008452145 1.93670481 3.828302443 2 (p85 beta) pik3r2 Phospholipase A-2-activating protein /// phospholipase A2- Xl2.33906.1.S1_at 0.02293398 0.199158879 1.148028835 activating protein plaa /// plaa

219 phosphoribosyl pyrophosphate

Xl2.16570.1.S2_at 0.039961571 0.228242549 1.171407104 synthetase 1 prps1 Xl2.23800.1.S1_at 0.036926685 -0.304324076 0.809821541 pim-3 oncogene pim3 Xl2.2305.1.S1_at 0.03960143 0.198024233 1.147126293 plastin 3 pls3 platelet derived growth factor Xl2.20029.1.S1_at 0.00908845 -0.553749662 0.681247218 receptor, alpha polypeptide pdgfra platelet-derived growth factor Xl2.841.1.S1_a_at 0.021981625 -1.658608161 0.31674458 alpha polypeptide pdgfa pleckstrin homology domain Xl2.8824.1.S1_at 0.023761575 -0.281391099 0.822797264 containing, family J member 1 plekhj1 poliovirus receptor-related 1 Xl2.27064.1.S1_at 0.03361927 -0.309943319 0.806673451 (herpesvirus entry mediator C) pvrl1 Xl2.12115.1.S1_at 0.019870252 2.300286861 4.925556938 polo-like kinase 2 plk2 Xl2.1271.1.S1_at 0.003776225 -0.326977259 0.797205042 poly (ADP-ribose) polymerase 1 parp1

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol poly (ADP-ribose) polymerase Xl2.49984.1.S1_at 0.004826871 3.697609543 12.97452253 family, member 3 parp3 polymerase (DNA directed) Xl2.16859.1.S2_at 0.004533151 2.935038284 7.647765484 kappa polk polymerase (DNA directed) Xl2.16859.1.S1_at 0.015124953 0.275070175 1.210052947 kappa polk polymerase (DNA directed), Xl2.7596.1.S1_a_at 0.020553906 -0.227201929 0.85429016 alpha 1, catalytic subunit pola1 220 polymerase (RNA) I polypeptide

Xl2.3359.1.S1_at 0.037166989 -0.352913397 0.783001298 C, 30kDa polr1c polymerase (RNA) II (DNA directed) polypeptide K, 7.0kDa, Xl2.17403.1.S1_at 0.025391497 -0.252430836 0.839480759 b polr2kb polypyrimidine tract binding Xl2.4377.1.S1_at 0.036342516 -0.297970818 0.813395651 protein 1 ptbp1 Xl2.12111.1.S1_s_at 0.026653219 0.494282328 1.40861986 prickle homolog 1 prickle1-b Xl2.7556.1.S1_a_at 0.044414658 0.370988362 1.2932385 prickle homolog 1 prickle1-a probable glutathione peroxidase Xl2.49093.1.S1_at 0.026720837 0.955097548 1.938710715 8-B gpx8-b

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol prosaposin (variant Gaucher disease and variant Xl2.23740.1.S1_at 0.04494452 0.222487375 1.166743455 metachromatic leukodystrophy) psap proteasome (prosome, macropain) 26S subunit, non- Xl2.20765.1.S1_at 0.008362989 1.339566094 2.530751923 ATPase, 14 psmd14 proteasome (prosome, Xl2.25803.1.S1_at 0.019858291 0.176862765 1.130423032 macropain) activator subunit 4 psme4

221 protein inhibitor of activated

Xl2.49143.1.S1_at 0.038751131 -0.201483948 0.86965558 STAT, 4 pias4 protein kinase, interferon- inducible double stranded RNA Xl2.852.1.S1_at 0.042394111 0.330675399 1.257601984 dependent activator prkra Xl2.54097.1.S1_at 0.00426106 -0.704608138 0.613609133 Protein odd-skipped-related 2-A osr2-a protein phosphatase 1, regulatory (inhibitor) subunit Xl2.3709.1.S1_at 0.041681053 0.218204186 1.163284671 15B ppp1r15b protein tyrosine phosphatase, Xl2.9691.1.A1_at 0.023818332 0.763015798 1.697034387 non-receptor type 3 ptpn3 Xl2.45977.1.A1_at 0.028901082 0.88567881 1.847633756 Pyruvate carboxylase MGC68971 pyruvate dehydrogenase Xl2.4211.1.S1_at 0.037961425 0.147318359 1.107508951 (lipoamide) beta pdhb

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol queuine tRNA-ribosyltransferase Xl2.15850.1.S1_at 0.007305839 0.105009154 1.075501215 domain containing 1 qtrtd1 RAB33A, member RAS oncogene Xl2.47462.1.S1_at 0.039852139 -0.005514023 0.996185265 family rab33a RAB33B, member RAS oncogene Xl2.42680.1.S1_at 0.01491865 -0.423960071 0.745375823 family rab33b RAB5A, member RAS oncogene Xl2.2484.1.S1_at 0.033089753 0.187990624 1.139175974 family rab5a 222 Xl2.292.1.S2_at 0.039418236 0.934427641 1.911132284 RAD51 homolog (RecA homolog) rad51

Xl2.1767.1.S2_at 0.019575073 0.434964536 1.351877601 RAN binding protein 9 ranbp9 RAN, member RAS oncogene Xl2.55052.1.A1_s_at 0.024833629 -0.091548028 0.938515172 family ran Xl2.50386.1.S1_at 0.04698287 -0.088800399 0.940304287 RAS protein activator like 2 rasal2 Xl2.3758.1.A1_at 0.026453014 0.336066676 1.26231037 Ras-related GTP binding A rraga Regulator of G-protein signaling Xl2.31883.2.S1_at 0.011450302 -1.200482273 0.4351298 1 rgs1 Xl2.6965.3.S1_x_at 0.007544618 -0.210586929 0.864185585 retinoic acid receptor, gamma rarg Xl2.6965.3.S1_s_at 0.026148891 -0.17539249 0.885526576 retinoic acid receptor, gamma rarg Xl2.3029.3.A1_a_at 0.02127903 -0.461734681 0.726112663 Retinoic acid-inducible gene 2 raig2 Xl2.396.1.S1_at 0.046489578 -0.16541451 0.891672291 retinoid X receptor, beta rxrb retinol dehydrogenase 10 (all- Xl2.47730.1.S1_at 0.001670914 -0.428847106 0.742855182 trans) rdh10-a

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.2582.1.S1_at 0.036286537 -0.336281646 0.792080164 RGM domain family, member A rgma Xl2.517.1.S2_a_at 0.020502373 0.899133004 1.864944896 Rho family GTPase 1 rnd1 Rho guanine nucleotide Xl2.47604.1.S1_at 0.032277302 1.26707536 2.406731765 exchange factor (GEF) 3 arhgef3 Xl2.53612.1.S1_at 0.027396922 -0.50805733 0.703168657 rhomboid domain containing 2 rhbdd2 Xl2.1365.1.S1_a_at 0.037423222 0.248765728 1.188190145 ribonucleotide reductase M1 rrm1 Xl2.29398.1.S1_at 0.019911272 -0.349309445 0.784959734 ribosomal protein L29 b rpl29b Xl2.8842.3.S1_a_at 0.023625802 -0.126819756 0.915848101 ribosomal protein L3 rpl3 223 Xl2.8225.2.S1_a_at 0.042473966 0.118813816 1.085841716 ribosomal protein S12 rps12 Xl2.6036.1.S1_a_at 0.012386048 -0.089071793 0.940127418 ribosomal protein S2 rps2 Xl2.47605.1.S1_at 0.000216254 0.048929707 1.034497176 ring finger protein 152 rnf152 Xl2.6274.1.S1_at 0.019406063 1.1434142 2.20903182 ring finger protein 168 rnf168 Xl2.33849.1.S1_at 0.009707765 2.098407966 4.282365591 RIO kinase 3 riok3 Xl2.52143.1.S1_s_at 0.038042273 1.474005412 2.777920692 RIO kinase 3 riok3 Xl2.33849.1.S2_at 0.039897377 1.611945188 3.056636909 RIO kinase 3 riok3 Xl2.24190.1.S2_at 0.005505496 0.951032332 1.933255519 RIO kinase 3 (yeast) riok3 Xl2.24190.1.S1_at 0.028706919 0.98070823 1.973433945 RIO kinase 3 (yeast) riok3 Xl2.16077.1.S1_at 0.001564255 -0.778134312 0.583120395 ripply2 homolog ripply2 RUN and FYVE domain Xl2.2726.3.S1_a_at 0.031671793 0.291371862 1.223803442 containing 1 rufy1

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol runt-related transcription factor 1; translocated to, 1 (cyclin D- Xl2.24705.1.S1_s_at 0.003050659 -0.740884414 0.59837242 related) runx1t1 Xl2.3005.1.S1_at 0.003986186 -0.237585877 0.848163393 sal-like 1 a sall1a Xl2.3005.3.S1_a_at 0.005413303 -0.471513602 0.721207546 sal-like 1 a sall1a secreted frizzled-related protein Xl2.49099.1.S1_at 0.008706069 -0.222538038 0.857056347 5 sfrp5 secreted protein, acidic, 224 Xl2.8379.1.S1_at 0.014914723 -0.883874761 0.541910024 cysteine-rich (osteonectin) sparc

secreted Xwnt8 inhibitor sizzled Xl2.620.1.S1_s_at 0.036578994 -0.498078731 0.708049078 /// sizzled szl /// szl serine threonine kinase 39 Xl2.48115.1.S1_at 0.044137302 0.228163991 1.17134332 (STE20/SPS1 homolog) stk39 Xl2.79.1.S1_at 0.00482297 2.332969442 5.038413199 sestrin 1 sesn1 Sestrin 1,nuclear factor XPA26- Xl2.56039.1.S1_at 0.000207045 3.189005867 9.119823272 T2 sesn1-A Xl2.6911.1.S1_at 0.012773597 0.390187986 1.310564162 sestrin 2 sesn2 Xl2.2090.1.S1_at 0.042190483 -0.270915767 0.828793293 seven in absentia homolog 1 siah1 SH3-binding domain protein 5- Xl2.6544.1.S1_at 0.023889294 -0.326047718 0.797718855 like sh3bp5l SHC SH2-domain binding protein Xl2.49137.1.S1_at 0.014258362 0.143590009 1.104650516 1 shcbp1

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol similar to B-cell translocation Xl2.6307.1.S1_x_at 0.025583973 -0.212257171 0.863185676 gene 1 MGC52780 similar to B-cell translocation Xl2.56973.1.A1_s_at 0.040340837 -0.529383671 0.692850662 gene 1 MGC52780 Xl2.5440.1.S1_at 0.00025143 2.537888502 5.80738428 similar to cyclin G1 MGC53060 similar to proliferating cell Xl2.3905.1.S1_at 0.047531081 0.462438097 1.377868394 nuclear antigen MGC53867 similar to serum-inducible 225 Xl2.8630.1.S1_at 0.002093682 4.3022989 19.72972442 kinase MGC53542

similar to serum-inducible MGC53542 /// Xl2.12115.1.S1_s_at 0.008728738 2.310794161 4.961561242 kinase /// polo-like kinase 2 plk2 Similar to splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing Xl2.2144.1.S1_at 0.029626966 -0.111095863 0.925884498 factor) Sfrs1 Xl2.4135.1.S1_x_at 0.022270569 0.122252616 1.08843301 similar to ubiquitin C MGC53081 Xl2.4135.4.S1_x_at 0.039473153 0.129696724 1.094063689 similar to ubiquitin C MGC53081 Xl2.627.1.S1_at 0.02504521 -1.934723184 0.261571419 SIX homeobox 3 six3 Xl2.620.1.S1_at 0.036808878 -0.8391961 0.558954944 Sizzled szl Xl2.2593.1.S1_at 0.000130623 -0.282249617 0.82230778 SMAD family member 6 smad6

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol solute carrier family 2 (facilitated glucose transporter), Xl2.24121.1.S1_at 0.04488454 0.37166181 1.293842323 member 1 slc2a1 solute carrier family 25 (mitochondrial carrier; citrate Xl2.8233.1.S1_at 0.027210098 -0.515344081 0.699626053 transporter), member 1 slc25a1 solute carrier family 25 (mitochondrial carrier;

226 Xl2.20806.1.S2_at 0.036689135 0.192796999 1.142977496 phosphate carrier), member 3 slc25a3

solute carrier family 3 (cystine, dibasic and neutral amino acid transporters, activator of cystine, dibasic and neutral amino acid transport), member Xl2.53873.1.S1_s_at 0.031907038 -0.627789764 0.647167128 1 slc3a1 solute carrier family 35 (CMP- sialic acid transporter), member Xl2.53388.1.S1_at 0.026736649 0.598958198 1.514622429 A1 slc35a1 solute carrier family 38, member Xl2.24047.1.S1_at 0.005406352 -0.683543176 0.622634241 2 slc38a2

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol solute carrier family 40 (iron- regulated transporter), member Xl2.19634.1.S1_at 0.009648958 3.364424296 10.29894245 1 slc40a1 solute carrier family 6 (neurotransmitter transporter, Xl2.15558.1.S1_at 0.014987548 1.162786934 2.2388951 GABA), member 13 slc6a13 Xl2.21860.1.S1_at 0.018927957 -1.872040856 0.273186698 SPARC protein MGC64258 sperm adhesion molecule 1 (PH- 227 20 hyaluronidase, zona pellucida

Xl2.482.1.S1_at 0.015618545 -1.812725164 0.284652729 binding) spam1 splA/ryanodine receptor domain Xl2.43090.1.S1_at 0.022874429 -1.059117758 0.479925456 and SOCS box containing 4 spsb4 splicing factor 3b, subunit 1, Xl2.2599.1.S2_at 0.03217606 -0.078164683 0.947261935 155kDa sf3b1 splicing factor, arginine/serine- Xl2.5499.1.A1_a_at 0.039010897 -0.097882704 0.934403314 rich 5 sfrs5 Xl2.16805.1.S1_at 0.021823498 1.221082611 2.331215882 SREBF chaperone scap SRY (sex determining region Y)- Xl2.188.1.S2_at 0.004227316 -0.483135978 0.715420827 box 2 sox2 SSU72 RNA polymerase II CTD Xl2.9702.1.S1_at 0.010984322 0.698917364 1.623286179 phosphatase homolog ssu72

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol SWI/SNF related, matrix associated, actin dependent regulator of chromatin, Xl2.34943.1.S1_s_at 0.008515008 0.216368586 1.161805517 subfamily a, member 2 smarca2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, Xl2.6286.1.S1_at 0.004007413 -0.245627501 0.843448858 subfamily c, member 1 smarcc1

228 synaptonemal complex protein

Xl2.12073.1.S1_at 0.040790372 0.431756037 1.348874417 2-like sycp2l synaptosomal-associated protein, Xl2.1430.1.S1_at 0.005121635 0.367307041 1.289942753 29kDa snap29 Xl2.222.1.S1_at 0.015056622 0.986230311 1.981001965 syndecan-2 sdc2-b Xl2.16785.1.S1_at 0.022927857 0.141471482 1.103029582 syntaxin binding protein 3 stxbp3 syntaxin binding protein 6 Xl2.5823.1.S1_at 0.017512575 0.308813428 1.238688498 (amisyn) stxbp6 Xl2.933.1.S1_at 0.011674564 -0.673485213 0.626990195 T, brachyury homolog, gene 2 t2 TAF11 RNA polymerase II, TATA box binding protein (TBP)- Xl2.8264.1.A1_at 0.037796586 0.163881568 1.120297245 associated factor, 28kDa taf11

