Notchless Interacts with Multiple Signaling Pathways During Mouse Peri-Implantation Development Chiao-Ling Lo Purdue University

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Fall 2013 Notchless Interacts with Multiple Signaling Pathways during Mouse Peri-Implantation Development Chiao-Ling Lo Purdue University

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This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance

This is to certify that the thesis/dissertation prepared

By Chiao-Ling Lo

Entitled Notchless Interacts with Multiple Signaling Pathways during Mouse Peri-Implantation Development

Doctor of Philosophy For the degree of

Is approved by the final examining committee:

Amy C. Lossie Chair Ann L. Kirchmaier

Shihuan Kuang

Yuk Fai Leung

To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.

Approved by Major Professor(s): ______Amy C. Lossie ______

Approved by: Christine A. Hrycyna 11/20/2013 Head of the Graduate Program Date i

NOTCHLESS INTERACTS WITH MULTIPLE SIGNALING PATHWAYS DURING

MOUSE PERI-IMPLANTATION DEVELOPMENT

A Dissertation

Submitted to the Faculty

Purdue University

Chiao-Ling Lo

In Partial Fulfillment of the

Requirements for the Degree i

Doctor of Philosophy

December 2013

Purdue University

West Lafayette, Indiana

ACKNOWLEDGEMENTS

I would like to thank my major advisor Dr. Amy Lossie, for her mentoring and all the support, financially and emotionally, throughout the course of my PhD. I could not have completed my thesis without her. Thanks to my committee members, Dr. Ann Kirchmaier, Dr. Shihuan Kuang, and Dr. Yuk Fai Leung for their advice and critical review. I also want to thank Dr. William Muir for teaching me NGS data analysis, and Dr. Joseph Garner for his sophisticated statistical analysis on our projects. I appreciate the help from all my former and present labmates: Jon, Giovana, Katherine, Missy, Jeremy, Feichen, Xiao, Jennifer and Muyi. Moreover, I want to thank my parents, my brothers and all of my friends and colleagues in Taiwan and Purdue for their encouragement and care. They let me know that I am loved and I am not alone. Last but not least, I thank the mice that were sacrificed for this project; truly, without them, I could not have accomplished this research.

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TABLE OF CONTENTS

Page LIST OF TABLES ...... vi LIST OF FIGURES ...... vii ABSTRACT ...... viii CHAPTER 1. INTRODUCTION ...... 1 1.1 Signaling During Peri-implantation Developmet………………………...... 1 1.2 NOTCH Signaling…………………………………………………………...... 5 1.3 Wnt Signaling……………………………………………………………...... 10 1.4 Ribosomal Biogenesis……………………………………………………… . 11 1.5 Generation and Phenotype of l11Jus1 and l11Jus4……………………… 14 1.6 NOTCHLESS in the ribosomal biogenesis pathway……………………... 16 1.7 P53 Regulation……………………………………………………………….. 17 1.8 Objective……………………………………………………………………… 20

CHAPTER 2. IDENTIFICATION OF 129S6/SVEVTAC-SPECIFIC iii

POLYMORPHISMS ON MOUSE CHROMOSOME 11 ...... 22 2.1 Introduction………………………..………………………..………………… 22 2.2 Methods………………………..………………………..…………………… . 25 2.3 Results…………………………………..…………………………………… . 28 2.4 Discussion………………………………………………………………… ..... 46 CHAPTER 3. ENU MUTAGENESIS REVEALS THAT NOTCHLESS HOMOLOG 1 (DROSOPHILA) AFFECTS CDKN1A AND SEVERAL MEMBERS OF THE WNT PATHWAY DURING MURINE PRE-IMPLANTATION DEVELOPMENT ... 50 3.1 Introduction………………………..………………………..………………… 50 3.2 Methods………………………..………………………..…………………… . 54

Page 3.2.1 Mouse Strains, Meiotic Mapping and Generation of Mutants ...... 54 3.2.2 Embryo Analysis ...... 55 3.2.3 Histology ...... 56 3.2.4 Candidate Gene interrogation ...... 56 3.2.5 Notch Pathway Expression of l11Jus1 mutants at E3.5 ...... 56 3.2.6 PCR Array Data Analysis for Gene Expression ...... 60 3.2.7 Caspase 3 Detection ...... 60 3.2.8 Quantitative RT-PCR by Taqman ...... 61 3.2.9 Statistical Methods ...... 62 3.3 Results………………………..………………………..……………………... 63 3.3.1 Phenotypic Analysis of l11Jus1 and l11Jus4 ...... 63 3.3.2 Positional Cloning of l11Jus1 and l11Jus4 ...... 66 3.3.3 The Nle1 locus ...... 68 3.3.4 Expression Analysis of the Notch Pathway in Mutant Embryos ...... 70 3.3.5 Confirmation of Cdkn1a in Different Stages in Pre-Implantation ...... 73 3.3.6 Apoptosis Occurs at E4.5 in Nle1 Mutants ...... 74 3.3.7 qRT-PCR Analysis of Trp53 in Mutant Embryos ...... 76 3.4 Discussion………………………..………………………..………………… . 77 CHAPTER 4. NLE1 MISSENSE MUTATIONS TIGGER TRP53 ACTIVATION iv

AND DISRUPTION IN RIBOSOMAL BIOGENESIS DURING MURINE PRE- IMPLANTATION DEVELOPMENT ...... 81 4.1 Introduction………………………..………………………..………………… 81 4.2 Methods………………………..………………………..…………………… . 86 4.2.1 Immunofluorescence ...... 86 4.2.2 Quantitative RT-PCR ...... 87 4.2.3 Sample Preparation for rRNASeq ...... 88 4.2.4 Data analysis ...... 89 4.3 Results………………………..………………………..……………………... 91 4.3.1 TRP53 is Activated in Mutant Embryos ...... 91