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol TBC1 domain family, Xl2.13727.1.S1_at 0.047392983 0.418959223 1.336962707 member 10A tbc1d10a Xl2.931.1.S1_at 0.02361102 -1.100207089 0.466449535 T-box 2 tbx2 Xl2.975.1.S1_at 0.029576384 -1.20108524 0.434947977 T-box 3 tbx3 TCF3 (E2A) fusion partner (in Xl2.44847.2.S1_s_at 0.027218541 -0.24442097 0.844154532 childhood Leukemia) tfpt tetratricopeptide repeat Xl2.50107.1.S1_at 0.047061667 0.8084581 1.751338675 domain 34 ttc34 229 Xl2.48968.1.S1_at 0.022625639 2.239234227 4.721463858 thioredoxin reductase 1 txnrd1

thymidine kinase 2, Xl2.2832.2.A1_at 0.018048808 0.575162365 1.489845125 mitochondrial tk2 trafficking protein particle Xl2.34882.1.S1_at 0.036643224 0.122111102 1.088326251 complex 2-like trappc2l transcription factor AP-2 beta (activating enhancer binding Xl2.6155.1.S1_at 0.025886042 2.312599102 4.967772485 protein 2 beta) tfap2b Xl2.15574.1.S1_at 0.037344276 -0.656249129 0.634525862 transcription factor CP2-like 1 b tfcp2l1b transcription factor Dp-2 (E2F Xl2.53295.1.S1_at 0.0182715 -0.560846561 0.677904259 dimerization partner 2) tfdp2 transcription factor IIA large Xl2.28611.2.S1_a_at 0.021853933 0.686093163 1.608920635 subunit-1 tfiiaa/b-1

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol transcription factor IIA large Xl2.28611.1.S2_x_at 0.026039164 0.659772179 1.579833127 subunit-1 tfiiaa/b-1 Transcription factor IIA large Xl2.28611.2.S1_x_at 0.03256313 0.610635267 1.52693142 subunit-1 TFIIAa/b-1 Xl2.25636.1.S1_s_at 0.040611318 0.285796467 1.219083096 transketolase-like 2 tktl2 Xl2.53918.1.S1_x_at 0.046138758 0.111630848 1.080448905 translation initiation factor SUI1 sui1 translocase of inner Xl2.20517.1.S1_at 0.045678349 0.194672937 1.144464678 mitochondrial membrane 13 timm13-b 230 Xl2.23894.1.S1_at 0.023580055 0.133373259 1.096855334 transmembrane protein 14C tmem14c

Xl2.5457.1.S1_at 0.017880265 0.446198421 1.362445406 transmembrane protein 168 tmem168 Xl2.31044.1.S1_at 0.02402228 0.103498933 1.074375963 transmembrane protein 56 tmem56 Transmembrane, prostate Xl2.54790.1.A1_at 0.033169747 -0.355591067 0.78154938 androgen induced RNA tmepai tsukushi small leucine rich Xl2.3858.1.S2_at 0.036948473 -0.25532585 0.837797887 proteoglycan homolog tsku tsukushi small leucine rich Xl2.3858.1.S1_at 0.044205989 -0.206874494 0.866412226 proteoglycan homolog tsku Xl2.22321.1.S1_at 0.001482728 -0.088056881 0.940789014 tubby like protein 3 tulp3 Xl2.52384.2.S1_s_at 0.011444156 -0.839629934 0.558786885 tubulin folding cofactor E-like tbcel Xl2.53906.1.S1_at 0.012319637 -0.975180471 0.50867621 tubulin folding cofactor E-like tbcel Xl2.394.1.S1_at 0.018016964 1.110297732 2.158901964 tumor protein p53 tp53

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol twist homolog 1 /// twist Xl2.879.1.S1_s_at 0.007285469 -1.027791966 0.49046022 homolog 2 twist1 /// twist2 Xl2.56708.1.S1_at 0.009828993 -1.806459166 0.285891738 twist homolog 2 twist2 Xl2.23050.1.S1_at 0.00669719 -0.916438579 0.529815303 type XVIII collagen alpha1 chain LOC398292 tyrosine 3- monooxygenase/tryptophan 5- monooxygenase activation Xl2.4573.1.S1_at 0.000772064 0.093737691 1.067131296 protein, zeta polypeptide ywhaz 231 U7 snRNA-associated Sm-like

Xl2.13277.1.S1_at 0.04974056 -0.537712751 0.688862166 protein Lsm11 lsm11 ubiquitin protein ligase E3 component n-recognin 7 Xl2.13116.1.S1_at 0.039206585 -0.24058803 0.846400257 (putative) ubr7 ubiquitin specific peptidase 5 Xl2.48731.1.S1_at 0.000594703 1.036771925 2.051631924 (isopeptidase T) usp5 ubiquitin-conjugating enzyme Xl2.49140.1.S1_at 0.044573455 0.160291552 1.117512952 E2D 2 (UBC4/5 homolog, yeast) ube2d2 ubiquitin-conjugating enzyme Xl2.12697.1.S1_at 0.025946965 0.715619813 1.642188586 E2F (putative) ube2f Xl2.122.1.S1_at 0.024773587 -0.189171902 0.877109032 upstream binding factor 1 ubtf-a Xl2.42472.1.S1_at 0.016317476 0.745649712 1.676729208 Vac14 homolog vac14

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol vascular endothelial zinc finger 1 /// vascular endothelial zinc Xl2.53359.1.S1_s_at 0.014944229 -0.441910072 0.736159317 finger 1 b vezf1 /// vezf1b vesicle amine transport protein Xl2.47269.1.S1_at 0.033448003 0.164055405 1.120432242 1 homolog vat1 v-myc myelocytomatosis viral oncogene homolog 1, lung Xl2.179.1.S2_at 0.038959155 -0.362108678 0.778026566 carcinoma derived mycl1-b

232 v-myc myelocytomatosis viral

oncogene homolog 1, lung Xl2.46111.1.S1_at 0.043087223 -0.466895831 0.723519682 carcinoma derived mycl1-a voltage-dependent anion Xl2.24385.1.S1_at 0.019655424 0.178548143 1.13174438 channel 2 vdac2 Xl2.524.1.S2_at 0.037102066 0.342104237 1.267604105 WD repeat domain 1 b wdr1-a Xl2.16441.1.S1_at 0.043452924 -0.711475946 0.610695049 WD repeat domain 16 wdr16 Xl2.7579.1.S1_at 0.037973606 -0.496853704 0.708650555 WD repeat domain 67 wdr67 Xl2.8563.1.S1_at 0.042953608 -0.538692186 0.688394661 WD repeat domain 77 wdr77 Xl2.16400.1.S1_at 0.029794567 -0.353250013 0.782818626 WD repeat domain 92 wdr92 Xl2.4222.1.S1_at 0.033792661 0.643623904 1.562248444 wee1 homolog wee1-b wingless-type MMTV integration Xl2.13686.1.S1_at 0.01112987 -0.810061814 0.57035742 site family, member 11 wnt11

TABLE A.2 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol wingless-type MMTV integration Xl2.332.1.S1_at 0.011992541 -0.878943458 0.543765506 site family, member 8B wnt8b wntless homolog Xl2.11487.1.S1_a_at 0.002626482 0.206631514 1.153990634 (Drosophila) wls-a Xl2.334.1.S1_at 0.011633218 -0.345503416 0.7870333 Xicl gene xicl Xl2.163.1.S1_at 0.024288905 0.82866601 1.776042383 Xp8 protein xp8 Xl2.5875.1.S1_at 0.034229204 -0.262619817 0.833572844 YTH domain family, member 1 ythdf1 Xl2.46155.1.S1_a_at 0.042108052 0.173602531 1.127871361 zinc finger protein 182 znf182 233 zinc finger protein 36, C3H type-

Xl2.476.1.S1_at 0.019265333 -0.559226655 0.67866586 like 2 zfp36l2 Xl2.47057.1.S1_at 0.007415693 -0.731914325 0.602104446 zinc finger protein 652-B znf652-b Xl2.3779.1.S1_at 0.049768782 -0.457361108 0.728317235 zinc finger, HIT type 2 znhit2

TABLE A.3

GENES DIFFERENTIALLY EXPRESSED (P<0.05) BETWEEN GAM1 MRNA AND H2O INJECTED EMBRYOS AT EARLY NEURULA.

log2 differential Probe ID p-value expression Fold Change Gene Title Gene Symbol Xl2.47144.1.S1_at 0.018368279 0.414646901 1.332972393 14-3-3 protein gamma-B ywhag-b 1-acylglycerol-3-phosphate O- Xl2.16684.1.S1_at 0.029298961 0.555658758 1.469839633 acyltransferase 3 agpat3

234 3'-phosphoadenosine 5'-

Xl2.24002.1.S2_at 0.03400703 0.248994846 1.18837886 phosphosulfate synthase 1 papss1 3'-phosphoadenosine 5'- Xl2.4799.1.S1_at 0.006825614 2.062704451 4.177687128 phosphosulfate synthase 2 papss2 Xl2.49747.1.S1_at 0.020185059 3.853773468 14.45777329 5'-nucleotidase, cytosolic III nt5c3 6-phosphofructo-2- kinase/fructose-2,6- Xl2.15223.1.S2_at 0.032266348 -0.291747043 0.816912213 biphosphatase 4 pfkfb4 A disintegrin and Xl2.53305.1.S1_at 0.017979674 -0.003810268 0.997362408 metalloproteinase domain 9 adam9-A Actin related protein 2/3 Xl2.5178.2.S1_at 0.017641056 0.150966855 1.110313325 complex, subunit 1A, 41kDa arpc1a activated leukocyte cell Xl2.57037.1.S1_at 0.030968639 -0.6890412 0.620265935 adhesion molecule Alcam

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol acyl-CoA synthetase medium- Xl2.50124.1.S1_at 0.002354596 -0.025523317 0.982464159 chain family member 3 acsm3 acyl-Coenzyme A binding Xl2.15549.1.S1_at 0.036614065 -0.619187908 0.651037293 domain containing 3 acbd3 Xl2.937.1.S1_at 0.032510463 0.281403196 1.215376411 adenosylhomocysteinase ahcy-a Xl2.780.1.S1_at 0.006619762 -0.514905567 0.69983874 ADP-ribosylation factor-like 4A arl4a Xl2.45519.1.S2_a_at 0.037295143 -0.035822543 0.975475442 akirin 1 akirin1 aldehyde dehydrogenase 2 235 Xl2.22158.1.S1_at 0.031556553 -0.589357697 0.664638745 family (mitochondrial) aldh2

aldehyde dehydrogenase 9 Xl2.4246.1.S1_at 0.039009574 0.18275851 1.135052092 family, member A1 aldh9a1 aldolase C, fructose- Xl2.8701.1.S1_at 0.038573555 0.400062346 1.319564934 bisphosphate Aldoc Xl2.32071.1.S1_at 0.040803171 -0.511686991 0.701401786 alkaline ceramidase 3 acer3 alkylglycerone phosphate Xl2.7726.1.S1_at 0.023420612 0.091633074 1.06557569 synthase Agps alpha- and gamma-adaptin Xl2.10581.1.S1_at 0.040232893 0.674534887 1.596082127 binding protein Aagab alveolar soft part sarcoma chromosome region, candidate Xl2.16681.1.S1_at 0.004260182 0.119323285 1.086225235 1 aspscr1 Xl2.23820.1.S1_at 0.032025758 -2.305249714 0.202325531 ankyrin repeat domain 11 ankrd11

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol ankyrin repeat, SAM and basic leucine zipper domain Xl2.47063.1.S1_at 0.043388568 1.435762884 2.7052518 containing 1 asz1 Xl2.25391.1.S1_at 0.043527725 1.34496714 2.540244105 apoptosis enhancing nuclease Aen apoptotic chromatin Xl2.52586.1.S1_x_at 0.03943424 -0.379132943 0.768899559 condensation inducer 1 acin1 apoptotic chromatin condensation inducer 1 /// acin1 /// 236 Xl2.52586.1.S1_s_at 0.002614471 -0.299778363 0.81237719 hypothetical protein LOC733423 LOC733423

Xl2.20033.1.S1_at 0.022052991 -0.056673344 0.961478602 Arg protein-tyrosine kinase arg-A Xl2.1242.1.S1_at 0.00512754 -0.410646058 0.752286414 arginase, liver arg1 Xl2.34147.2.A1_x_at 0.000228652 0.536440409 1.450389523 aspartoacylase (aminocyclase) 3 acy3 Xl2.34147.1.A1_a_at 0.00045753 0.459041513 1.374628249 aspartoacylase (aminocyclase) 3 acy3 AT hook containing transcription Xl2.1139.1.S1_at 0.032930387 -0.28757044 0.819280601 factor 1 ahctf1 Xl2.6373.1.A1_at 0.048581401 0.518914767 1.432876995 ataxin 10 atxn10 ATP synthase, H+ transporting, mitochondrial F0 complex, Xl2.2639.1.S1_a_at 0.049887865 0.089761104 1.064193948 subunit C3 (subunit 9) atp5g3 ATPase, H+ transporting, Xl2.43120.2.S1_a_at 0.00632846 0.266500769 1.202886707 lysosomal 13kDa, V1 subunit G1 atp6v1g1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol ATPase, H+/K+ transporting, Xl2.2659.1.S1_at 0.017156028 -1.381877385 0.383719135 nongastric, alpha polypeptide b atp12ab ATPase, Na+/K+ transporting, Xl2.21686.1.S1_at 0.032762935 -0.127897397 0.915164252 alpha 1 polypeptide atp1a1 ATPase, Na+/K+ transporting, Xl2.17577.1.S1_at 0.006498249 4.61647689 24.53002654 beta 2 polypeptide atp1b2 Xl2.10807.1.S1_at 0.007554305 0.358952572 1.282494441 ATR interacting protein Atrip baculoviral IAP repeat- 237 Xl2.8352.1.S1_at 0.004705855 0.377088104 1.298717911 containing 5, gene 1 birc5.1-a

baculoviral IAP repeat- birc5.1-a /// Xl2.34932.1.S1_s_at 0.007235473 0.366265337 1.289011681 containing 5, gene 1 /// survivin LOC398389 BAH domain and coiled-coil Xl2.12374.1.A1_at 0.012625286 -0.469115811 0.722407206 containing 1 bahcc1 basic helix-loop-helix Xl2.26468.2.S1_a_at 0.021298063 0.136805839 1.09946817 transcription factor XMad4-236 xmad4 BCL2/adenovirus E1B 19kDa Xl2.29375.1.S1_at 0.000899881 0.432410516 1.349486473 interacting protein 1 bnip1 breast cancer anti-estrogen Xl2.32813.1.S1_at 0.018100938 -1.121913603 0.459483958 resistance protein 3 homolog bcar3 BRF2, subunit of RNA polymerase III transcription Xl2.4665.1.S1_at 0.023654449 0.226176501 1.169730761 initiation factor, BRF1-like brf2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.21809.1.S1_at 0.00763531 0.121641578 1.087972113 calcium modulating ligand camlg calcium/calmodulin-dependent protein kinase (CaM kinase) II Xl2.693.20.S1_x_at 0.027976188 -0.609673022 0.655345215 gamma camk2g Xl2.3403.1.S1_s_at 0.038242726 -0.550636618 0.682718799 calpastatin Cast carboxypeptidase X (M14 Xl2.56776.1.A1_at 0.002936453 1.690836895 3.228439283 family), member 2 cpxm2 Xl2.29275.1.S1_at 0.016364888 0.270699072 1.206392255 carnitine acetyltransferase Crat 238 Xl2.553.1.S1_a_at 0.017406708 0.595858024 1.511371187 casein kinase 1, epsilon csnk1e

CASP8 and FADD-like apoptosis Xl2.46704.1.S2_a_at 0.016326623 -0.0264923 0.981804511 regulator Cflar Xl2.7945.1.S1_at 0.036295252 0.460789158 1.376294449 catalase Cat CCHC-type zinc finger, nucleic Xl2.985.1.S1_a_at 0.011318547 -0.21619144 0.860834951 acid binding protein cnbp CCR4-NOT transcription Xl2.4277.1.S1_at 0.025865019 0.230406398 1.173165376 complex, subunit 8 cnot8 CCR4-NOT transcription Xl2.4277.1.S2_at 0.035214549 0.230795689 1.173481981 complex, subunit 8 cnot8 CDC23 (cell division cycle 23, Xl2.16640.1.S1_at 0.02118207 0.241332359 1.182083837 yeast, homolog) cdc23 CDC28 protein kinase regulatory Xl2.3190.1.A1_at 0.020883287 0.401992789 1.321331801 subunit 1B-A cks1ba

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol CDK5 regulatory subunit Xl2.12738.2.S1_at 0.003624689 -0.02087086 0.98563756 associated protein 1 cdk5rap1 Xl2.45543.1.S1_at 0.043031636 -0.318442428 0.801935201 cell division cycle 16 homolog cdc16 cell division cycle 25 homolog A /// tyrosine phosphatase cdc25a /// Xl2.7523.2.S1_s_at 0.029611052 -0.402248871 0.756677855 Cdc25A LOC398141 Xl2.25641.1.S2_at 0.009096865 -0.322048617 0.799933171 cell division cycle 6 homolog cdc6 Xl2.7817.1.S2_at 0.003249723 0.292617861 1.224860849 centrin Xcen 239 Xl2.55781.1.S1_at 0.035795606 -0.608124574 0.656048976 centromere protein T cenpt

Xl2.142.2.S1_a_at 0.005631312 -0.778363875 0.583027616 c-ets-1b proto-oncogene c-ets-1b Xl2.142.1.S1_at 0.008903411 -0.638685012 0.642298124 c-ets-1b proto-oncogene c-ets-1b chaperonin containing TCP1, Xl2.7645.1.S1_a_at 0.041077268 -0.223848583 0.85627815 subunit 2 (beta) cct2 Xl2.6837.1.S1_at 0.014746477 -0.728273535 0.603625837 chitobiase, di-N-acetyl- Ctbs Xl2.248.1.S1_s_at 0.042354754 -0.482569856 0.715701617 chloride channel 3 clcn3 cholinergic receptor, nicotinic, Xl2.13873.1.S1_at 0.035854778 1.35594822 2.559652957 alpha 5 chrna5 Chromatin assembly factor 1 Xl2.7155.1.S1_at 0.027442655 -0.641143486 0.641204526 p150 subunit LOC398222 Xl2.21401.1.S1_at 0.047452923 0.338274601 1.26424371 chromatin modifying protein 2A chmp2a chromosome 1 open reading Xl2.55440.1.S1_at 0.003613423 0.15557193 1.113863101 frame 123 c1orf123

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol chromosome 1 open reading Xl2.48693.1.S1_at 0.038455768 0.474085692 1.389037638 frame 212 c1orf212 chromosome 10 open reading Xl2.52870.1.S1_at 0.03430198 -0.962936534 0.51301164 frame 140 c10orf140 chromosome 10 open reading Xl2.25073.1.S1_at 0.046061417 0.480690589 1.395411462 frame 57 c10orf57 chromosome 10 open reading Xl2.6886.1.S1_at 0.00991153 0.204848345 1.152565184 frame 58 c10orf58 240 chromosome 10 open reading

Xl2.47907.1.S1_at 0.001755397 0.495403026 1.409714513 frame 78 c10orf78 chromosome 11 open reading Xl2.23846.1.S1_at 0.018383894 0.287407743 1.220445391 frame 10 c11orf10 chromosome 15 open reading Xl2.50378.1.S1_at 0.029886595 0.318232006 1.246801681 frame 40 c15orf40 chromosome 16 open reading Xl2.11439.1.S1_at 0.010908746 -0.299535556 0.812513925 frame 48 c16orf48 chromosome 16 open reading Xl2.9881.1.S1_at 0.001486177 0.56209128 1.476407816 frame 57 c16orf57 chromosome 17 open reading Xl2.50833.1.S1_at 0.007912182 -0.39234894 0.761888117 frame 56 c17orf56 chromosome 17 open reading Xl2.14404.1.A1_at 0.003546183 -0.567449498 0.674808711 frame 58 c17orf58

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol chromosome 18 open reading Xl2.7564.1.S1_at 0.03204574 0.772180335 1.707848895 frame 19 c18orf19 chromosome 19 open reading Xl2.15831.1.S1_at 0.002449897 0.318271416 1.246835741 frame 40 c19orf40 chromosome 22 open reading Xl2.7463.1.S1_at 0.030800103 0.23814432 1.179474575 frame 13 c22orf13 chromosome 3 open reading Xl2.19250.1.S1_at 0.02855574 1.075811913 2.107908016 frame 63, gene 2 c3orf63.2 241 chromosome 5 open reading

Xl2.24558.1.S1_at 0.011817426 0.752845993 1.685113765 frame 28 c5orf28 chromosome 5 open reading Xl2.2220.1.S1_at 0.02387055 -0.396038649 0.75994207 frame 44 c5orf44 chromosome 9 open reading Xl2.52141.1.A1_at 0.00357196 0.860261112 1.815366842 frame 125 c9orf125 chromosome 9 open reading Xl2.47795.1.A1_s_at 0.000373542 0.661436013 1.581656171 frame 6 c9orf6 chromosome 9 open reading Xl2.46798.1.S1_at 0.041451189 -1.185722209 0.439604418 frame 9 c9orf9 chromosome X open reading Xl2.55525.1.A1_at 0.026584728 0.40231993 1.321631456 frame 57 cxorf57 ciliary neurotrophic factor Xl2.17571.1.S1_at 0.03602651 -0.594706147 0.662179317 receptor cntfr

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.7040.1.S1_at 0.017285674 0.245988947 1.185905414 clathrin, light chain (Lcb) Cltb Xl2.54911.1.S1_at 0.000252289 0.159238274 1.116697379 clathrin, light polypeptide A a Cltaa Xl2.23515.1.S1_at 0.046846909 -1.811875372 0.284820448 claudin 1 cldn1 cleavage stimulation factor, 3' Xl2.54485.1.S1_at 0.042933482 0.266704302 1.203056419 pre-RNA, subunit 3, 77kDa cstf3 coenzyme Q9 homolog /// ubiquinone biosynthesis protein Xl2.14922.1.S1_s_at 0.008495386 0.41813347 1.33619769 COQ9-B, mitochondrial coq9 /// coq9-b 242 coiled-coil domain containing

Xl2.3532.1.S1_at 0.032071175 0.482109911 1.396784942 124 ccdc124 coiled-coil domain containing Xl2.18510.1.S1_at 0.001350872 -0.379094645 0.768919971 149 ccdc149 coiled-coil-helix-coiled-coil-helix Xl2.18790.1.S1_at 0.047984872 0.239678436 1.180729458 domain containing 3 chchd3 Xl2.5123.1.S2_at 0.002395146 4.586845424 24.03134388 complement component 9 c9 Xl2.5123.1.S1_at 0.023681041 3.575754319 11.92365241 complement component 9 c9 Xl2.5152.1.S1_at 0.042808721 1.355224538 2.558369311 complement factor B LOC397725 COP9 constitutive photomorphogenic homolog Xl2.33935.1.S1_at 0.006417081 0.456338432 1.372055112 subunit 4 cops4 copper metabolism (Murr1) Xl2.10925.1.S1_a_at 0.005490852 0.259419119 1.196996654 domain containing 1 commd1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Core promoter element binding Xl2.25185.1.A1_at 0.04415242 -0.463650468 0.725149082 protein copeb core promoter element binding Xl2.48835.1.S1_s_at 0.011371557 -0.500861189 0.706684814 protein /// Kruppel-like factor 6 copeb /// klf6 core-binding factor, beta Xl2.25702.1.S1_at 0.014989073 -0.391352883 0.762414317 subunit Cbfb COX18 cytochrome c oxidase Xl2.26154.1.S1_at 0.022435917 -0.343769234 0.787979917 assembly homolog cox18 243 Xl2.4103.1.S1_at 0.015518953 0.545873724 1.459904223 creatine kinase, brain Ckb

Xl2.4103.1.S1_x_at 0.041715441 0.468413288 1.383586927 creatine kinase, brain Ckb cryptochrome 2 (photolyase- Xl2.12080.1.S1_at 0.036825107 0.84508967 1.796376395 like) cry2 Xl2.263.1.S1_at 0.025306447 -0.037317731 0.974464997 c-src tyrosine kinase csk-A Xl2.49810.1.S1_at 0.015735959 -0.002798335 0.998062222 CTP synthase Ctps Xl2.12976.1.S1_at 0.040399237 -0.213101497 0.862680651 cyclin D1 b ccnd1b Xl2.55726.1.A1_at 0.009579273 2.380009846 5.205402948 cyclin G1 ccng1 Xl2.55582.1.A1_at 0.022458497 2.5459262 5.839829304 cyclin G1 ccng1 Xl2.1768.1.S1_at 0.033066184 -0.030354758 0.979179488 cyclin-dependent kinase 7 cdk7 Xl2.7046.1.S1_at 0.004806432 -0.013772954 0.990498741 cytochrome c, somatic A cycs-a cytochrome P450, family 17, Xl2.725.1.S1_at 0.020919135 0.145276419 1.105942531 subfamily A, polypeptide 1 cyp17a1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol cytochrome P450, family 26, Xl2.55931.1.S1_at 0.020843173 -1.272435415 0.413960376 subfamily C, polypeptide 1 cyp26c1 dapper, antagonist of beta- Xl2.7602.1.S2_at 0.033604429 -0.444456294 0.73486121 catenin dact1-b Xl2.31912.1.S1_at 0.04287734 -0.162661179 0.893375639 DAZ interacting protein 1 dzip1 Xl2.14461.1.S1_at 0.045856689 0.499887172 1.414102966 DCN1-like protein 3 MGC83887 DDB1 and CUL4 associated Xl2.11079.1.S1_at 0.015302466 0.190337503 1.141030616 factor 10 dcaf10 244 DDB1 and CUL4 associated

Xl2.56013.1.A1_at 0.021075999 0.803208909 1.744978075 factor 5 dcaf5 DEAD (Asp-Glu-Ala-Asp) box Xl2.42212.1.S1_at 0.007957052 -0.151970672 0.900020226 polypeptide 52 ddx52 DEAD (Asp-Glu-Ala-Asp) box Xl2.157.1.S1_at 0.02933197 -0.111803221 0.925430645 polypeptide 6 ddx6 Xl2.29785.1.S2_at 0.029590535 0.894286427 1.858690329 dead end homolog 1 dnd1 death effector domain Xl2.35319.1.S1_at 0.029859727 -1.026439591 0.490920191 containing 2 dedd2 Xl2.4673.1.S1_at 0.020751202 0.174125122 1.128279987 decapping enzyme, scavenger Dcps deiodinase, iodothyronine, type Xl2.862.1.S1_at 0.042299158 -0.831900463 0.56178871 3 dio3 delta/notch-like EGF repeat Xl2.17677.1.S1_at 0.027561247 -0.084022273 0.943423686 containing dner

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.54916.1.A1_s_at 0.005871488 -0.282372833 0.822237552 delta-like 1 dll1 Xl2.14759.1.S1_at 0.021080984 -0.384738448 0.765917842 delta-like 1 dll1 diacylglycerol kinase, epsilon Xl2.48666.1.S1_at 0.011618946 -0.023796702 0.983640674 64kDa Dgke DiGeorge syndrome critical Xl2.15714.1.S1_at 0.007252698 -0.236160488 0.849001797 region gene 14 dgcr14 Xl2.5231.1.S1_at 0.019354725 0.162209512 1.118999593 dipeptidyl-peptidase 9 dpp9 Xl2.721.1.S1_at 0.012830166 -1.687310462 0.310505244 distal-less homeobox 2 dlx2 245 DnaJ (Hsp40) homolog,

Xl2.23335.1.S1_at 0.046491792 0.746708019 1.677959645 subfamily B, member 9 dnajb9 DnaJ (Hsp40) homolog, Xl2.23489.1.S1_at 0.048065614 0.22181704 1.166201465 subfamily C, member 19 dnajc19 Xl2.18853.1.S1_at 0.017678528 -0.746111805 0.59620823 DPY30 domain containing 1 dydc1 Xl2.1243.1.S1_x_at 0.033278955 -0.348707387 0.785287377 dual specificity phosphatase 1 dusp1 Xl2.1243.1.S1_at 0.043181653 -0.351584602 0.783722813 dual specificity phosphatase 1 dusp1 dual specificity phosphatase 1 /// MAP kinase phosphatase dusp1 /// Xl2.2803.1.A1_s_at 0.003308801 -0.217470207 0.860072268 XCL100(beta) protein LOC398254 Xl2.53897.1.S1_at 0.037810388 -0.003488231 0.997585063 dynamin 2 dnm2 E74-like factor 1 (ets domain Xl2.21868.1.S1_at 0.023239113 -0.410615747 0.75230222 transcription factor) elf-1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol ecto-NOX disulfide-thiol Xl2.51428.1.S1_s_at 0.03703278 -0.021193276 0.985417313 exchanger 1 enox1 ELAV (embryonic lethal, abnormal vision)-like 2 (Hu antigen B) /// ELAV (embryonic lethal, abnormal vision, Xl2.34955.1.A1_x_at 0.001366578 1.807916897 3.501363625 Drosophila)-like 2 (Hu antigen B) elavl2 /// elavl2-a ELAV (embryonic lethal,

246 abnormal vision)-like 2 (Hu

antigen B) /// ELAV (embryonic lethal, abnormal vision, Xl2.1648.2.S1_s_at 0.007027713 1.393299185 2.626786936 Drosophila)-like 2 (Hu antigen B) elavl2 /// elavl2-a ELOVL family member 7, elongation of long chain fatty Xl2.5611.1.S1_at 0.022534723 -0.21321249 0.862614283 acids elovl7 embryonal Fyn-associated Xl2.30266.1.S1_at 0.033058658 -0.642596129 0.640559225 substrate Efs enhancer of mRNA decapping 3 Xl2.30613.1.S1_at 0.048104474 -0.169528449 0.88913325 homolog edc3 enhancer of split related Xl2.585.1.S1_at 0.028770758 -0.373267038 0.77203222 epidermal protein-6 esr-6e

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol enhancer of split related protein Xl2.12444.1.S1_at 0.038347896 -0.360031568 0.779147531 9b esr9b epidermis specific serine Xl2.909.1.A1_at 0.011383909 -0.770893032 0.586054593 protease xepsin Xl2.6176.1.S1_a_at 0.000962445 -0.257777273 0.836375511 epithelial membrane protein 2 emp2 Xl2.17492.1.S1_a_at 0.015210806 0.448606109 1.364721065 ethylmalonic encephalopathy 1 ethe1 eukaryotic translation elongation factor 1 delta 247 (guanine nucleotide exchange

Xl2.1171.1.S1_at 0.00903479 -0.001806614 0.998748535 protein) eef1d eukaryotic translation initiation Xl2.51779.1.S1_at 0.046811008 0.12995142 1.094256854 factor 3, subunit E eif3e-b Xl2.5428.1.S1_at 0.013371992 -0.196225713 0.872831024 exosome component 2 exosc2 Xl2.8074.1.S1_at 0.032466509 -0.335947154 0.792263831 exosome component 4 exosc4 Xl2.30779.1.S1_at 0.0274332 -0.701928984 0.614749694 extended synaptotagmin-2-A esyt2-a family with sequence similarity Xl2.6930.1.S1_at 0.046575774 0.328217182 1.255460973 113, member A fam113a family with sequence similarity Xl2.8193.1.S1_a_at 0.027386348 -0.801130081 0.573899459 161, member A fam161a family with sequence similarity Xl2.29791.1.S1_at 0.019665413 0.432957261 1.349997991 173, member B fam173b

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol family with sequence similarity Xl2.13086.1.A1_at 0.018376739 1.919683458 3.783400381 46, member A fam46a family with sequence similarity Xl2.46795.1.S1_at 0.039988202 0.763576138 1.69769364 53, member C fam53c family with sequence similarity Xl2.5000.1.S1_at 0.020164822 -0.406299224 0.754556468 55, member B fam55b family with sequence similarity Xl2.48344.1.S1_at 0.008441835 1.988358021 3.967851469 69, member A fam69a 248 farnesyl diphosphate synthase