Page 4.3.2 Altered Gene Expression in Cell Cycle and Apoptosis Pathways .... 93 4.3.3 rRNA processing is disrupted in the Nle1 mutants ...... 97 4.4 Discussion………………………..………………………..……………… .. 102 CHAPTER 5. DISCUSSION ...... 105 5.1 NLE1 and Its Potential Biological Function in Ribosomal Biogenesis .. 105 5.2 The rRNASeq study………………………..………………………………. 110 5.3 Mutations in NLE1 Affect Wnt Signaling…………………………………. 112 5.4 Altered Nucleolar Structure and p53 Activation in the Mutant………….114 5.5 Model for Signaling Regulation in Nle1 Mutants…………………………116 LIST OF REFERENCES ...... ……….………. ………………. 119 VITA ……….………. ……………………………………….……….……………...141

LIST OF TABLES

Table ...... Page Table 1.1 Mutant lethality / phenotypes of the NOTCH signaling in mice ...... 9 Table 2.1 Primers used for this study ...... 26 Table 2.2 Polymorphisms in the Nle1 locus ...... 31 Table 2.3 Mettl16 polymorphisms ...... 34 Table 2.4 Psmd11 polymorphisms ...... 36 Table 2.5 Evi2a polymorphisms ...... 36 Table 2.6 Cct6b polymorphisms ...... 37

Table 2.7 Rffl polymorphisms………………………………………………………...39

Table 2.8 Ap2b1 polymorphisms……………………………..………………….…….41 Table 2.9 Summary of polymorphism analysis …………………………………….44 Table 3.1 Primers used for this study ...... 59 Table 3.2 Time of death for Nle1 mutants ...... 65 Table 3.3 X2 analysis of selected timed matings ...... 66 vi

Table 3.4 Logistic multiple regression analysis of caspase 3 detection ...... 76 Table 4.1 Primers used for this study ...... 87 Table 4.2 Logistic multiple regression analysis of active TRP53 detection ...... 93 Table 4.3 Average coverage of each rRNA subunits ...... 98

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LIST OF FIGURES

Figure ...... Page Figure 1.1 Mouse pre-implantation development ...... 2 Figure 1.2 NOTCH signaling pathway ...... 5 Figure 1.3 Ribosomal biogenesis ...... 13 Figure 2.1 Represnetative chromatographs ...... 29 Figure 2.2 Density map of polymorphisms located in the domain of study ...... 45 Figure 3.1 Postional cloning of l11Jus1 and l11Jus4 ...... 58 Figure 3.2 Mutant phenotypes ...... 63 Figure 3.3 NLE1 protein sequence alignment ...... 69 Figure 3.4 Expression of Nle1l11Jus1 embryos using a Notch-specific PCR array 73 Figure 3.5 Caspase 3 detection in Nle1l11Jus1 mutant embryos ...... 75 Figure 4.1 Mammalian rRNA prcessing ...... 89 Figure 4.2 Acety-K378 TRP53 expression in Nle1 embryos ...... 92

Figure 4.3 Altered gene expressions in Nle1 mutants ...... 96 vii

Figure 4.4 RNASeq analysis of rRNA processing ...... 98 Figure 4.5 Immunofluorescent images of nucleolar structure ...... 100 Figure 5.1 Proposed model for the biological function of NLE1 ...... 110 Figure 5.2 Proposed model for the regulatory network in Nle1 mutants ...... 118

viii

ABSTRACT

Lo, Chiao-Ling. PhD, Purdue University, December 2013. Purdue University, Notchless Interacts with Multiple Signaling Pathways during Mouse Peri- Implantation Development. Major Professor: Amy C. Lossie.

During peri-implantation, the embryo transitions from a suspension environment in the fallopian tubes to an adherent system within the uterus. Successful transition requires maternal and fetal signaling cascades that establish maternal- fetal boundaries. Failure is common, as ~ 25% of all human pregnancies terminate during these steps. A large-scale mutation study in mice produced two mutants (l11Jus1 and l11Jus4) that are excellent models of this transition. l11Jus1 and l11Jus4 contain missense mutations in the Notchless homolog 1 (Drosophila) (Nle1) gene. NLE1 is thought to signal via the canonical NOTCH pathway in vertebrates. Although in invertebrates and lower vertebrates, NOTCH

signaling directs cell fate prior to gastrulation, it is dispensable for gastrulation in viii mice. Moreover, in yeast and plants, which lack NOTCH signaling, Nle1 is crucial for ribosome biogenesis. These seemingly contradictory data led me to hypothesize that mutation of Nle1 causes a lethal trauma to the embryo that disrupts multiple signaling pathways during peri-implantation development. I present data that: 1. Refute the presumption that Nle1 functions as a negative regulator of NOTCH during pre-implantation development; 2. Demonstrate that mutations in Nle1 lead to mis-expression of several members of the Wnt pathway; and 3. Show that mutant embryos enter cell cycle arrest; when that fails, they undergo p53-mediated programmed cell death. To understand the trauma(s) that

ix precipitated these lethal cascades, I discovered that Nle1 mutants display delays in ribosomal RNA processing and nucleogenesis. These results uncover novel functions for NLE1 in the ribosomal biogenesis, TRP53 and WNT pathways during mammalian embryonic development.

CHAPTER 1. INTRODUCTION

1.1 Signaling During Peri-implantation Development

During peri-implantation, the embryo transitions from a free-flowing suspension environment (i.e. pre-implantation development) in the oviduct (mice) or fallopian tubes (humans) to an adherent system within the uterus. Pre-implantation development occurs between fertilization and the blastocyst stage. After fertilization, the totipotent zygote undergoes a series of cell divisions as well as morphological changes whereby it reaches the morula and blastocyst stage (Fig. 1.1). In mice, embryos usually become blastocysts by embryonic day 3.5 (E3.5), where E0.5 is 12 hours post coitus. A blastocyst is characterized by the presence of an inner cell mass (ICM) toward one end and a fluid-filled cavity (blastocoel) on the other (Doetschman et al., 1985; Gardner and Johnson, 1972). The inner cell mass and blastocoel are surrounded by the trophectoderm. The ICM differentiates into two cell layers, called the epiblast and primitive endoderm. The 1 epiblast is slated to become the embryo property, while the primitive endoderm forms the yolk sac. The trophectoderm develops into the embryonic portion of the placenta (Tarkowski et al., 2010). The zona pellucida (ZP) surrounds the entire blastocyst, and is produced by the oocyte during maturation to prevent polyspermy and protect the embryo through early development until the blastocyst stage (Bleil et al., 1981).