(farnesyl pyrophosphate synthetase, dimethylallyltranstransferase, Xl2.50432.1.S2_a_at 0.014629775 0.481350673 1.396050058 geranyltranstransferase) Fdps Xl2.9265.1.S1_at 0.04877178 1.575748277 2.980900618 F-box protein 44 fbxo44 Xl2.8959.1.S1_at 0.022751162 -0.458799801 0.727591301 ferredoxin reductase Fdxr Xl2.8760.1.S1_at 0.046074444 -0.030508853 0.979074907 FGF receptor 3 fgfr3 Xl2.1179.1.S1_at 0.025213791 -0.820633756 0.566193167 Fibroblast growth factor-3 fgf3-A Xl2.15054.2.S1_a_at 0.048938596 -0.749583207 0.594775363 fibulin 1 fbln1 FK506 binding protein 11, 19 Xl2.23956.1.S1_at 0.000112592 0.287631464 1.220634662 kDa fkbp11 Xl2.3150.1.S1_at 0.006616789 -0.433905772 0.740254996 follistatin-related protein xFRP Xl2.182.1.S1_at 0.006059462 -0.818600611 0.566991648 forkhead box F1 foxf1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.2074.1.S2_at 0.015327166 1.656771676 3.153101641 forkhead box O3 foxo3 Xl2.2074.1.S1_at 0.024991515 0.914192232 1.884513633 forkhead box O3 foxo3 Xl2.53881.1.S1_a_at 0.004740843 -1.170120451 0.444384237 forkhead box P4 foxp4 Xl2.54939.1.S1_at 0.001475665 -0.802229009 0.573462475 forkhead box protein F1-B foxf1-b Xl2.8097.1.S1_at 0.04756273 0.539085165 1.453050824 fracture callus 1 homolog fxc1 Xl2.4558.1.S1_at 0.049635898 0.311828033 1.241279524 fragile X mental retardation 1 fmr1-A fragile X mental retardation, Xl2.331.6.S1_s_at 0.039380849 0.062254726 1.04409626 autosomal homolog 1 /// FXR1 fxr1 /// fxr1-a 249 Xl2.559.1.S2_at 0.007158649 -0.657786981 0.633849844 frizzled homolog 10 fzd10-b Xl2.559.1.S1_at 0.012809721 -0.737577594 0.599745531 frizzled homolog 10 fzd10-b Xl2.1040.1.S1_at 0.032679681 -0.606796753 0.656653066 frizzled homolog 10 fzd10-a Xl2.633.2.S1_at 0.043455951 -0.617536737 0.651782835 frizzled homolog 7 fzd7 fucosyltransferase 1 (galactoside 2-alpha-L-fucosyltransferase, H Xl2.24073.1.S1_at 0.02136886 0.274098018 1.20923783 blood group) fut1 Xl2.25994.1.S1_at 0.035624118 0.251078735 1.190096644 FUN14 domain containing 1 fundc1 furin (paired basic amino acid Xl2.6432.1.S3_at 0.048125711 -0.525495025 0.694720693 cleaving enzyme) Furin Xl2.331.3.S1_x_at 0.00071278 -0.007780776 0.994621295 FXR1 fxr1-a Xl2.331.4.S1_x_at 0.016099531 0.107983392 1.077720739 FXR1 fxr1-a Xl2.8053.1.S1_at 0.02179487 0.393130606 1.313240006 G patch domain and KOW motifs gpkow

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.18687.1.S1_s_at 0.047222983 0.323637049 1.251481573 G patch domain and KOW motifs gpkow Xl2.47648.1.S1_at 0.048878347 0.612635036 1.529049419 G protein-coupled receptor 155 gpr155 G protein-coupled receptor Xl2.11463.1.S1_at 0.032021144 0.470591615 1.385677585 kinase interacting ArfGAP 2 git2 GABA(A) receptor-associated Xl2.11101.1.S1_at 0.002292448 0.647880353 1.566864423 protein-like 2 gabarapl2 Xl2.9656.1.S1_at 0.013530967 -0.958098915 0.514734749 galactosidase, beta 1-like 2 glb1l2 Xl2.16466.1.S2_at 0.006314812 -0.714411638 0.60945363 galectin-related protein hspc159 250 Xl2.16466.1.S1_at 0.031209233 -1.896407853 0.268611345 galectin-related protein hspc159

gametogenetin binding protein Xl2.15356.1.S1_at 0.028768672 -0.204617005 0.867769025 2 ggnbp2 Xl2.7290.1.S1_at 0.018168715 -0.339774627 0.790164739 gamma-glutamyl carboxylase Ggcx Xl2.792.1.S2_at 0.044066737 -0.545309766 0.685244256 GATA binding protein 2 gata2 Xl2.183.1.S1_at 0.024615982 -0.310668539 0.806268051 GATA binding protein 6 gata6-b Xl2.23009.2.S1_a_at 0.034073924 -0.922486886 0.527598773 GATA binding protein 6 gata6-a Xl2.860.1.S1_at 0.002418678 1.763843355 3.396016233 general transcription factor 3A gtf3a general transcription factor IIA, Xl2.29248.1.S1_at 0.000478451 0.83427404 1.782959633 1, 19/37kDa gtf2a1 general transcription factor IIA, Xl2.29766.1.S1_at 0.036724611 2.273234206 4.834056068 1-like gtf2a1l

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol GINS complex subunit 4 (Sld5 Xl2.1783.1.S1_at 0.01262197 0.177148476 1.130646922 homolog) gins4 glucosamine-6-phosphate Xl2.51720.1.S1_s_at 0.022349464 0.373040884 1.295079699 deaminase 2 gnpda2 glucose-fructose oxidoreductase Xl2.21885.1.S1_at 0.005191726 0.524376827 1.438312166 domain containing 2 gfod2 Xl2.51310.1.S1_at 0.003322395 -0.002623868 0.998182926 glucosidase, alpha; neutral AB ganab Xl2.18860.1.S1_at 0.019048696 0.175965065 1.129719858 glucoside xylosyltransferase 1 gxylt1 251 glutathione peroxidase 4

Xl2.23492.1.S1_s_at 0.019904492 0.35671677 1.280508447 (phospholipid hydroperoxidase) gpx4 glutathione peroxidase 4 a Xl2.25745.1.S1_at 0.007433057 0.260570537 1.197952361 (phospholipid hydroperoxidase) gpx4a Xl2.18511.1.S1_at 0.010325811 0.346149258 1.271163195 glycerol kinase 5 (putative) gk5 glycerophosphodiester phosphodiesterase domain Xl2.16435.1.S1_at 0.019245125 -0.910664838 0.531939901 containing 1 gdpd1 Xl2.19313.1.A1_at 0.028182858 -0.00307453 0.997871167 GPP34-related protein golph3l Xl2.318.1.S1_at 0.009945484 -0.312078537 0.805480442 gremlin 1 grem1 Xl2.34033.1.S1_at 0.022779 -1.465260294 0.362170189 growth arrest-specific 6 gas6 Xl2.41658.1.S1_at 0.04855603 -0.174820998 0.885877427 GTP binding protein 4 gtpbp4 guanine monphosphate Xl2.11147.2.S1_a_at 0.041257768 -0.362524144 0.777802543 synthetase gmps

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol guanine nucleotide binding protein (G protein), alpha 11 (Gq Xl2.884.1.S2_at 0.038735463 0.131926678 1.095756076 class) gna11 guanine nucleotide binding Xl2.13669.1.S1_at 0.016508511 -0.446476682 0.733832812 protein (G protein), alpha 14 gna14 guanine nucleotide binding Xl2.14093.2.S1_at 0.011910176 -0.538918042 0.6882869 protein (G protein), alpha 14 a gna14 guanine nucleotide binding

252 protein (G protein), q

Xl2.49085.1.S1_at 0.000107528 0.432749213 1.349803325 polypeptide gnaqb guanosine monophosphate Xl2.12819.1.S1_at 0.036478385 -0.620965338 0.650235696 reductase 2 gmpr2 Xl2.17187.1.S2_at 0.024709674 0.349547564 1.274160981 H2A histone family, member Y2 h2afy2 Xl2.1764.1.S1_at 0.028896543 -0.411723799 0.751724641 hairy and enhancer of split 4 hes4-b heat shock 60kDa protein 1 Xl2.23194.1.S1_at 0.021392708 0.25920795 1.19682146 (chaperonin) hspd1 Xl2.4111.1.S1_at 0.026183384 0.092199781 1.065994342 heat shock cognate protein 70 hsc70 Xl2.34774.1.S1_at 0.044486542 0.355590497 1.279509179 Heat shock protein gp96 TRA1 Xl2.34797.1.A1_x_at 0.012306318 5.062921765 33.42653168 hemoglobin, gamma A hbg1 heparan sulfate 2-O- Xl2.46199.1.S3_at 0.042279631 -0.438600644 0.737849946 sulfotransferase 1 hs2st1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol heterogeneous nuclear Xl2.7820.2.A1_a_at 0.047049126 -0.115835214 0.922847896 ribonucleoprotein A1 hnrnpa1 Xl2.43505.3.S1_a_at 0.039739408 0.12414402 1.089860905 high mobility group HMG-17 hmg-17 Xl2.3421.3.S1_a_at 0.02456422 0.235843696 1.1775952 histone H3.3 MGC52708 Xl2.54004.1.S1_at 0.007837789 0.92443418 1.89793973 HMG box domain containing 3 hmgxb3 HMG box mitochondrial Xl2.8873.1.S1_at 0.0263981 0.127169349 1.092148738 transcription factor mttfa-A HMG box mitochondrial 253 Xl2.8873.1.S1_x_at 0.04634895 0.167004541 1.122724956 transcription factor mttfa-A

Xl2.9542.1.S1_at 0.016393367 1.489371243 2.807665844 HMG-box transcription factor 1 hbp1 Xl2.15455.1.S1_at 0.036983378 -0.647686785 0.638302948 HNF1 homeobox B hnf1b Xl2.266.1.S1_a_at 0.007312713 -0.869124148 0.547479121 homeobox A11 hoxa11 Xl2.18197.1.S1_at 0.025364881 -0.553972095 0.681142192 homeobox B6 hoxb6 Xl2.1213.1.S1_at 0.01466365 -0.866484272 0.548481828 homeobox C5 hoxc5 Xl2.1209.3.S1_a_at 0.027838495 -0.715153577 0.609140285 homeobox C6 hoxc6 Xl2.1209.3.S1_x_at 0.034196432 -0.708613228 0.611908045 homeobox C6 hoxc6 homeobox C8 /// homeobox Xl2.207.1.S1_s_at 0.045301581 -0.52696481 0.694013288 protein pXhoxc8 hoxc8 /// xhoxc8 Xl2.3370.1.S1_at 0.036410317 -0.356805389 0.780891823 homeobox D1 hoxd1 Xl2.207.1.S1_at 0.043749496 -0.645767407 0.639152718 homeobox protein pXhoxc8 xhoxc8

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol homocysteine-inducible, endoplasmic reticulum stress- inducible, ubiquitin-like domain Xl2.5778.1.S1_at 0.049198746 0.30224447 1.233061254 member 1 herpud1-a Xl2.3085.1.S1_at 0.029617837 0.834641789 1.783414175 homolog of rat pragma of Rnd2 sgk223 hydroxyacid oxidase (glycolate Xl2.3534.1.S1_at 0.020182828 -0.387346628 0.764534426 oxidase) 1 hao1 Xl2.5446.1.S1_at 0.001526634 -0.45332959 0.730355317 hypothetical LOC494716 LOC494716 254 hypothetical LOC494805 ///

ubiquitin-conjugating enzyme LOC494805 /// Xl2.49130.1.S1_s_at 0.027547719 1.716700798 3.286839018 E2E 2 (UBC4/5 homolog) ube2e2 Xl2.17925.1.S1_at 0.008434693 0.832040689 1.780201673 hypothetical LOC495146 LOC495146 Xl2.49116.1.S1_at 0.000230107 0.379318334 1.300727123 hypothetical LOC495170 LOC495170 Xl2.49052.1.S1_at 0.035476483 -0.900220256 0.535804924 hypothetical LOC495233 LOC495233 Xl2.25637.1.S1_at 0.021911288 0.143000658 1.104199351 hypothetical LOC495269 LOC495269 Xl2.49178.1.S1_at 0.027450976 -0.015044428 0.989626181 hypothetical LOC495273 LOC495273 Xl2.17988.1.S1_at 0.045220068 1.039692599 2.055789571 hypothetical LOC495517 LOC495517 Xl2.49094.1.S1_at 0.008067429 1.578417032 2.98641991 hypothetical LOC495519 LOC495519 Xl2.49836.1.S1_at 0.008896502 -0.036459906 0.975044585 hypothetical LOC495825 LOC495825 Xl2.49911.1.S1_at 0.020180297 0.31229543 1.241681733 hypothetical LOC495975 LOC495975 Xl2.50004.1.S1_at 0.044388489 -0.253328011 0.83895887 hypothetical LOC496148 LOC496148

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.16601.1.S1_at 0.010278366 -0.348376016 0.78546777 hypothetical LOC496289 LOC496289 Xl2.5507.1.S1_at 0.047994641 0.620768748 1.537694332 hypothetical LOC496339 LOC496339 Hypothetical protein Xl2.37592.1.S1_at 0.003298054 1.232012721 2.348944654 LOC100036828 LOC100036828 Hypothetical protein Xl2.50224.2.A1_at 0.03721317 -0.000421127 0.999708139 LOC100036864 LOC100036864 hypothetical protein Xl2.15918.3.A1_at 0.001309048 -0.002648514 0.998165874 LOC100036929 LOC100036929 255 hypothetical protein

Xl2.13981.2.A1_at 0.02566478 0.740858857 1.671170415 LOC100037016 LOC100037016 Hypothetical protein Xl2.51228.2.A1_at 0.015535222 -0.026950405 0.981492804 LOC100037115 LOC100037115 hypothetical protein Xl2.54213.1.S1_at 0.042405816 0.428480753 1.345815604 LOC100049103 LOC100049103 Hypothetical protein Xl2.51218.1.S1_at 0.037696667 0.125423782 1.090828109 LOC100101270 LOC100101270 hypothetical protein Xl2.31867.1.S1_at 0.043061673 -0.424883736 0.744898759 LOC100101333 LOC100101333 Hypothetical protein Xl2.7022.1.S1_at 0.018911267 0.171238476 1.126024701 LOC100126631 LOC100126631 hypothetical protein Xl2.8333.1.A1_at 0.038549952 0.376070727 1.297802389 LOC100126636 LOC100126636

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol hypothetical protein Xl2.16209.1.S1_at 0.014733283 0.430862668 1.348039404 LOC100126638 LOC100126638 hypothetical protein Xl2.44884.3.S1_at 0.037949694 -0.488287398 0.712870834 LOC100126652 LOC100126652 hypothetical protein Xl2.48567.2.S1_at 0.031079311 0.336890687 1.263031558 LOC100127246 LOC100127246 Hypothetical protein Xl2.2891.2.S1_at 0.014458603 -0.002370814 0.998358027 LOC100127261 LOC100127261 256 hypothetical protein

Xl2.21140.1.S1_at 0.027337001 -0.987502255 0.504350204 LOC100137623 LOC100137623 hypothetical protein Xl2.51712.3.S1_a_at 0.016607245 0.423638832 1.341306397 LOC100137672 LOC100137672 Hypothetical protein Xl2.40560.1.S1_at 0.041991935 0.200113621 1.148788825 LOC100158318 LOC100158318 Hypothetical protein Xl2.12269.3.S1_a_at 0.002351268 -0.027368025 0.98120873 LOC100158326 LOC100158326 hypothetical protein Xl2.53009.1.S1_at 0.014065903 0.455095221 1.370873283 LOC100158370 LOC100158370 hypothetical protein Xl2.34769.1.S1_at 0.010904549 1.907684782 3.752064898 LOC100158389 LOC100158389 hypothetical protein Xl2.41863.1.A1_at 0.030993023 -0.722108727 0.606210721 LOC100158415 LOC100158415

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Hypothetical protein Xl2.5159.1.A1_at 0.023324099 0.931022209 1.906626441 LOC100158435 LOC100158435 Hypothetical protein Xl2.54310.1.S1_at 0.031711159 0.044712754 1.031477788 LOC100174796 LOC100174796 hypothetical protein LOC100189564 /// 71 kDa LOC100189564 protein /// hypothetical protein /// LOC780751 Xl2.3352.1.S1_a_at 0.036068238 0.616810299 1.533481008 MGC79993 /// MGC79993

257 Hypothetical protein

Xl2.55917.1.S1_at 0.045270657 -0.003721802 0.997423568 LOC100189575 LOC100189575 Xl2.11979.1.S1_a_at 0.036677351 0.300862953 1.231881047 Hypothetical protein LOC398481 LOC398481 Xl2.14521.2.S1_a_at 0.021177792 -0.514560529 0.700006135 hypothetical protein LOC398688 LOC398688 Xl2.14521.1.S1_at 0.026407193 -0.501716435 0.706266007 hypothetical protein LOC398688 LOC398688 Xl2.15353.1.S1_a_at 0.015426681 3.18486314 9.093673082 hypothetical protein LOC432271 LOC432271 Xl2.41921.1.S1_at 0.00673055 0.464209522 1.37956126 Hypothetical protein LOC443600 LOC443600 Xl2.47633.1.S1_at 0.012802678 0.712264753 1.638374031 hypothetical protein LOC443613 LOC443613 Xl2.29343.1.S1_at 0.027064218 -0.211546805 0.863610803 hypothetical protein LOC443680 LOC443680 Xl2.48365.1.S1_at 0.00525892 0.281307967 1.215296189 hypothetical protein LOC445878 LOC445878 Xl2.8104.1.S1_at 0.038224165 -0.725797567 0.604662676 hypothetical protein LOC446305 LOC446305 Xl2.11377.1.S1_at 0.024551121 0.209299653 1.156126813 hypothetical protein LOC733166 LOC733166 Xl2.51518.1.S1_a_at 0.006360419 1.640042185 3.116749452 hypothetical protein LOC733187 LOC733187

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.49701.1.S1_s_at 0.006060356 2.152397798 4.44566056 Hypothetical protein LOC733253 LOC733253 Xl2.22789.1.S1_at 0.006880346 -0.387887318 0.76424795 hypothetical protein LOC733255 LOC733255 Xl2.3525.1.S1_at 0.002880273 0.444390153 1.360738792 hypothetical protein LOC733328 LOC733328 Xl2.28646.1.S1_at 0.000867379 -0.507533343 0.703424094 hypothetical protein LOC733423 LOC733423 Xl2.47703.1.A1_s_at 0.014089742 -0.232519068 0.851147418 Hypothetical protein LOC733436 LOC733436 Xl2.21410.1.S1_at 0.017231093 -0.205301748 0.867357255 Hypothetical protein LOC733436 LOC733436 hypothetical protein Xl2.16117.1.S1_at 0.028424604 -0.228422998 0.853567412 MGC115313 MGC115313 258 hypothetical protein Xl2.33172.1.S1_at 0.041092681 -0.416752906 0.749108758 MGC115544 MGC115544 hypothetical protein Xl2.7910.1.S1_at 0.042587096 -0.360643702 0.77881701 MGC115585 MGC115585 hypothetical protein Xl2.53880.1.S1_at 0.039636174 0.280387851 1.21452135 MGC116545 MGC116545 hypothetical protein Xl2.46291.1.S1_at 0.04983781 0.447183332 1.363375849 MGC130871 MGC130871 Hypothetical protein Xl2.55143.1.A1_at 0.007879787 -0.025928097 0.982188546 MGC131328 MGC131328 Hypothetical protein Xl2.48488.1.S1_at 0.043293897 0.707620164 1.633107958 MGC132029 MGC132029 hypothetical protein Xl2.27263.2.S1_s_at 0.029262502 -0.844074437 0.557068082 MGC154351 MGC154351

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol hypothetical protein Xl2.27263.1.A1_s_at 0.037834487 -0.66283906 0.631634089 MGC154351 MGC154351 hypothetical protein Xl2.1619.1.A1_s_at 0.026461824 -0.663502998 0.631343473 MGC154492 MGC154492 hypothetical protein Xl2.1619.2.S1_s_at 0.043451084 -0.779456008 0.582586426 MGC154492 MGC154492 Xl2.3966.1.S1_at 0.037018946 -0.50363244 0.705328656 hypothetical protein MGC52932 MGC52932 Xl2.8199.1.S1_at 0.033192947 -0.25983043 0.835186079 hypothetical protein MGC52980 MGC52980 259 Xl2.5427.1.S1_at 0.026293931 0.395292361 1.315209259 hypothetical protein MGC53066 MGC53066

Xl2.23093.1.S1_at 0.019003115 -0.705327545 0.613303231 hypothetical protein MGC53111 MGC53111 Xl2.2924.1.S1_at 0.020972174 -0.605144834 0.657405379 hypothetical protein MGC53311 MGC53311 Xl2.5227.1.S1_at 0.043317533 0.24043121 1.181345704 hypothetical protein MGC53617 MGC53617 Xl2.3951.1.S1_at 0.010581594 -1.009809054 0.496611972 hypothetical protein MGC53794 MGC53794 Xl2.21906.1.S1_at 0.027714192 -0.436050963 0.739155104 hypothetical protein MGC53997 MGC53997 Xl2.45569.1.S1_at 0.016214914 0.419827381 1.337767481 hypothetical protein MGC64382 MGC64382 Xl2.28478.2.S1_at 0.013240046 0.14618517 1.106639381 Hypothetical protein MGC64431 MGC64431 Xl2.34820.1.S1_at 0.002267364 0.755220395 1.687889425 hypothetical protein MGC68516 MGC68516 Xl2.12639.1.S1_at 0.020064973 -0.22063483 0.858187725 hypothetical protein MGC68614 MGC68614 Xl2.2686.1.S1_at 0.038774463 -0.274701349 0.826621419 hypothetical protein MGC68893 MGC68893 Xl2.13893.1.S1_at 0.047390551 -0.568050963 0.674527439 hypothetical protein MGC68927 MGC68927

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol hypothetical protein MGC68999 /// tight junction protein 2 (zona MGC68999 /// Xl2.8126.1.S1_at 0.03213832 0.206227173 1.153667252 occludens 2) tjp2 Xl2.2782.1.S1_at 0.027433452 0.178912972 1.132030613 hypothetical protein MGC69166 MGC69166 Xl2.16662.1.S1_at 0.016267286 0.405309252 1.324372768 hypothetical protein MGC69176 MGC69176 Xl2.47019.1.S1_at 0.038520566 -0.97341948 0.509297493 hypothetical protein MGC78844 MGC78844 Xl2.17857.1.S1_at 0.043191685 -0.415616044 0.749699298 hypothetical protein MGC78985 MGC78985 Xl2.47051.1.S1_at 0.04267576 -0.00296931 0.997943948 hypothetical protein MGC80011 MGC80011 260 Xl2.46807.1.S1_at 0.014369515 0.323789473 1.251613802 hypothetical protein MGC81039 MGC81039 Xl2.16763.1.S1_at 0.039537791 0.105007663 1.075500103 hypothetical protein MGC81183 MGC81183 Xl2.1295.1.S1_at 0.044238318 1.129570559 2.187936031 hypothetical protein MGC81217 MGC81217 Xl2.32858.1.S1_at 0.048253521 -0.025770783 0.982295651 hypothetical protein MGC81434 MGC81434 Xl2.15758.1.S1_at 0.037631341 -0.279044179 0.824136847 hypothetical protein MGC81482 MGC81482 Xl2.46730.1.S1_a_at 0.002375746 -0.002230029 0.998455456 hypothetical protein MGC83092 MGC83092 hypothetical protein MGC84860 /// hypothetical protein MGC84860 /// Xl2.8526.1.S1_s_at 0.027927233 0.116340404 1.083981702 MGC85186 MGC85186 Xl2.4460.1.S1_at 0.003988922 1.033535189 2.047034176 hypothetical protein MGC84886 MGC84886 Xl2.13767.1.S1_at 0.005402998 -0.553608813 0.681313731 hypothetical protein MGC85058 MGC85058 Xl2.24520.1.S1_at 0.033527979 0.246592997 1.186402051 hypothetical protein MGC85143 MGC85143

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol IKAROS family zinc finger 2 Xl2.21586.1.S1_at 0.043485141 -0.024864971 0.98291259 (Helios) ikzf2 inositol polyphosphate-5- Xl2.14338.1.S1_at 0.019511718 0.534012425 1.447950642 phosphatase K inpp5k Xl2.48844.1.S1_at 0.028409521 -0.164093617 0.892489055 insulin induced gene 2 insig2 insulin-like growth factor 1 Xl2.269.1.S1_at 0.027773836 -0.309096372 0.807147155 receptor igf1r insulin-like growth factor 2 261 Xl2.47110.1.S1_at 0.007596811 -1.712731463 0.305081909 (somatomedin A) igf2

Xl2.14733.1.A1_at 0.042357255 -0.887347471 0.540607161 insulinoma-associated 1 insm1 Xl2.24778.1.S1_at 0.024833352 -0.114656297 0.92360232 integral membrane protein 2A b itm2ab Xl2.15899.3.S1_at 0.028881115 -0.003295599 0.997718272 integrator complex subunit 7 ints7 Xl2.48308.1.S1_at 0.040364104 0.34814389 1.272921886 integrin-linked kinase Ilk intelectin 1 (galactofuranose Xl2.6266.1.S1_at 0.01920116 -0.710597644 0.611066949 binding) itln1 Xl2.415.1.S1_at 0.029941212 1.406494179 2.650921922 interleukin 1, beta il1b interleukin enhancer binding Xl2.3647.2.A1_at 0.039723679 -0.088737231 0.940345459 factor 3, 90kDa ilf3 Xl2.8258.1.S1_at 0.024448183 -0.045433104 0.968998875 isopeptidase T Isot Xl2.11712.1.S1_at 0.031298616 0.594563583 1.510015735 Josephin domain containing 2 josd2 Xl2.542.1.S1_at 0.029485268 0.214799848 1.160542896 jun oncogene Jun

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol karyopherin alpha 3 (importin Xl2.47059.1.S1_at 0.040471283 0.077533583 1.055212517 alpha 4) kpna3 karyopherin alpha 4 (importin Xl2.41126.1.S1_at 0.039884216 -0.12326175 0.918109574 alpha 3) kpna4 Xl2.48452.1.S1_at 0.015177929 -0.643114067 0.640329301 kelch-like 20 klhl20 Xl2.23326.1.S1_at 0.04911555 -0.348159784 0.785585505 keratin Krt Xl2.55480.1.S1_at 0.030633342 -0.529337455 0.692872857 keratin 18 krt18 Xl2.5100.3.S1_s_at 0.01837482 -0.685321363 0.621867289 keratin 19 krt19 262 Xl2.5482.1.S1_at 0.040326946 -0.719607998 0.607262422 keratin 19 krt19

Xl2.1642.2.S1_a_at 0.042045122 -0.220467912 0.858287022 keratin 5, gene 7 krt5.7 Xl2.4511.1.A1_s_at 0.007326783 -0.167039262 0.890668661 keratin 8 krt8 keratinocyte associated protein Xl2.21819.1.S1_at 0.027523546 0.267923198 1.204073281 3 krtcap3 KH domain containing, RNA binding, signal transduction Xl2.25540.1.S1_at 0.035235665 -0.0907313 0.939046627 associated 1 khdrbs1 Xl2.34254.1.S1_at 0.027080079 -0.251896387 0.839791804 KIAA1012 kiaa1012 Xl2.23923.1.S1_at 0.001060724 0.644219312 1.562893327 KIAA1715 kiaa1715 Xl2.16672.1.S1_at 0.035909474 -0.364102088 0.776952288 KIT ligand Kitlg Xl2.16840.1.S1_at 0.014940251 1.145814721 2.212710518 Kruppel-like factor 11 klf11

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol kynurenine 3-monooxygenase Xl2.52644.1.S1_at 0.001365304 2.314504572 4.97433811 (kynurenine 3-hydroxylase) Kmo Xl2.204.1.S1_at 0.025882797 -0.397505247 0.759169929 lamin B receptor Lbr Xl2.52554.1.S1_at 0.032616663 -0.045946667 0.968653997 leucine rich repeat containing 68 lrrc68 Xl2.10574.1.S1_at 0.002077147 1.711168444 3.274258995 ligase IV, DNA, ATP-dependent lig4 Xl2.32655.1.S1_at 0.030356848 -0.937995672 0.52195753 LIM homeobox 1 lhx1 Xl2.50566.1.S1_at 0.037954705 -0.712193704 0.610391297 LIM homeobox protein 1b Lmx1b Xl2.24771.1.S1_at 0.04293649 0.587483642 1.502623574 lipin 2 lpin2 263 lipoma HMGIC fusion partner- Xl2.34718.1.S1_at 0.031598769 0.114481318 1.082585761 like 3 lhfpl3 low density lipoprotein receptor Xl2.8355.1.S1_at 0.02114982 0.312647993 1.241985209 adaptor protein 1 ldlrap1 low molecular weight neuronal Xl2.992.1.S1_at 0.035174511 1.395626276 2.631027405 intermediate filament Nif LysM, putative peptidoglycan- Xl2.8367.1.S1_at 0.007992488 0.898197353 1.86373579 binding, domain containing 2 lysmd2 lysophosphatidylcholine Xl2.6751.1.S1_at 0.038419406 -0.321888219 0.800022112 acyltransferase 3 lpcat3 Xl2.53980.1.S1_at 0.03622009 -0.00498148 0.996553056 lysyl oxidase-like 3 loxl3 Xl2.6714.1.S1_at 0.044935882 0.435446859 1.352329637 MACRO domain containing 2 macrod2 MAD2 mitotic arrest deficient- Xl2.8200.1.S1_at 0.044540911 -0.202256581 0.869189962 like 2 mad2l2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.47015.1.S2_at 0.040788317 -0.107603094 0.928128783 Mad2 protein mad2 major facilitator superfamily Xl2.30347.1.S1_at 0.031631404 -0.927422978 0.525796711 domain containing 6-like mfsd6l-b malate dehydrogenase 1, NAD Xl2.26072.1.S1_a_at 0.046580398 0.156473166 1.114559137 (soluble) mdh1 Xl2.2971.1.S1_at 0.017546781 -0.21666119 0.860554704 Malectin-B mlec-b mannosyl (alpha-1,3-)- glycoprotein beta-1,4-N- 264 acetylglucosaminyltransferase,

Xl2.51709.1.S1_at 0.000272176 -1.02021735 0.493042067 isozyme A mgat4a Xl2.1244.1.S1_at 0.004417256 0.648307713 1.567328635 maternal B9.10 protein b9-a Xl2Affx.103.1.S1_at 0.017816321 0.470882069 1.385956588 MAX dimerization protein 1 mxd1 Xl2.12095.1.S2_at 0.01425925 -0.155334417 0.89792421 mediator complex subunit 26 med26 Xl2.53912.1.S1_s_at 0.012536132 -0.361661031 0.778268013 mediator complex subunit 4 med4 Xl2.452.2.S1_at 0.046648549 -0.622674951 0.649465614 Meis homeobox 3 meis3-a Meis homeobox 3 /// Meis meis3-a /// Xl2.452.1.S1_s_at 0.046672911 -0.724388562 0.605253507 homeobox 3 meis3-b membrane-associated ring Xl2.51293.1.S1_at 0.030319719 -0.028794048 0.98023934 finger (C3HC4) 6 mar6 membrane-associated ring Xl2.8458.1.S1_at 0.01036495 0.581339326 1.496237637 finger (C3HC4) 8 mar8