During the late blastocyst stage, the blastocoel expands, causing hydrostatic pressure on the zona pellucida, which nicks this protective shell. Helped by

2 enzymatic degradation, the embryo gradually egresses from the zona pellucida in a process called hatching (Bergstrom, 1972; Seshagiri et al., 2009). The hatched blastocyst is now ready for implantation. Mouse embryos implant between E4.5 to E5.5. At this time point, the blastocyst attaches itself to the maternal cells and the trophectoderm invades the luminal endothelium of the uterus (Johnson and McConnell, 2004; Red-Horse et al., 2004).

After implantation, the embryo undergoes gastrulation, which is the generation of three germ layers from the epiblast: ectoderm, mesoderm, and endoderm. The ectoderm becomes the epithelium and nervous system. The mesoderm forms somites and notochord that eventually produce muscles, as well as the circulatory and excretory systems. The endoderm develops into all the digestive and respiratory tracks of the body (Keller, 1981; Sweeton et al., 1991; Williams et al.,Figure 2012 ;1 Yamanaka Mouse pre-implantation et al., 2007). development

Hatched Zygote 2-Cell stage 4-Cell stage Morula Blastocyst blastocyst

Zona Polar body ICM Epiblast pellucida Trophectoderm 2 Primitive endoderm

Blastocoel Embryonic Day ! E0.5 E1.5 E2 E2.5-3.5 E3.5 E3.5-E4.5 Days post fertilization

Figure 1.1 Mouse pre-implantation development Early embryonic stages from the zygote (fertilized egg) to a hatched blastocyst are shown. Embryos are surrounding by the zona pellucida until the late blastocyst stage. The polar body is the by-product of the second meiotic division and can be found in the embryo until the 4-cell stage. E0.5 is denoted as 12 hours after fertilization. At E3.5, the embryo shows distinct cell specification of

3 the inner cell mass (ICM) and trophectoderm. The ICM differentiates into the epiblast and primitive endoderm.

Successful transition of the embryo to the uterine environment requires maternal and fetal signal transduction cascades that establish and/or maintain maternal- fetal boundaries. Studies in humans underscore the importance of these events, as 25% of all pregnancies are predicted to end in miscarriage by this transition (Rai and Regan, 2006). According to the American Pregnancy Association (http://americanpregnancy.org/), miscarriage is the spontaneous loss of pregnancy before the fetus is viable, and includes all pregnancy loss before or at 24 weeks of gestation. The numbers are staggering, as 25% of women experience at least one miscarriage in their lives (Stephenson and Kutteh, 2007). Although 15-20% of clinically recognized pregnancies will end in miscarriage, upwards of 50% of all pregnancies are predicted to end in miscarriage (Rai and Regan, 2006).

Although most miscarriage is attributed to sporadic chromosomal deficits, infection, structural abnormalities, and endocrine problems that often cannot be detected or controlled in a timely manner, a significant number of women suffer from recurrent miscarriage, which may have a genetic component (Rai and

Regan, 2006). Recurrent miscarriage is defined as three or more consecutive 3 pregnancy losses and occurs in 1-5% of couples (Jaslow et al., 2010; Stephenson et al., 2002). Several causes can lead to miscarriage. Some of the most common cases during the first trimester, and in particular in recurrent miscarriage, are caused from genetic abnormalities (Nathan, 2007). Peri- implantation is one critical window where genetic, epigenetic, and environmental factors coordinate embryonic and maternal gene regulation. Correct gene expression and timing are critical for establishing maternal-fetal interactions, and failures during this stage often lead to lethality.

The regulation of peri-implantation development involves multiple pathways, such as NOTCH, WNT and ribosomal biogenesis. Although disruption of NOTCH signaling in mice indicates that NOTCH signaling is not necessary for implantation, all NOTCH receptors and their ligands are detected during pre- implantation development. And recent studies that knocked down γ-secretase, which cleaves the intracellular domain of NOTCH so that it can translocate to the nucleus to initiate downstream target genes, indicate that γ-secretase levels are important for blastocyst competency (Chu et al., 2011). This study contradicts gene-targeting reports (Shi et al., 2005), and suggests that γ-secretase levels could be imperative during pre-implantation development. Although gene- targeting studies indicate that the canonical Wnt pathway is not necessary during pre-implantation development (de Vries et al., 2004), non-canonical Wnt signaling is critical for cross-talk between the blastocyst and the uterine environment (de Vries et al., 2004; Mohamed et al., 2004; Xie et al., 2008), and successful implantation requires an activated blastocyst and a receptive uterine niche. While uterine receptivity depends primarily on estrogen and progesterone (Dey et al., 2004), a variety of factors are crucial for blastocyst activation, including the cannabinoid, MAPK and Erb1/2 receptors (Wang and Dey, 2006). Ribosome biogenesis is the process by which a 47S pre-rRNA is systematically cleaved into mature rRNAs that will be incorporated into ribosomal subunits. 4

Mutations in genes involved in ribosome biogenesis have deleterious effects during pre-implantation development (Lerch-Gaggl et al., 2002; Yamamura et al., 2008; Romanova et al., 2006; Zhang et al., 2008; Ding et al., 2010; Gallenberger et al., 2011; Vogt et al., 2012). These studies all show disruption in the nucleolus and increased cell apoptosis, which suggests that nucleolar proteins are important for embryo development.