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol mesoderm specific transcript Xl2.50661.1.S1_at 0.032066785 -8.31E-05 0.999942367 homolog mest Xl2.29629.1.S1_at 0.007495562 -0.366964851 0.775412098 metastasis suppressor 1 mtss1 Xl2.46689.1.S1_at 0.048947608 -0.262090128 0.833878948 metaxin 3 mtx3 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate 265 cyclohydrolase,

formyltetrahydrofolate Xl2.3693.1.S1_at 0.00525779 -0.33164278 0.794631131 synthetase mthfd1 AFFX-Xl-gapdh-5_a_at 0.003925907 1.182150196 2.269147194 mg:bb02e05 mg:bb02e05 AFFX-Xl-gapdh- M_a_at 0.00724485 1.153190699 2.224052276 mg:bb02e05 mg:bb02e05 Xl2.23223.1.S1_a_at 0.007247167 1.255638515 2.387728024 mg:bb02e05 mg:bb02e05 AFFX-Xl-gapdh-3_a_at 0.014208778 1.150129142 2.219337599 mg:bb02e05 mg:bb02e05 Xl2.16769.1.S1_at 0.045469485 0.928975533 1.903923528 MGC80034 protein MGC80034 Xl2.47361.1.S1_at 0.023665975 -0.659581462 0.633061927 MGC80203 protein MGC80203 Xl2.47581.1.S1_at 0.009021359 -0.46651779 0.723709297 MGC80400 protein MGC80400 Xl2.7809.1.S2_at 0.020252999 2.173828013 4.512190611 MGC81892 protein MGC81892 Xl2.23708.1.S1_at 0.021136623 -0.211435905 0.863677191 MGC81939 protein MGC81939 Xl2.23700.2.S1_x_at 0.030333232 -0.161339259 0.894194602 MGC82151 protein MGC82151

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.19790.1.S1_at 0.040769719 1.69092188 3.228629468 MGC82544 protein MGC82544 Xl2.47602.1.S1_at 0.010847891 -0.408079114 0.753626127 MGC82760 protein MGC82760 Xl2.47626.1.S1_at 0.005738868 -0.288959341 0.818492248 MGC82764 protein MGC82764 Xl2.23043.1.S1_at 0.041659723 0.318078621 1.246669131 MGC82833 protein MGC82833 Xl2.14433.1.S1_at 0.034467356 -0.313535468 0.804667423 MGC82975 protein MGC82975 Xl2.25723.2.A1_at 0.010197537 -0.044080831 0.969907567 MGC83400 protein MGC83400 Xl2.47551.1.S1_at 0.023019138 1.036812269 2.051689297 MGC84110 protein MGC84110 Xl2.47427.1.S1_at 0.019866598 0.899245518 1.865090347 MGC84280 protein MGC84280

266 Xl2.15713.1.S1_at 0.046265454 0.921610622 1.894228826 MGC84460 protein MGC84460 Xl2.48977.1.S1_at 0.020270689 1.322251568 2.500560599 MGC84470 protein MGC84470 microfibrillar-associated protein Xl2.56225.1.S1_at 0.03834316 -0.416237754 0.749376294 3 mfap3 microtubule associated Xl2.3201.1.S1_at 0.028378363 -0.485740913 0.714130225 serine/threonine kinase-like mastl microtubule associated tumor Xl2.23255.2.S1_a_at 0.012561897 -0.781126862 0.581912095 suppressor 1 mtus1 microtubule associated tumor Xl2.23255.1.A1_at 0.012757521 -0.678558168 0.624789378 suppressor 1 mtus1 microtubule-associated protein Xl2.14237.1.S1_at 0.022551344 1.52849278 2.884842947 1 light chain 3 alpha map1lc3a

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol mitochondrial ribosomal protein Xl2.19285.1.S1_at 0.047553668 0.096805228 1.069402703 L15 mrpl15 mitochondrial ribosomal protein Xl2.10444.1.S1_at 0.014735406 0.199391569 1.148214014 L18 mrpl18 mitochondrial ribosomal protein Xl2.5698.1.S1_at 0.03326085 0.408138124 1.32697218 L28 mrpl28 mitochondrial ribosomal protein Xl2.25722.1.S1_at 0.041339547 0.134537027 1.097740483 L44 mrpl44 267 mitogen-activated protein

Xl2.12756.1.S1_at 0.015370767 0.560654335 1.474938024 kinase 12 mapk12 mitogen-activated protein Xl2.10790.1.S1_at 0.03393866 -0.305423977 0.809204374 kinase kinase 7 map2k7 Xl2.47180.1.S1_at 0.035095023 -0.201616015 0.869575974 MLX interacting protein mlxip MOB1, Mps One Binder kinase Xl2.5955.1.S1_at 0.048453742 0.170296287 1.125289562 activator-like 3 mobkl3 Xl2.46038.1.S1_at 0.000666901 0.123754452 1.089566651 MON2 homolog mon2 Xl2.45216.1.S1_at 0.028571627 -0.447684869 0.733218519 msh homeobox 1 msx1 Xl2.31078.1.S1_at 0.002925125 -0.623995799 0.648871273 msh homeobox 2 msx2 Xl2.17369.1.S1_a_at 0.013150762 0.021172375 1.014783787 mu/m-calpain large subunit LOC398288 Xl2.51183.1.S1_at 0.001256241 4.590665804 24.09506527 myeloid zinc finger 1 mzf1 myoblast determination protein Xl2.823.1.S1_at 0.0379651 -0.404021045 0.75574894 1 homolog B mf25

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol MyoD family inhibitor domain Xl2.757.1.S1_at 0.047996956 0.330415685 1.257375611 containing mdfic Xl2.5485.1.S1_at 0.015566547 0.558921522 1.473167548 myosin ID myo1d Xl2.1285.2.S3_a_at 0.014523334 1.029817171 2.041765487 Myosin light chain, regulatory A mylc2a myosin regulatory light chain Xl2.49524.1.S1_at 0.008026882 0.850118692 1.802649225 interacting protein mylip Xl2.29888.1.S1_at 0.010789024 -0.002958442 0.997951465 myotubularin-related protein 3 mtmr3 Na+/K+-transporting ATPase 268 Xl2.6045.1.S1_s_at 0.047613047 -0.126202427 0.916240076 beta subunit atpb-3

NADH dehydrogenase (ubiquinone) 1 alpha Xl2.6970.1.S1_at 0.003490877 1.317031442 2.49152913 subcomplex, 8, 19kDa ndufa8 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH-coenzyme Q Xl2.13717.1.S1_at 0.010535801 0.180803103 1.133514702 reductase) ndufs7 NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa (NADH-coenzyme Q Xl2.33470.1.S1_at 0.041891633 0.252750889 1.191476823 reductase) ndufs8 Xl2.2279.1.S1_at 0.005408902 0.686425769 1.609291607 NDRG family member 2 ndrg2 Xl2.15874.1.S1_at 0.016104216 0.378407385 1.299906075 neurocalcin delta ncald

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.370.1.S2_at 0.009068132 -0.777572704 0.583347435 neurogenin 2 neurog2-b NIMA (never in mitosis gene a)- Xl2.4275.2.S1_a_at 0.028415507 -0.535373728 0.689979915 related kinase 2 nek2 NIMA (never in mitosis gene a)- Xl2.18997.1.S1_at 0.045967861 -0.216427897 0.860693872 related kinase 4 nek4 NIMA (never in mitosis gene a)- Xl2.14397.1.S2_at 0.011473545 0.754072463 1.686546929 related kinase 6 nek6 Xl2.4683.1.S1_at 0.023844067 0.469559534 1.384686648 nipsnap homolog 3A nipsnap3a 269 Xl2Affx.79.1.S1_s_at 0.046323713 -0.590099736 0.664296982 NK6 homeobox 2 nkx6-2

NmrA-like family domain Xl2.9998.1.S1_at 0.042306825 0.985427564 1.979899999 containing 1 nmral1 non-metastatic cells 4, protein Xl2.32033.1.S1_at 0.008177532 0.203169367 1.151224633 expressed in b nme4b non-SMC condensin I complex, Xl2.23106.1.S1_at 0.011516478 0.205598846 1.153164913 subunit H ncaph non-SMC condensin II complex, Xl2.6337.1.S1_at 0.016009366 0.125313904 1.090745033 subunit D3 ncapd3 NOP56 ribonucleoprotein Xl2.3788.1.S1_at 0.045075267 -0.19376791 0.874319263 homolog nop56 NOP58 ribonucleoprotein Xl2.47829.1.S1_at 0.036636201 -0.199208891 0.871028065 homolog nop58

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol nuclear factor of activated T- cells, cytoplasmic, calcineurin- Xl2.40993.1.S1_at 0.029939085 -0.707909449 0.61220662 dependent 1 nfatc1 nuclear factor, interleukin 3 Xl2.48950.1.S1_at 0.012138215 -0.475331597 0.719301443 regulated nfil3 Xl2.1954.1.S1_at 0.022681157 -0.174878348 0.885842213 nucleolar protein 10 nol10 nudix (nucleoside diphosphate Xl2.14286.1.S1_at 0.018327821 0.508372312 1.422444449 linked moiety X)-type motif 17 nudt17 270 nudix (nucleoside diphosphate

Xl2.444.1.S1_a_at 0.030482271 0.402773975 1.322047466 linked moiety X)-type motif 6 nudt6 oocyte-specific histone mRNA Xl2.5571.1.S1_at 0.034750787 0.533976282 1.447914368 stem-loop binding protein slbp2 Xl2.847.1.S1_at 0.030078046 -1.076193663 0.474278488 otogelin Otog Xl2.847.1.S2_at 0.044883382 -1.019267621 0.493366744 otogelin Otog outer dense fiber of sperm tails Xl2.5460.1.S1_at 0.038670388 -0.815601623 0.568171502 2-like odf2l Xl2.35424.1.S1_at 0.00736499 1.298187515 2.459197349 oviduct protein p20 LOC407840 Xl2.3829.1.S1_at 0.041247008 -0.255651364 0.837608876 oxysterol binding protein-like 2 osbpl2 Xl2.34499.1.S1_s_at 0.040484065 -1.24689165 0.421355058 p51/p63 LOC399036 Xl2.45665.1.S1_at 0.047594865 -0.2017957 0.869467677 P70 S6 kinase p70s6k-A

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol paired-like homeodomain 2 /// paired-like homeodomain Xl2.103.1.S1_s_at 0.04024809 -2.002614738 0.249547311 transcription factor 2 pitx2 /// pitx2-A Xl2.11081.1.S1_at 0.039683232 -2.088028997 0.235201799 palmdelphin palmd Xl2.12677.1.S1_at 0.006253775 0.218725135 1.163704802 palmitoyl-protein thioesterase 2 ppt2 Xl2.3219.2.S1_at 0.01650753 -1.146807155 0.451623619 paralemmin Palm Xl2.19495.1.S1_x_at 0.008902463 0.258327117 1.196090968 paraoxonase 2 pon2 Xl2.2356.1.A1_at 0.047461208 0.624871763 1.542073749 PDZ and LIM domain 5 pdlim5 271 Xl2.16816.1.A1_at 0.031032741 -2.239647081 0.211738118 PDZ domain containing 3 pdzd3 Xl2.14729.1.S1_at 0.028099634 -0.508727488 0.702842098 peripheral myelin protein 22 pmp22 Xl2.2129.1.S1_at 0.028268731 0.278345822 1.2128035 peroxiredoxin 6 prdx6 peroxisomal biogenesis factor Xl2.15224.1.S1_at 0.048060256 0.240535551 1.181431146 19 pex19 Xl2.35430.2.S1_a_at 0.00062194 2.950421492 7.729748594 pheromone receptor-like xV2R1 phosphatidylinositol-3,4,5- Xl2.34826.1.S1_at 0.026196766 -0.00038663 0.999732044 trisphosphate 5-phosphatase 1 inpp5d Xl2.44834.1.A1_at 0.033243599 0.281131845 1.215147837 phosphatidylserine synthase 2 ptdss2 Xl2.4347.1.S1_at 0.026762351 -0.346546093 0.786464694 phosphofructokinase, muscle Pfkm phosphoinositide-3-kinase, regulatory subunit, polypeptide Xl2.1475.1.A1_at 0.016439243 1.765014265 3.398773601 2 (p85 beta) pik3r2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.31928.1.S1_at 0.049520215 0.168712659 1.124055024 phospholipase A2, group XIIA pla2g12a phospholipase C, beta 3 Xl2.39175.2.A1_s_at 0.011051662 0.094521233 1.067711023 (phosphatidylinositol-specific) plcb3 phosphoribosyl pyrophosphate Xl2.16570.1.S2_at 0.031647738 -0.20061992 0.870176572 synthetase 1 prps1 phosphoribosyl transferase Xl2.7409.1.S1_at 0.004275446 0.174147289 1.128297322 domain containing 1 prtfdc1 platelet derived growth factor 272 Xl2.20029.2.S1_a_at 0.005398928 -0.480451337 0.716753358 receptor, alpha polypeptide pdgfra

platelet derived growth factor Xl2.20029.1.S1_at 0.016337652 -0.843891385 0.557138769 receptor, alpha polypeptide pdgfra Xl2.28141.2.S1_at 0.021055088 -0.188091045 0.877766402 Polo-like kinase (Drosophila) Plk Xl2.12115.1.S1_at 0.010843945 1.90934018 3.756372617 polo-like kinase 2 plk2 Xl2.7020.1.S1_s_at 0.036985014 -0.453219625 0.730410988 polo-like kinase 4 plk4 poly (ADP-ribose) polymerase Xl2.48213.1.S1_at 0.007666298 0.460850096 1.376352583 family, member 11 parp11 poly (ADP-ribose) polymerase Xl2.49984.1.S1_at 0.021280631 3.746877521 13.42525439 family, member 3 parp3 poly(A) binding protein, Xl2.8945.1.S1_at 0.012441192 -0.149903321 0.90131086 cytoplasmic 1 pabpc1-a Xl2.341.1.S2_at 0.043527006 0.075427916 1.053673519 poly(A) polymerase alpha papola Xl2.19450.1.A1_at 0.003058471 -0.023745665 0.983675472 Poly(rC) binding protein 2 pcbp2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol polymerase (DNA directed) Xl2.16859.1.S2_at 0.001251302 3.651819613 12.56918858 kappa Polk Xl2.56.1.S1_at 0.041094232 -0.593380714 0.662787954 polymerase (DNA directed), beta Polb polymerase (RNA) I polypeptide Xl2.15770.1.S1_at 0.0382661 -0.224182871 0.856079764 B, 128kDa polr1b post-GPI attachment to proteins Xl2.48708.1.S1_at 0.045020509 -0.338983792 0.790597998 3 pgap3 potassium inwardly-rectifying 273 Xl2.29882.1.S1_at 0.046779934 -0.663804112 0.631211716 channel, subfamily J, member 15 kcnj15

Xl2.2681.1.S1_at 0.027579186 -0.291098136 0.817279733 PPAN-P2RY11 readthrough ppan-p2ry11 Xl2.19035.1.S1_at 0.032751228 -0.780530013 0.582152884 PR domain containing 12 prdm12 Xl2.15323.2.S1_a_at 0.04439064 -0.16601214 0.891302996 pre-B-cell leukemia homeobox 2 pbx2 Xl2.55576.1.S1_at 0.012000338 0.278083104 1.212582666 prefoldin subunit 5 pfdn5 Xl2.1253.1.S1_at 0.004073452 0.070535346 1.050106278 presenilin 1 psen1 Xl2.988.1.S1_at 0.012699778 -0.314308298 0.804236491 prevents mitotic catastrophe 2 rexo4 Xl2.19729.1.A1_at 0.000567683 -0.531680041 0.691748712 prickle homolog 3 prickle3 PRKR interacting protein 1 (IL11 Xl2.23753.1.S1_at 0.044509864 -0.459222476 0.727378165 inducible) prkrip1 probable glutathione peroxidase Xl2.49093.1.S1_at 0.030628888 1.134806345 2.195890851 8-B gpx8-b Xl2.34965.1.S1_at 0.045077629 0.38754959 1.308169597 proliferating cell nuclear antigen Pcna

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol prostaglandin reductase 1, gene Xl2.23763.1.S1_at 0.038094061 0.352396892 1.276679941 2 ptgr1.2 prostaglandin-endoperoxide synthase 1 (prostaglandin G/H Xl2.6116.1.A1_at 0.017640693 0.506166283 1.420271048 synthase and cyclooxygenase) ptgs1 proteasome (prosome, macropain) 26S subunit, non- Xl2.20765.1.S1_at 0.044513566 0.737425981 1.667198611 ATPase, 14 psmd14

274 proteasome (prosome,

macropain) 26S subunit, non- Xl2.45017.1.S1_at 0.027321874 0.136619285 1.099326008 ATPase, 8 psmd8 Xl2.5997.1.S1_at 0.033562622 0.356613013 1.280416358 Protein FAM54B-B fam54b-b Xl2.3877.1.S1_at 0.042432076 0.255795117 1.193993611 Protein GTLF3B gtlf3b protein kinase, interferon- inducible double stranded RNA Xl2.852.1.S1_at 0.014380225 0.293769266 1.225838793 dependent activator prkra protein phosphatase, Xl2.14351.1.S1_at 0.021895916 0.523988694 1.437925264 Mg2+/Mn2+ dependent, 1D ppm1d protein tyrosine phosphatase Xl2.17950.1.S1_at 0.029699689 0.424497567 1.34210502 type IVA, member 1 ptp4a1 protein tyrosine phosphatase, Xl2.922.1.S1_s_at 0.038691935 -0.275914018 0.825926887 receptor type, A ptpra

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol protein-L-isoaspartate (D- Xl2.6342.1.S1_at 0.003372295 0.479153864 1.393925894 aspartate) O-methyltransferase pcmt1 Xl2.8877.1.S1_at 0.002014573 -0.182481918 0.881185758 prothymosin, alpha ptma-a Xl2.24523.1.S1_at 0.018941812 -0.278761176 0.824298528 prothymosin, alpha ptma-b Xl2.315.1.S1_at 0.029606899 -1.006597203 0.497718803 protocadherin 7 pcdh7 PRP4 pre-mRNA processing Xl2.10066.1.S1_at 0.003194446 -0.671026981 0.628059445 factor 4 homolog B prpf4b Xl2.47664.1.S1_at 0.016933319 0.35055465 1.275050731 pyrophosphatase (inorganic) 2 ppa2 275 pyruvate dehydrogenase E1

Xl2.48839.1.S1_at 0.021713087 0.61059122 1.526884802 alpha 1 pdha1-b Xl2.8592.1.S1_s_at 0.041613727 0.098488423 1.070651105 pyruvate kinase, muscle pkm2 RAB11B, member RAS oncogene Xl2.3255.1.S1_a_at 0.022378854 0.379617753 1.300997106 family, gene 1 rab11b.1 RAB27A, member RAS oncogene Xl2.49803.1.S1_at 0.020895698 -0.538449004 0.688510707 family rab27a rab2b, member RAS oncogene Xl2.2749.1.S1_at 0.00925586 0.2080784 1.155148556 family rab2b RAB8A, member RAS oncogene Xl2.12142.1.A1_at 0.039132765 -0.151679606 0.900201825 family rab8a Xl2.15449.1.S1_at 0.020598774 0.17852138 1.131723386 rab-like protein 3 MGC82648 Xl2.292.1.S1_at 0.00945287 1.109292509 2.157398235 RAD51 homolog (RecA homolog) rad51 Xl2.292.1.S2_at 0.025620669 1.279891543 2.428207218 RAD51 homolog (RecA homolog) rad51

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.284.2.S1_a_at 0.039659469 0.540941061 1.454921244 rad51 protein rad51-b Xl2.53870.1.S1_at 0.003853145 0.234165501 1.176226175 RAD52 homolog rad52 Rap guanine nucleotide Xl2.48638.1.S1_at 0.035646272 -0.386956967 0.76474095 exchange factor (GEF) 2 rapgef2 Xl2.29260.1.S1_a_at 0.000464658 1.373835918 2.591587165 RAP1 GTPase activating protein rap1gap ras homolog gene family, Xl2.47494.1.S1_at 0.002409777 -0.002400852 0.99833724 member J Rhoj Xl2.21562.1.S1_at 0.006501988 1.591906114 3.014473641 RAS, dexamethasone-induced 1 rasd1 276 Xl2.3855.1.S1_at 0.009626309 0.823380289 1.769547256 receptor accessory protein 6 reep6

Xl2.13.1.S2_at 0.019678753 -0.340192247 0.789936042 receptor tyrosin kinase epha4-a regulator of G-protein signaling Xl2.46296.1.S1_at 0.017782117 -1.191159906 0.437950613 2, 24kDa rgs2 reticulocalbin 2, EF-hand Xl2.20666.1.S1_at 0.004226302 0.702502317 1.627324899 calcium binding domain rcn2 retina and anterior neural fold Xl2.186.1.S1_at 0.007478704 -2.595678456 0.165433297 homeobox rax-a retinal pigment epithelium- Xl2.16557.1.S1_at 0.046658265 -0.663749064 0.631235801 specific protein 65kDa rpe65 Xl2.6965.3.S1_x_at 0.020232203 -0.162380733 0.89354932 retinoic acid receptor, gamma Rarg Xl2.517.1.S2_a_at 0.002593212 1.258303219 2.392142311 Rho family GTPase 1 rnd1 Rho GTPase activating protein Xl2.13839.1.A1_at 0.03043072 -0.630544052 0.645932783 23 arhgap23

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Rho GTPase activating protein Xl2.48203.1.S1_at 0.047831146 -0.00085294 0.999408962 29 arhgap29 Rho guanine nucleotide Xl2.47604.1.S1_at 0.003103545 2.983234008 7.90756773 exchange factor (GEF) 3 arhgef3 Xl2.53612.1.S1_at 0.039225883 -0.389155259 0.763576571 rhomboid domain containing 2 rhbdd2 Xl2.47524.1.S1_a_at 0.025811019 0.522960859 1.436901192 rhombotin-1 lmo1 Xl2.47360.1.S1_at 0.04798253 0.185517169 1.137224566 ribosomal protein S9 rps9 ribosomal RNA processing 1 277 Xl2.3220.1.S1_at 0.007264641 -0.317836174 0.802272264 homolog B rrp1b

rieske [2Fe-2S] domain Xl2.25762.1.A1_at 0.041133242 -0.877034473 0.544485497 containing protein (5K704) MGC154819 Xl2.6274.1.S1_at 0.01649479 1.234246003 2.35258362 ring finger protein 168 rnf168 Xl2.47379.1.S1_at 0.004454598 -0.721968611 0.6062696 ring finger protein 219 rnf219 Xl2.33849.1.S1_at 0.010951297 2.599950518 6.062658325 RIO kinase 3 riok3 Xl2.24190.1.S2_at 0.004745516 1.123068661 2.178097689 RIO kinase 3 (yeast) riok3 Xl2.24190.1.S1_at 0.031634195 1.10143943 2.145686689 RIO kinase 3 (yeast) riok3 Xl2.12294.1.S1_x_at 0.032918131 -0.289582087 0.818139019 RNA binding motif protein 16 rbm16 Xl2.24054.1.S1_at 0.020003198 -0.336281923 0.792080012 RNA binding motif protein 34 rbm34 Xl2.20922.1.A1_at 0.005693058 -0.698268496 0.616311452 RNA binding motif protein 9 rbm9 RNA guanylyltransferase and 5'- Xl2.612.1.S1_at 0.011289631 0.330713169 1.257634909 phosphatase rngtt

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.47349.1.S1_at 0.010504494 -0.012351784 0.991474942 SAC3 domain containing 1 sac3d1 Xl2.3005.1.S1_at 0.004633425 -0.203874828 0.868215553 sal-like 1 a sall1a Xl2.3005.3.S1_a_at 0.011742093 -0.342848337 0.788483059 sal-like 1 a sall1a Xl2.8637.1.S1_at 0.019787167 0.185734009 1.137395506 SAP30 binding protein sap30bp Xl2.48056.3.S1_x_at 0.002418258 -0.809218853 0.570690775 Scc2-1B scc2-1 Xl2.48056.1.S1_s_at 0.004798162 -0.859471864 0.551154285 Scc2-1B scc2-1 Xl2.39237.1.S1_at 0.025967353 0.147661404 1.107772327 sec1 family domain containing 1 scfd1 Xl2.7305.1.S1_at 0.018000112 0.254018364 1.192524052 SEC11 homolog A sec11a

278 SEC22 vesicle trafficking protein homolog B (S. cerevisiae) Xl2.15170.1.S1_at 0.040173921 -0.487542589 0.713238957 (gene/pseudogene) sec22b Xl2.46419.1.A1_at 0.022884517 0.199244903 1.148097291 selenophosphate synthetase 2 sephs2 sema domain, immunoglobulin domain (Ig), short basic domain, Xl2.19183.1.A1_at 0.02954993 -0.993147371 0.502380589 secreted, (semaphorin) 3A sema3a sema domain, transmembrane domain (TM), and cytoplasmic Xl2.22993.1.S1_at 0.003678697 -0.421682272 0.746553589 domain, (semaphorin) 6D sema6d Xl2.31476.1.S1_at 0.029887556 -0.783531228 0.5809431 septin 12 sep12 Xl2.731.1.S1_at 0.041950853 0.192160465 1.142473312 septin 2 sep2 Xl2.15477.1.A1_s_at 0.015375371 -0.174961709 0.885791029 septin 5 sep5

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.2688.1.S1_at 0.046174747 1.961732875 3.895295765 serine dehydratase-like Sdsl serine palmitoyltransferase, long Xl2.13570.2.S1_s_at 0.007931751 -0.039515587 0.97298159 chain base subunit 2 sptlc2 serine palmitoyltransferase, long Xl2.2338.1.S1_at 0.049544006 0.38665943 1.307362689 chain base subunit 2 sptlc2 Xl2.4061.1.S1_at 0.000305173 0.784668608 1.722696565 serine/threonine kinase 31 stk31 Xl2.79.1.S1_at 0.008522112 2.677284718 6.396508878 sestrin 1 sesn1 Sestrin 1,nuclear factor XPA26- 279 Xl2.56039.1.S1_at 0.000750274 3.173657564 9.023315084 T2 sesn1-A

Xl2.2090.1.S1_at 0.010079508 -0.221589071 0.857620282 seven in absentia homolog 1 siah1 Xl2.379.1.S1_at 0.016413927 0.710496346 1.636366998 SH2/SH3 adaptor protein Nck Xl2.379.1.S2_at 0.016561809 -0.00217698 0.998492171 SH2/SH3 adaptor protein Nck SH3 domain binding glutamic Xl2.16385.1.S1_at 0.031133346 -0.152361184 0.899776639 acid-rich protein like 2 sh3bgrl2 SH3 domain containing ring Xl2.47112.1.S1_at 0.008703431 2.412903999 5.325452069 finger 2 sh3rf2 Xl2.8663.1.S1_at 0.000606791 0.6641183 1.584599552 short coiled-coil protein Scoc Xl2.52393.1.S1_at 0.021245097 0.322746275 1.2507091 shugoshin-like 1 sgol1 signal sequence receptor, gamma (translocon-associated Xl2.8245.1.S1_at 0.031808735 0.091968712 1.065823621 protein gamma) ssr3

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol signal transduction regulatory Xl2.49091.1.A2_at 0.048692463 -0.490324205 0.711865108 protein SAP-2 LOC779362 Xl2.20900.1.S1_at 0.034595886 -0.16590255 0.891370703 similar to CD63 antigen LOC398555 Xl2.5440.1.S1_at 0.003853707 1.924008662 3.794760044 similar to cyclin G1 MGC53060 Xl2.2991.1.S1_at 0.043099175 0.223605325 1.16764792 similar to dynamin 1-like MGC53884 similar to enhancer of split Xl2.8440.1.S1_at 0.009775457 0.241507569 1.182227405 related MGC53782 similar to nucleolar protein 280 Xl2.3622.1.S1_at 0.021899581 -0.15346408 0.89908905 NOP5/NOP58 LOC398558

similar to proliferating cell Xl2.3905.1.S1_at 0.022443795 0.292270852 1.224566271 nuclear antigen MGC53867 similar to serum-inducible Xl2.8630.1.S1_at 0.002404566 2.948469917 7.719299409 kinase MGC53542 similar to serum-inducible MGC53542 /// Xl2.12115.1.S1_s_at 0.006904288 1.770933189 3.412746346 kinase /// polo-like kinase 2 plk2 small nuclear ribonucleoprotein Xl2.5196.1.S1_at 0.037339997 0.073557694 1.052308486 D3 polypeptide 18kDa snrpd3 solute carrier family 13 (sodium/sulfate symporters), Xl2.7988.1.S1_at 0.000755213 0.312238225 1.241632499 member 4 slc13a4

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol solute carrier family 16, member 1 (monocarboxylic acid Xl2.6563.1.S1_at 0.030412998 0.125234819 1.090685243 transporter 1) slc16a1 solute carrier family 18 (vesicular monoamine), member Xl2.21973.1.S1_at 0.046740515 -0.747902641 0.595468608 1 slc18a1 solute carrier family 2 (facilitated glucose transporter),

281 Xl2.46932.1.S1_at 0.039769184 -2.353907311 0.195615513 member 12 slc2a12

solute carrier family 25 (mitochondrial carrier; citrate Xl2.8233.1.S1_at 0.014507129 -0.356278898 0.78117685 transporter), member 1 slc25a1 solute carrier family 27 (fatty Xl2.15221.1.S1_at 0.04659234 -0.415915173 0.749543871 acid transporter), member 2 slc27a2 solute carrier family 38, member Xl2.24047.1.S1_at 0.034368282 -0.60131119 0.659154612 2 slc38a2 solute carrier family 38, member Xl2.24047.1.S2_at 0.048689357 -0.402679195 0.756452189 2 slc38a2 solute carrier family 40 (iron- regulated transporter), member Xl2.19634.1.S1_at 0.000463604 4.216209163 18.58683425 1 slc40a1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol solute carrier family 41, member Xl2.12490.1.S1_at 0.023430761 -0.553751837 0.681246191 3, gene 2 slc41a3.2 solute carrier family 44, member Xl2.47305.1.S1_at 0.040362907 -0.549592913 0.683212884 4 slc44a4 solute carrier family 6 (neurotransmitter transporter, Xl2.15558.1.S1_at 0.001234654 0.884877466 1.846607775 GABA), member 13 slc6a13 solute carrier family 7 (cationic

282 amino acid transporter, y+

Xl2.47170.1.S1_at 0.024582526 -0.383266581 0.766699646 system), member 4 slc7a4 Xl2.23345.1.S1_at 0.019573801 0.446093829 1.362346635 sorting nexin family member 30 snx30 Xl2.26163.1.S2_at 0.034178119 0.725195962 1.653125166 speckle-type POZ protein-like spopl Xl2.44940.1.S1_at 0.010532289 -0.634689534 0.644079405 spermatogenesis associated 17 spata17 S-phase kinase-associated Xl2.20950.2.S2_at 0.006903245 0.252258815 1.191070505 protein 1 skp1 splicing factor, arginine/serine- Xl2.14672.1.S1_at 0.038422009 -0.14141983 0.906626459 rich 6 sfrs6 SSU72 RNA polymerase II CTD Xl2.9702.1.S1_at 0.017102608 1.149377243 2.218181232 phosphatase homolog ssu72 Xl2.45199.1.S1_at 0.021082619 -0.207051395 0.866305994 stannin Snn structural maintenance of Xl2.19399.1.S1_at 0.042541017 -1.326879561 0.398629515 chromosomes 5 smc5

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol succinate dehydrogenase [ubiquinone] flavoprotein subunit A, mitochondrial Xl2.9291.1.S1_at 0.014643018 0.144036091 1.104992128 precursor MGC68518 succinate-CoA ligase, alpha Xl2.48496.1.S1_a_at 0.028263096 0.413767472 1.332160096 subunit suclg1 SUMO1/sentrin specific Xl2.7870.1.S2_at 0.003036672 -0.465459143 0.724240548 peptidase 1 senp1