1.2 NOTCH Signaling

The NOTCH signaling pathway, first identified in Drosophila and conserved across species (Weinmaster et al., 1991), is involved in a wide range of biological processes from cell fate specification to tissue homeostasis (Artavanis-Tsakonas et al., 1999; Bolos et al., 2007). The name NOTCH comes from the appearance of notched wings in mutant fruit flies. The epidermal growth factor-like (EGF) repeats of the ligand’s extracellular domain interact with the EGF repeats 11 and 12 of the NOTCH receptors’ extracellular domain to initiate the signaling. This interaction causes the activation of A Disintegrin And Metalloproteinase (ADAM) 10 or 17 (Figure 1.2). The ADAMs cleave the extracellular domain of NOTCH, which is then cleaved by the S3 cleavage to release the NOTCH intracellular domain (NICD). NICD is the substrate of proteolytic gamma-secretase complex. The C-terminal of NOTCH translocates to the nucleus. The NOTCH intracellular domain does not contact DNA directly, that is, it needs to associate with a transcriptional factor. The well- known NOTCH associated transcriptional factor is RBP-Jk (CSL), which contains the CGTGGGA recognition motif (Reichrath and Reichrath, 2012). The interaction between NICD and RBP-Jk recruits the coactivator Mastermind-Like (MAML) and the histone acetyltransferease p300. Kinase CDK8 later on phosphorylates NICD to become a substrate for ubitiquination degradation (Fryer 5 et al., 2004). The NICD translocates to the nucleus, where it disrupts the repressor complex, leading to the activation of downstream target genes (Schroeter et al., 1998). NOTCH ligands are transmembrane proteins of the DSL (Delta/Serrate/LAG-2 like family). NOTCH 1, 2, 3, and 4 are the four transmembrane receptors on the surface of signal receiving cell that can be activated by one of the five canonical NOTCH ligands (Jagged 1, 2; Delta-like 1,

Dll 3, and 4 in mammals).

Signal sending cell

ligand Cell membrane EGF repeats receptor cleavage by ADAMs cleavage by Υ-Secretase Signal receiving cell NICD

CoR HDAc MAML HAc CoR HDAc

RBPJ RBPJ Repressed Activated nucleus

Cell membrane

Figure 1.2 NOTCH signaling pathway The red protein located on the signal sending cell is the ligand of NOTCH signaling, JAG1, 2 and DLL 1, 3, 5. The NOTCH receptor contains an extracellular domain that interacts with the ligand, a transmembrane domain, and an intracellular domain (NICD). After the binding of the NOTCH ligand on the receptor, NOTCH receptor is cleaved by ADAMs to release the extracellular domain and then follows by the cleavage by Υ-secreatase complex to release NICD. NICD is then translocated to the nucleus, displaces the co-repressors of NOTCH downstream target genes and histone deacetylase, and then recruits coactivator of NOTCH and histone acetyltransferease to activate downstream gene expression.

Despite the simple ligand/receptor activation transduction, NOTCH also elicits another complex regulation that involves a posttranslational modification. Common posttranslational modifications are ubiquitination of the ligand, glycosylation and proteolytic cleavages of receptor, and the inhibiting ubiquitination of the receptor by the E3 ubiquitin ligase Numb (Lai, 2004). The most well known NOTCH target genes are members of the Hairy/Enhancer of Split family (Hes1, Hes5, Hes7) and the Hairy/Enhancer of Split related with YRPW motif family (Hey1, Hey2, Hey7). Hes genes are important for somitogenesis (i.e. mesoderm specification) and generation of the nervous system. The Hey family is critical for the development of the cardiovascular system (Fischer and Gessler, 2007). Gene regulation by NOTCH can be mediated by interactions between NICD and other partners. The most well- characterized regulator is a DNA-binding protein called CSL (CBF1 in human, Su(H) in fruit fly, and LAG1 in C. elegans). CSL is also called RBPJ. CSL can both activate and suppress NOTCH target genes by associating with different binding partners (Persson and Wilson, 2010).

The NOTCH signaling components are detected from the zygote stage throughout gestation (Ahnfelt-Ronne et al., 2012; Cormier et al., 2004b; Del Monte et al., 2007; Yoshikawa et al., 2006). Cormier et al. (Cormier et al., 2004b) 7 examined the expression of genes that were directly or indirectly involved in the NOTCH pathway in the pre-implantation embryo development. Notch1-2, Jag1-2, Dll-3, Rbpj, and Dtx2 are expressed from unfertilized oocytes to late blastocyst stages; Notch 4 and Dll-4 transcripts are detected from two-cell stage to the hatched blastocyst stages. Notch 3, Dll1, and Dtx1 mRNA are found in two-cell embryos and in hatched blastocysts, but are not detectable at the morula stage. These results suggest that the NOTCH signaling pathway may be active during pre-implantation development. Using microarray, Wang et al. also uncovered that other genes of the NOTCH pathway, such as Fringe, Srrt, Psen1 and Dtx1 are expressed in mouse pre-implantation embryos (Wang et al., 2004).

NOTCH signaling is essential in regulating mid-gestation processes that include somitogenesis, lymphopoiesis, and vascular development in a CSL-dependant process (Conlon et al., 1995; Irvine, 1999; Lai, 2004; Palmeirim et al., 1997). However, NOTCH might act in a CSL-independent manner before gastrulation. Several lines of evidence demonstrate that NOTCH signaling is dispensable for gastrulation in mice. (Table 1.1) Single gene and compound knockout studies of the Notch receptors and ligands result in either viable animals or embryonic lethality at mid-gestation (Conlon et al., 1995; Dunwoodie et al., 2002; Hamada et al., 1999; Hrabe de Angelis et al., 1997; Jiang et al., 1998a; Jiang et al., 1998b; Jiang et al., 1998c; Krebs et al., 2004; Krebs et al., 2000; Krebs et al., 2003; McCright et al., 2001; Swiatek et al., 1994). Similarly, deletion of genes that block NOTCH signaling, such as Pofut1 and members of the Υ-secretase complex, leads to embryonic failure after gastrulation and midline formation (Shi et al., 2005b). This suggests that another regulator may play a similar role as CSL to direct the NOTCH regulation during pre- and peri-implantation stage.

Table 1.1 Mutant lethality / phenotypes of the NOTCH signaling in mice

Gene Mutation Phenotype Interaction References Notch 1 KO Lethal Notch 4 (fail to turn Swiatek et al., 1994 ~E11.5 at E9.5) Notch 2 KO Lethal Hamada et al., 1999 ~E11.5 Notch 3 KO Normal None with Notch 1 Krebs et al., 2003 Notch 4 KO Normal Notch 4 (fail to turn Krebs et al., 2003 at E9.5) Delta KO Lethal ~E12 Hrabe de Angelis et Like-1 al., 1997 Delta KO Skeletal Dunwoodie et al., Like-3 Malformatio 2002 ns Delta KO Lethal ~E12 Krebs et al., 2004 Like-4 Jagged 1 KO Lethal Xue et al., 1999 ~E11.5 Nle1 Missense Lethal

1.3 Wnt Signaling Wnt signaling is conserved from Drosophila to human, and it regulates a myriad of processes, including embryogenesis, cell fate specification, cell proliferation, and homeostasis in adult tissues (Logan and Nusse, 2004). Canonical Wnt signaling in mammals, which is mediated by β-catenin, includes 19 Wingless (WNT) ligands, 10 G-protein coupled frizzled receptors (FZD) and two low- density lipoprotein receptor-related protein co-receptors (LRP-5 and 6) (Wodarz and Nusse, 1998). When a WNT ligand binds to the cysteine-rich domain of its cognizant FZD receptor, signaling induces heterodimerization of FZD-LRP and recruits Dishevelled (DSH) to the FZD-LRP dimmer, forming a complex called a signalosome (Bilic et al., 2007). β-catenin accumulates in the cytoplasm. β- catenin translocates into the nucleus where it co-regulates transcription. The non-canonical pathway is poorly characterized and involves diverse receptors and downstream targets. Wnt/planar cell polarity (PCP) and Wnt/Ca2+ pathway are the two well-characterized non-canonical pathways. Binding of the non- canonical WNT ligands to FZD results in DSH activation without the incorporation of LRP as a co-receptor (Sheldahl et al., 2003). Signaling then passes down to different downstream targets depending upon the interacting pathways.

WNT signaling plays important roles in both embryo and the uterus to during 10

implantation. In situ studies of the Frizzled (Fzd) genes indicate that Fzd5, Fzd7, and Fzd10 are detected in pre-gastrulation embryos (Kemp et al., 2005; Kemp et al., 2007), as well as in the uterus during peri-implantation (Hayashi et al., 2007; Hayashi et al., 2009a; Hayashi et al., 2009b; Miller and Sassoon, 1998). At the blastocyst stage, several Wnt genes, such as Wnt5a, Wnt7a, and Wnt11 are expressed at higher levels than in the morula stage (Mohamed et al. 2004). In addition, expression levels of Wnt7a, Wnt7b, Wnt11, and Wnt16 are much higher in the implantations sites of maternal uterus compared to non-implantation sites (Hayashi et al., 2009b). Moreover, in embryos where Wnt3 has been deleted, embryos do not form a primitive streak, and fail prior to gastrulation (Barrow et al.,

2007). However, depletion of both maternal and zygotic β-catenin does not disrupt blastocyst development (de Vries et al., 2004), indicating that canonical Wnt pathway is dispensable during pre-implantation development.

Therefore, the non-canonical signaling may play a role in pre-implantation development. For example, overexpression of Dishevelled proteins, which transduce divergent Wnt pathways, disrupts cell-cell adhesion during pre- implantation (Na et al., 2007). Dishevelled proteins regulate cell polarity and cell adhesion through non-canonical Wnt signaling (Fanto and McNeill, 2004; Moon et al., 1993). These data suggest that non-canonical Wnt pathway(s) may be active during pre-implantation development. Nevertheless, the canonical Wnt pathway becomes critical at implantation. Xie et al. (2008) showed that inactivation of zygotic nuclear Wnt-β-catenin signaling through activating Dkk1, a potent inhibitor of β-catenin, limits the competency of blastocysts to implant (Xie et al., 2008). This cascade initiates in the trophectoderm, which expands during implantation to invade the maternal decidua and form a protective environment for the developing embryo. As a whole, these studies suggest that Wnt signaling undergoes a switch from the non-canonical pathway in the early pre-implantation blastocyst to canonical signaling as the hatched blastocyst creates its protective niche within the uterine wall where it interacts with new signaling molecules in the 11

maternal tissue (Xie et al., 2008).

1.4 Ribosomal Biogenesis During early development, there is an increased demand in protein synthesis (Schultz et al., 1990). To meet the high protein requirements, the embryo must improve its translational capacity by up-regulating ribosomal biogenesis. Ribosomal biogenesis is a key process that governs protein synthesis and requires factors for processing rRNA, assembly, and transporting of ribosomal subunits from nucleolus to cytoplasm (Tschochner and Hurt, 2003). Alterations in this process can cause defects in protein translation and therefore disrupt

12 embryogenesis or lead to detectable disease (Baserga et al., 2010; Fukuda et al., 2008; Gallenberger et al., 2011; Zhang et al., 2008)

Ribosomal biogenesis is a well-coordinated and evolutionarily conserved process that takes place in the nucleolus (Fromont-Racine et al., 2003). During mouse early embryo development, the nucleolus is exclusively maternally inherited. The maternal nucleolus disassembles after pronuclear fusion, and later on, the zygotic nucleolus is assembled when the embryonic genome becomes activated. The oocyte-originated nucleolar components are crucial for serving as the precursor materials that are required to reassemble the functional zygotic nucleoli (Ogushi et al., 2008). When the nucleolus is removed from the oocytes, about 80% of enucleolated oocytes can reach second metaphase (MII) and are fertilized, which suggests that the nucleolus is not required for fertilization. However, the embryos that originate from enucleolated oocytes never progress to the 4-cell stages and show an abnormality of higher chromatin organization in both female and male pronuclei. After the reinjecting the nucleolus in the oocytes, these oocytes are fertilized and develop to full term. While reinjection of the nucleolus at the pronucleus stage can rescue the embryos, it highly reduces the rate of blastocyst formation (Ogushi and Saitou, 2010). This data indicates that the nucleolus of oocytes is critical for the early step of pronuclear organization. 12

Ribosome biogenesis in eukaryotic cells starts with the transcription of the ribosomal DNA (rDNA) into a 47S (45S in yeast) polycistronic precursor by RNA polymerase I in the nucleolus (Fig.1.3). The precursor contains the mature 18S, 5.8S and 28S rRNA interspersed with non-coding internal transcribed sequences ITS1 and ITS2, and is flanked 5’ and 3’ by external transcribed spacers (ETS). Hundreds of r-proteins, non-ribosomal proteins, and small nucleolar RNAs (snoRNAs) associate with the 47S pre-rRNA, forming the 90S pre- ribonucleoprotein particle (90S pre-RNP) (Cisterna and Biggiogera, 2010; Fromont-Racine et al., 2003; Tschochner and Hurt, 2003). After cleaving by the

13 exo- and endonulceolytic ribonucleases within the ITS1, the 90S precursor splits into the pre-40S small subunit and the pre-60S large subunit, which further become mature 40S and 60S subunits, respectively. The 40S subunit consists of the 18S rRNA, while the 60S subunit contains the 5.8S and 28S rRNAs as well as the 5S rRNA that is transcribed independently by RNA polymerase III (Eichler and Craig, 1994; Venema and Tollervey, 1999). The 40S and 60S subunits are then exported from the nucleolus to the cytoplasm, where final maturation takes place.