283 Xl2.19656.1.S1_at 0.007803724 0.067714537 1.048055079 suppressor of IKBKE 1 sike1

Xl2.23755.1.S1_at 0.009325359 0.076707932 1.054608794 suppressor of Ty 4 homolog 1 supt4h1 Xl2.7354.1.S1_at 0.031417321 -0.735152615 0.600754471 synaptotagmin-like 2 sytl2 Xl2.34456.1.S1_at 0.045785385 -0.176280411 0.884981737 syndecan-1 xsyn-1 Xl2.222.1.S1_at 0.049950744 0.821176147 1.766845815 syndecan-2 sdc2-b Xl2.52330.1.S1_at 0.001701153 0.243026756 1.18347297 syntaxin 5 stx5 Xl2.17307.1.S1_at 0.018956569 0.262790409 1.199797067 syntaxin 8 stx8 Xl2.933.1.S1_at 0.031349336 -0.602209712 0.658744213 T, brachyury homolog, gene 2 t2 TAF1 RNA polymerase II, TATA box binding protein (TBP)- associated factor, 250kDa, gene Xl2.49485.1.S1_at 0.034271556 -0.079179066 0.946596133 2 taf1.2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol TAF11 RNA polymerase II, TATA box binding protein (TBP)- Xl2.8264.1.A1_at 0.02041406 0.211564311 1.157943059 associated factor, 28kDa taf11 Xl2.50035.1.S1_at 0.018803382 -0.054982571 0.962606072 talin 1 tln1 TBC1 domain family, member Xl2.13727.1.S1_at 0.012092746 1.205966146 2.306917076 10A tbc1d10a Xl2.228.1.S1_at 0.019841625 0.21692912 1.162257004 TBP-like 1 tbpl1 Xl2.21520.1.S1_x_at 0.043249797 -0.003077224 0.997869304 TCR VDJ BV4 BJ10 LOC443733 284 Xl2.15616.1.S1_at 0.013063938 -1.235233296 0.424773805 tektin 2 (testicular) tekt2

Xl2.9755.1.S1_at 0.036439629 -0.234828097 0.849786249 tetraspanin 13 tspan13 tetratricopeptide repeat domain Xl2.50107.1.S1_at 0.013747767 0.854510335 1.808144949 34 ttc34 tetratricopeptide repeat domain Xl2.47050.1.S1_at 0.028380005 0.310166763 1.239851007 35 ttc35-a Xl2.3544.1.S1_at 0.021247231 0.118205903 1.085384268 TFIIS elongation factor xTFIIS.oB Xl2.48968.1.S1_at 0.015879594 3.352774309 10.21611177 thioredoxin reductase 1 txnrd1 Xl2.4321.1.S1_at 0.042195654 0.111977021 1.080708189 THO complex 7 homolog thoc7 thymidine kinase 2, Xl2.2832.2.A1_at 0.014394234 0.963141173 1.949550022 mitochondrial tk2 Xl2Affx.69.1.S1_at 0.037960617 -0.211337186 0.863736292 thymine-DNA glycosylase Tdg thyroid hormone up-regulated Xl2.46780.1.S1_at 0.049188689 0.448274003 1.364406945 protein (gene 16) thul16

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol TIP41, TOR signaling pathway Xl2.3036.1.S1_at 0.021400262 0.388901286 1.309395827 regulator-like Tiprl trafficking protein particle Xl2.34882.1.S1_at 0.018652905 0.197202567 1.146473149 complex 2-like trappc2l trafficking protein particle Xl2.53607.1.S1_s_at 0.018534395 0.172696561 1.127163313 complex 6B trappc6b TRAF-interacting protein with Xl2.49742.1.S1_at 0.00595014 -0.029340635 0.979868031 forkhead-associated domain Tifa 285 TRAF-type zinc finger domain

Xl2.15937.1.S1_at 0.00700348 0.647445159 1.566391844 containing 1 trafd1 Xl2.47638.1.S1_at 0.04958536 0.516163353 1.430146909 trans-2,3-enoyl-CoA reductase Tecr transcription factor 25 (basic Xl2.23789.1.S1_at 0.046453849 -0.139027292 0.908131238 helix-loop-helix) tcf25 transcription factor 7 (T-cell Xl2.3962.1.S1_at 0.008475745 -0.27400464 0.827020709 specific, HMG-box) tcf7 transcription factor AP-2 gamma (activating enhancer binding Xl2.14588.1.S1_a_at 0.029285871 -0.952943704 0.516577351 protein 2 gamma) tfap2c Xl2.15574.1.S1_at 0.002913392 -1.228325548 0.426812535 transcription factor CP2-like 1 b tfcp2l1b transcription factor Dp-2 (E2F Xl2.25544.1.A1_at 0.046994384 -0.410466723 0.752379934 dimerization partner 2) tfdp2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Transcription factor IIA large Xl2.28611.2.S1_x_at 0.038662947 0.470221586 1.385322225 subunit-1 TFIIAa/b-1 transcription factor IIA large Xl2.28611.2.S1_a_at 0.049088283 0.566889137 1.481325962 subunit-1 tfiiaa/b-1 transcription factor IIA small Xl2.25790.1.S1_a_at 0.000306877 0.25926143 1.196865827 subunit Tfiiag Xl2.579.1.S1_at 0.040740121 -0.54043035 0.687565781 transcription factor xGATA-5b gata5-b Xl2.3233.1.S1_at 0.038852687 -0.182423876 0.88122121 transducin (beta)-like 3 tbl3 286 transforming growth factor beta

Xl2.8009.1.S1_at 0.033392045 -0.260025675 0.835073058 regulator 4 tbrg4 transforming, acidic coiled-coil Xl2.580.1.S1_a_at 0.037932354 0.43597416 1.352824001 containing protein 3 tacc3 Xl2.25636.1.S1_at 0.033167318 0.546555084 1.460593873 transketolase-like 2 tktl2 Xl2.25636.1.S1_s_at 0.035544919 0.432579562 1.349644607 transketolase-like 2 tktl2 Xl2.53918.1.S1_at 0.010980269 0.042298034 1.02975279 translation initiation factor SUI1 sui1 Xl2.53918.1.S1_x_at 0.048668513 0.053800464 1.037995698 translation initiation factor SUI1 sui1 translocase of inner Xl2.20517.1.S1_at 0.029135445 0.282229236 1.216072495 mitochondrial membrane 13 timm13-b transmembrane 9 superfamily Xl2.16276.1.S1_at 0.043110475 -0.199959727 0.870574865 member 2 tm9sf2 transmembrane 9 superfamily Xl2.23926.1.S1_at 0.032120198 -0.249740435 0.84104772 protein member 4 tm9sf4

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol transmembrane and tetratricopeptide repeat Xl2.48375.1.S1_at 0.033970249 -4.69E-05 0.999967471 containing 2 tmtc2 Xl2.47453.1.S1_at 0.043411096 0.435141436 1.352043376 transmembrane protein 106B tmem106b Xl2.21883.1.S1_at 0.011851813 -0.106044689 0.929131893 transmembrane protein 111 tmem111 Xl2.34728.1.S1_at 0.025702896 0.156947594 1.114925718 transmembrane protein 111 tmem111.2 Xl2.3173.1.S1_at 0.00365128 0.325244847 1.252877052 transmembrane protein 147 tmem147 Xl2.23894.1.S1_at 0.023579805 0.103694664 1.074521734 transmembrane protein 14C tmem14c 287 Xl2.19645.1.S1_at 0.040945822 0.333473247 1.260043244 transmembrane protein 167B tmem167b Xl2.52391.1.S1_s_at 0.030653318 0.412203311 1.330716558 transmembrane protein 18 tmem18 Xl2.16113.1.S1_at 0.033421354 0.123283227 1.089210826 transmembrane protein 30A tmem30a Xl2.16096.1.A1_at 0.042421834 -0.379592699 0.768654566 transmembrane protein 45B tmem45b Xl2.45218.1.S1_at 0.005882717 -0.250014864 0.840887752 transmembrane protein 52 tmem52 Xl2.17394.1.S1_at 0.00628783 -0.397729473 0.759051947 TRH4 protein trh4 trimethylguanosine synthase Xl2.33099.1.S1_at 0.028945449 -0.169321941 0.889260531 homolog tgs1 trimethyllysine hydroxylase, Xl2.54368.1.S1_at 0.004253675 0.32994684 1.256967057 epsilon tmlhe Xl2.9359.1.S1_s_at 0.042429814 -0.27700348 0.825303418 tripartite motif-containing 28 trim28 Xl2.9051.1.S1_at 0.021879733 -0.546005718 0.684913776 tripartite motif-containing 29 trim29 Xl2.18306.1.S1_at 0.043483379 0.561020573 1.475312495 tubulin folding cofactor E Tbce

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol Xl2.53906.1.S1_at 0.030218771 -1.486947249 0.356766671 tubulin folding cofactor E-like tbcel Xl2.52384.2.S1_s_at 0.036065458 -1.213246745 0.431296901 tubulin folding cofactor E-like tbcel tubulin, gamma complex Xl2.48886.1.S1_at 0.037775685 -0.321002742 0.800513289 associated protein 4 tubgcp4 tubulin, gamma complex Xl2.621.1.S1_at 0.015753354 -0.183891964 0.880324934 associated protein 6 tubgcp6 tumor necrosis factor receptor Xl2.35367.1.S1_at 0.023148537 0.883140797 1.844386226 superfamily, member 10b tnfrsf10b 288 Xl2.18435.1.S1_at 0.023563137 0.434146462 1.351111242 tumor protein p63 regulated 1 tprg1

Xl2.56708.1.S1_at 0.020172491 -0.616789127 0.652120679 twist homolog 2 twist2 Xl2.5776.1.S1_at 0.016036668 -0.480367505 0.716795008 TWIST neighbor twistnb Xl2.47599.1.S1_at 0.001863537 1.490131957 2.809146681 tyrosylprotein sulfotransferase 1 tpst1 Ubiquinol-cytochrome C Xl2.8388.1.S1_at 0.019204929 0.397551039 1.317269959 reductase complex uqcrc2 ubiquinol-cytochrome c Xl2.29395.1.S1_at 0.041211199 0.293115359 1.225283303 reductase core protein II uqcrc2 ubiquitin carboxyl-terminal esterase L3 (ubiquitin Xl2.4744.1.S1_at 0.018662233 0.269357514 1.205270956 thiolesterase) uchl3 ubiquitin fusion degradation 1 Xl2.16477.1.S1_at 0.020357327 0.244404257 1.184603503 like ufd1l Xl2.47043.1.S1_at 0.015514918 -0.029364051 0.979852127 ubiquitin protein ligase E3B ube3b

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol ubiquitin specific peptidase 5 Xl2.48731.1.S1_at 0.021779407 1.042029783 2.059122675 (isopeptidase T) usp5 ubiquitin-conjugating enzyme E2 Xl2.12019.1.S1_at 0.016631101 0.097157382 1.06966377 variant 2 ube2v2 ubiquitin-conjugating enzyme Xl2.45490.1.S2_at 0.031776831 0.054487148 1.038489873 E2D 3 (UBC4/5 homolog) ube2d3 ubiquitin-conjugating enzyme Xl2.25541.1.S1_a_at 0.048975089 -0.046470726 0.968302198 E2L 3 ube2l3 289 ubiquitin-fold modifier

Xl2.11450.1.S1_at 0.015520279 0.085733997 1.061227523 conjugating enzyme 1 ufc1 Xl2.34999.1.S1_at 0.001876127 0.266227383 1.202658785 UBX domain protein 2A ubxn2a-b UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase Xl2.3386.1.S1_at 0.021723716 -0.476794232 0.718572569 6 (GalNAc-T6) galnt6 UEV and lactate/malate Xl2.12325.1.S2_at 0.004038407 -0.016008592 0.988965027 dehyrogenase domains uevld unc-5 homolog B /// unc-5-like Xl2.20739.1.S1_at 0.008284987 -0.717837472 0.608008133 protein B unc5b /// unc5b Xl2.122.1.S1_at 0.001934127 -0.280119589 0.82352275 upstream binding factor 1 ubtf-a

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol UTP6, small subunit (SSU) processome component, Xl2.16002.1.S1_at 0.046643822 -0.397731165 0.759051056 homolog utp6 Xl2.42472.1.S1_at 0.039376702 0.818076738 1.763054091 Vac14 homolog vac14 vacuolar protein sorting 37 Xl2.47224.1.S1_at 0.01094163 0.873205945 1.831728834 homolog A vps37a Xl2.5981.1.S1_at 0.043301698 -0.182877337 0.880944272 vang-like 2 (van gogh) vangl2-b Xl2.38014.1.S1_a_at 0.014023004 0.102354753 1.073524229 variant histone H2A.Zl1 LOC397949 290 Xl2.15156.1.S1_at 0.035611955 -0.667000335 0.629814844 villin-like Vill

Xl2.127.1.S1_at 0.02473552 -0.28872379 0.818625896 vimentin Vim v-myb myeloblastosis viral Xl2.23371.1.S1_at 0.038066652 -0.159409593 0.895391425 oncogene homolog-like 2 mybl2 v-myc myelocytomatosis viral oncogene homolog 1, lung Xl2.46111.1.S2_at 0.022471882 -0.131229829 0.913052785 carcinoma derived mycl1-a v-myc myelocytomatosis viral oncogene homolog 1, lung Xl2.179.1.S2_at 0.025041422 -0.394519515 0.760742697 carcinoma derived mycl1-b voltage-dependent anion Xl2.6273.1.S1_at 0.040956781 0.228529964 1.171640496 channel 1 vdac1 voltage-gated potassium Xl2.16370.1.S1_at 0.02685372 0.465792478 1.381075776 channel subunit MiRP4.1 kcne5.1

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol voltage-gated potassium Xl2.16370.1.S2_at 0.037040769 0.399220593 1.318795247 channel subunit MiRP4.1 kcne5.1 v-yes-1 Yamaguchi sarcoma viral Xl2.876.1.S1_at 0.033209327 -0.320415866 0.800838997 related oncogene homolog Lyn Xl2.53449.1.S1_at 0.012446036 -0.058427103 0.960310526 WD repeat domain 34 wdr34 WD repeat domain phosphoinositide-interacting Xl2.10287.1.S1_at 0.040763905 0.287890054 1.220853469 protein 2 wipi2 291 WD repeat domain,

Xl2.48216.1.S1_at 0.014226559 0.219172885 1.164066022 phosphoinositide interacting 1 wipi1 wingless-type MMTV integration Xl2.332.1.S1_at 0.000140628 -1.147469558 0.451416308 site family, member 8B wnt8b wntless homolog Xl2.11487.1.S1_a_at 0.04476032 0.156514203 1.114590841 (Drosophila) wls-a Xl2.16656.1.A1_at 0.04543101 0.537116385 1.451069263 Xpo protein Xpo Zic family member 5 (odd-paired Xl2.1043.1.A1_at 0.043286466 -1.251367996 0.420049718 homolog) zic5 zinc finger and BTB domain Xl2.25378.1.S1_at 0.047373859 -0.352981735 0.782964209 containing 17 zbtb17 zinc finger and BTB domain Xl2.2709.1.S1_at 0.009896617 -0.572132858 0.67262166 containing 2 zbtb2

TABLE A.3 (CONTINUED)

log2 differential Probe Set ID p-value expression Fold Change Gene Title Gene Symbol zinc finger and BTB domain Xl2.47944.1.A1_s_at 0.022856473 -0.40420775 0.755651141 containing 2 zbtb2 zinc finger and BTB domain Xl2.15152.1.S1_at 0.015678916 -0.328024145 0.796626764 containing 44 zbtb44 zinc finger E-box binding Xl2.958.1.S2_at 0.02447558 -0.326540676 0.797446326 homeobox 2 zeb2 zinc finger protein 161 Xl2.52495.1.S1_at 0.008220248 -0.52460219 0.695150765 homolog zfp161 292 Xl2.32184.1.S1_at 0.015994434 0.133711542 1.097112555 zinc finger protein 507 znf507

Xl2.47057.1.S1_at 0.047219182 -0.782950706 0.581176911 zinc finger protein 652-B znf652-b Xl2.7756.1.S1_at 0.025405852 -0.246213853 0.843106126 zinc finger protein 750 znf750 zinc finger protein clone Xl2.23505.1.S1_at 0.012116233 -0.00101572 0.999296204 XLcGF48.2 lcgf48.2-a Xl2.51.1.S1_s_at 0.02472459 -0.432483267 0.740985252 zinc finger protein, X-linked Zfx Xl2.3322.1.S1_at 0.020028848 -0.297364649 0.813737483 zinc fingers and homeoboxes 3 zhx3 zinc metallopeptidase (STE24 Xl2.7419.1.S1_at 0.015501499 0.240310666 1.181247 homolog) zmpste24 Zwilch, kinetochore associated Xl2.47135.1.S1_at 0.044181242 -0.092376328 0.937976493 homolog zwilch

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