5’ETS 18S 5.8S 28S 3’ETS Nucleus 47S pre-rRNA ITS1 ITS2

pre-90S subunit 47S

45S Nucleolus

18S 5.8S ITS2 28S 5’ETS ITS1 32S

5.8S ITS2 21S 12S

5S pre-40S subunit 5.8S 28S Cleavage site 18S pre-60S subunit 13

Ribosomal biogenesis proteins

Small nucleolar Ribonucleoproteins Nuclear Cytoplasm 40S pore 60S subunit subunit Ribosomal proteins

Figure 1.3 Ribosomal biogenesis Ribosomal biogenesis occurs in the nucleolus, where the 47S pre-rRNA is transcribed by RNA polymerase I. The 47S pre-rRNA is then being processed by ribosomal proteins, ribonucleoproteins, and ribosomal proteins to generate the mature 28S, 18S, and 5.8S rRNA. The 18S rRNA will be incorporated into the pre-40S ribosomal subunit, while 28S, 5.8S and 5S rRNA, and ribosomal proteins form the pre-60S subunits. The pre-40S and pre-60S subunits transport from nucleolus to cytoplasm and then initiate the final maturation.

Several studies have used gene-targeting techniques to identify the role of nucleolar proteins during early embryogenesis. Most cases have shown defects in embryo development when the protein function was disrupted. For instance, in mouse mutants for proteins involved in ribosomal rRNA synthesis and processing, such as pescadillo-1 (PES-1) (Lerch-Gaggl et al., 2002), RNA polymerase 1 (Yamamura et al., 2008), SURF6 (Romanova et al., 2006), RBM19 (Zhang et al., 2008), EMG1 (Ding et al., 2010), WDR36 (Gallenberger et al., 2011), LIN28 (Vogt et al., 2012) and so on, mutant embryos are arrested at the morula stage. For the Tif1a (a gene mediating the processing of rRNA transcription) mutant (Yuan et al., 2005), and the S6 (Ribosomal protein S6) mutant (Volarevic et al., 2006), phenotypes appear after implantation. In addition, knocking out of nulceophosmin, a nucleolar protein that processes rRNA and segregates chromosome, mutants fail between embryonic day E11.5 and E16.5 (Grisendi et al., 2005). Although these mutants die at different developmental time points, they all show disruption in the nucleolus and increased cell apoptosis, which suggests that nucleolar proteins are important for embryo development.

1.5 Generation and Phenotype of l11Jus1 and l11Jus4 Mouse chromosome (Mmu Chr) 11 shares significant synteny conservation with regions of six different human (Hsa) chromosomes: 22, 7, 2, 5, 1, and 17 (Boles 14

et al., 2009). The largest domain of synteny conservation between mouse and human occurs on distal Mmu Chr11, which is entirely syntenic with Hsa chr 17 (Hentges et al., 2007). The gene-rich domain flanked by Trp53 and Wnt3 in this region of synteny conservation contains 2545 gene structures, including 1597 predicted protein-coding genes, 450 processed RNAs, and 498 pseudogenes (Boles et al., 2009).

15 al., 2003). Functional analysis of 785 total pedigrees from this ENU mutagenesis screen resulted in the discovery of a variety of mutant phenotypes, including infertility, craniofacial abnormalities, neurological defects, and lethality (Kile et al., 2003). Subsequent studies detailed the embryonic lethal phenotypes of 45 mutant lines that fell into 40 complementation groups (Hentges et al., 2006b; Kile et al., 2003). Resequencing efforts led to the identification of causative or putatively causative lesions in 31 genes in 17 lethal lines (Boles et al., 2009).

Although many mutations were identified in the sequencing study, the lesions in the l11Jus1 and l11Jus4 complementation group have yet to be identified. These two mutants survive through implantation but arrest prior to embryonic day (E) 6.5 (Fig. 1.4) (Hentges et al., 2006b; Kile et al., 2003). Since these two mutants fail during this critical window, we undertook a positional cloning strategy to identify the causative mutations in this complementation group. We identified that both mutant alleles have non-conservative missense mutations in the Notchless homolog 1 (Drosophila) gene, Nle1 (Lossie et al., 2012). Moreover, targeted disruption of Nle1 in mice (Cormier et al., 2006a) results in an embryonic lethal phenotype that is remarkably similar to l11Jus1 and l11Jus4, providing further supporting evidence that Nle1 is disrupted in both mutant alleles.

Previous studies have determined that Nle1 is a key regulator in the NOTCH pathway (Royet et al., 1998b). NLE1 was first discovered in Drosophila and belongs to the WD40 repeat family of proteins. It can either reduce or enhance NOTCH activities in a complex manner, depending upon the developmental stage and the species. Studies in Drosophila and Xenopus demonstrate that NLE1 signals via the canonical NOTCH pathway (Cormier et al., 2006a; Royet et al., 1998b). In invertebrates and lower vertebrates, the NOTCH pathway is critical for directing cell fate prior to gastrulation, and also plays important but varied roles in germ layer boundary formation. At the 4-cell stage in C. elegans, NOTCH signaling dictates an ectodermal cell fate in ABp daughter cells by repressing

16 expression of TBX-37 and TBX-38 (Good et al., 2004). In sea urchins, the NOTCH pathway impacts the development and differentiation of the secondary mesenchymal cells, which are fated to produce mesodermal cells (Sherwood and McClay, 1999, 2001). In contrast, in X. laevis, induction of NOTCH signaling leads to an increase in endoderm-specific and a decrease in mesoderm-specific markers, while suppression of NOTCH signaling has the opposite consequence (Contakos et al., 2005).

Figure 1.4 Nle1 mutant phenotype

H&E stained sections of implantation sites from the WT and mutant embryos

1.6 NOTCHLESS in the ribosomal biogenesis pathway 16

For the species that lack NOTCH signaling, such as yeast and plants, NLE1 is integral to ribosomal biogenesis (Chantha et al., 2007b; de la Cruz et al., 2005a; Matton and Chantha, 2007). NLE1 is structurally similar to a protein called WDR12, in that they both contain an Nle domain and WD40 repeats (Nal et al., 2002). In yeast, the WDR12 homolog Ytm1 and the NLE1 homolog Rsa4 both interact with the AAA+-ATPase Rea1 in directing rRNA processing (Bassler et al., 2010; Bottcher et al., 2009). Rea1 is a conserved pre-ribosomal factor that was identified as a component of the pre-60S particle located in the nucleoplasm (Galani et al., 2004; Nissan et al., 2004). Rea1 is required for the formation of 60S and processing of ITS2, being specifically involved in a late step generating

17 the mature 5.8S rRNA from the 7S pre-rRNA (Galani et al., 2004). Electron microscopy showed that Rea1 contains two major domains, an AAA motor domain that binds to the pre-60S particles, and a long flexible tail harboring a metal ion-dependent adhesion site (MIDAS) (Bottcher et al., 2009). The MIDAS of Rea1 directly but sequentially contacts the N-terminal NLE domain of Ytm1 and Rsa4 during 60S subunit biogenesis; therefore, the NLE is also called MIDAS interacting domain (MIDO) (Bassler et al., 2010; Bottcher et al., 2009). A conserved glutamate in the MIDO has been found to be essential for interacting with MIDAS. Disruptions of both yeast NLE1 and WDR12 lead to the blocking of the processing from 35/27S precursor rRNA to the mature 25S rRNA and 5.8S rRNA (in mammal, corresponding to 36/32S pre-rRNA to 28S rRNA and 5.8S rRNA) (Bassler et al., 2010). In addition, mutating the conserved glutamate in this domain in yeast WDR12 causes an early block at the transition of 60S from the nucleolus to the nucleoplasm (Bassler et al., 2010). The glutamate mutation in yeast NLE1 leads to inhibition of exporting the pre-60S particles from the nucleoplasm to the cytoplasm (Bottcher et al., 2009). Because all these proteins are conserved across species, it is possible that in mouse, the Rea1 homolog (MDN1) drives a similar interaction between WDR12 and NLE1 during 60S ribosomal biogenesis.

1.7 P53 Regulation The tumor suppressor gene p53, which governs the regulation of cell proliferation and apoptosis (Levine and Oren, 2009), also plays an important role in the normal development. The function of p53 and the p53 family members (p63 and p73) is mostly determined by cell context, i.e., the cell’s physiological background (Vousden and Lane, 2007). For example, when cells are under stress caused by radiation, hypoxia, metabolite shortages, oncogene dysregulation or viral infections, the p53 clan members, and in particular p53 itself, can be activated to restrain damaged cells from transformating by inducing cell cycle arrest, senescence, apoptosis, and terminal differentiation (Hu et al., 2008; Levine and

Oren, 2009; Vousden and Lane, 2007). In contrast, lack of proper function of p53 leads to cancer progression (Molchadsky et al., 2010).

P63 and p73 belong to the p53 clan family. They play similar but slightly different roles than p53. P63 is usually overproduced in tumor cells, while most of these over-produced p63 are the isoforms that are able to bind DNA, but incapable of transactivation because they do not contain the transactivation domain (Candi et al., 2007). Therefore, p63 is thought to act in a dominant negative way toward transactivation. Like p53, p63 can direct cells to apoptosis in response to DNA damaging (Gressner et al., 2005). Similarly, p73 is a transcriptional factor activated by damaging stress and can induce many of the classical p53 target genes (e.g., p21, Puma, Bax) as well as p53 itself (Wang and El-Deiry, 2006).

Since the p53 response can lead to cell death, it is crucial to maintain appropriate p53 activity levels. One of the major ways in which p53 is regulated is through a rapid protein turnover. In normal cells, p53 is expressed at very low levels with a short half life (Shadfan et al., 2012). The p53 protein is constantly produced and rapidly degraded by ubiquitylation mediated by MDM2, allowing the quick removal or buildup of p53. P53 undergoes various post-translational modifications, including phosphorylation, acetylation, methylation, and 18

ubiquitination (Appella and Anderson, 2001; Carr et al., 2012; Dai and Gu, 2010).

After cellular stress, p53 is activated and stabilized through acetylation and phosphorylation, thereby inducing cell cycle arrest or apoptosis. The reversible acetylation of lysine residues is widely accepted for activating p53 function (Tang et al., 2008b). Many studies have described how DNA damage can induce p53 acetylation either through activating of acetyltransferases or by recruitment of those acetyltransferases by phosphorylating p53 (Jenkins et al., 2012; Meek and Anderson, 2009; Sykes et al., 2006). P300 and CBP are the two highly related histone acetyltransferases that acetylate the core domain as well as six lysines

19 on the C-terminal of p53. These six lysines are K367, K369, K370, K378, K379, and K383 in mouse, equivalent to the human K370, K372, K373, K381, K382, and K386, respectively (Feng et al., 2005; Krummel et al., 2005). The same lysine residues are targeted by MDM2 for ubiquitylation. Since acetylation and ubiquitylation of the same lysine are mutually exclusive, acetylation on p53 leads to blocking of ubiquitylation and thereby suppressing the degradation of p53 by MDM2 (Li et al., 2002). Acetylation of these residues prevents p53 translocation from the nucleus and inhibits subsequent degradation (Tang et al., 2008b). Acetylation also increases the DNA-binding ability of p53, and consequently activates the transcription of its target genes, leading to the induction of cell cycle arrest or apoptosis, depending on the cell types and the nature of cellular stress (Gu and Roeder, 1997; Luo et al., 2004; Wang et al., 2003).

An active p53 can be induced by cellular stress. One of the major types of stress responses that initiates a p53 response is DNA damage (Shadfan et al., 2012). After DNA damage, proteins are recruited to the damage site and activate the p53 pathway that leads to cell cycle arrest in order to repair the damage. If the damage cannot be reversed, the cell will undergo permanent cell cycle arrest or programmed cell death (apoptosis) (Molchadsky et al., 2010). One of the key activators in this p53 activation is Ataxia Telangiectasia Mutated (ATM), which 19

acts on the p53 inhibitor MDM2 (Chun and Gatti, 2004; Maya et al., 2001). ATM kinase directly phosphorylates MDM2 at S395, which changes the activity of MDM2 ligase to prevent p53 degradation and export (Mayo and Donner, 2001). Another kinase, DNA-PK (DNA-activated Protein Kinase), also phosphorylates MDM2 at its p53-binding domain that leads to reduced affinity of p53 and MDM2 (Mayo et al., 1997). Therefore, the amount of stabilized p53 is increased.

Another type of cellular stress that activates p53 is ribosomal stress (Deisenroth and Zhang, 2011). Several studies link p53 activation to the interaction between MDM2 and ribosomal proteins such as L5, L11, L23, L37, S7, S15, and S20

(Daftuar et al., 2013; Dai and Lu, 2004; Jin et al., 2004; Lohrum et al., 2003; Zhang et al., 2003; Zhu et al., 2009). These studies determined that during misregulation of ribosomal biogenesis, ribosomal proteins can activate p53 by binding to and inhibiting MDM2. Moreover, studies have shown that ribosomal stress induces degradation of MDMX (also known as MDM4) (Gilkes et al., 2006). MDM2 and MDMX are binding partners and have been shown to regulate p53 activity as a complex (Joseph et al., 2010). When cells are under ribosomal stress, ribosomal protein L11 promotes MDMX degradation by binding to MDM2 and causes p53 activation (Gilkes and Chen, 2007; Gilkes et al., 2006). 5S rRNA has been found to stabilize MDMX, which may be important to control the activity of MDMX under normal cell conditions (Li and Gu, 2011).

1.8 Objective According to the American Pregnancy Association, miscarriage is the most common type of pregnancy loss, and 15-20% of clinically recognized pregnancies terminate in miscarriage (Rai and Regan, 2006). Given the high number of miscarriage occur in human population, further research in embryonic development is necessary. The long-term goal of my research is to understand how genetic and epigenetic events work together to regulate early mammalian development. This information can be used to develop therapeutic strategies to 20

reduce the incidence of miscarriage in the U.S. The phenotype of the Nle1 mutant mimics early miscarriage. Mutant animals survive through implantation but fail prior to gastrulation. NLE1 is a member of the WD40-repeat protein family, and is thought to signal via the canonical Notch pathway. In invertebrates and lower vertebrates, the Notch pathway directs cell fate prior to gastrulation. However, gene-targeting studies demonstrate that Notch signaling is dispensable for gastrulation in mice. Interestingly, reports in yeast and plants (which lack NOTCH signaling) demonstrate a role for NLE1 in ribosomal biogenesis. In addition, previous studies in our laboratory have demonstrated that Nle1-/- animals do not exhibit a general disruption in the downstream target gene

21 expression, but do show mis-expression of Cdkn1a and several members of the Wnt pathway, suggesting that NLE1 interacts with other signaling pathways prior to gastrulation in mammals. Therefore, the objective of my project is to elucidate the role(s) of Nle1 during early embryonic development. I hypothesize that prior to gastrulation, Nle1 functions in multiple signaling pathways. The rationale is that the investigation of the genetic regulatory network of Nle1 would provide opportunities for developing enhanced diagnostic strategies and potential treatments to detect and prevent miscarriage.

CHAPTER 2. IDENTIFICATION OF 129S6/SVEVTAC-SPECIFIC POLYMORPHISMS ON MOUSE CHROMOSOME 11

The contents in this chapter have been published in DNA Cell Biol. 2012 Mar; 31 (3):402-14

2.1 Introduction The laboratory mouse is the major animal model used for human disease research because of its high relevance to human biology and striking genetic similarities. Understanding the polymorphisms in the laboratory mouse will provide insights into the evolutionary history of the species and increase knowledge of the relationship between genotypic and phenotypic differences (Guenet, 2005). Polymorphisms are genetic variants arise from spontaneous mutations, and by random genetic drift that occur with a population frequency greater than 1%, eventual reach steady-state in a population (Schork et al., 2000). Several types of polymorphisms are found within mammalian genomes, 22 including single nucleotide variants (SNVs), insertions and deletions (Indels), variable number tandem repeats (VNTRs), copy number variants (CNVs) and microsatellite repeats.

Different types of polymorphisms are generally created by different kinds of alteration events. For example, SNVs, the most common types of polymorphisms, result from a single base substitution. The most common SNV in the mammalian genome is a C to T transition, whereby a methylated cytosine is spontaneously deaminated to form a thymine base (Miller et al., 2001). Since this change is not recognized by DNA repair enzymes, the SNV segregates within the population

23 unless it is under selection, is lost or by chance randomly fixates. SNV variation in protein-coding genes and in other functionally constrained regions of the genome, such as promoters, enhancers, miRNAs and other non-coding sequences contributes significantly to phenotypic variation (Bartel, 2009; Morin PA, 2004; Sethupathy et al., 2007). Indels are often, but not always, present in highly repetitive sequences (Chen et al., 2009).They are abundantly distributed across the genome, but not as common as SNVs (Vali et al., 2008). Both of these types of genetic variations can be used as tools in genotyping, as well as discovery of disease- or trait-related genes by directly sequencing SNVs or identifying Indels based on size separation or sequence.

Many mouse polymorphism databases are available, such as the MGI (Mouse Genome Informatics) Strains, SNPs & Polymorphisms database (http://www.informatics.jax.org/strains_SNPs.shtml) (Blake et al., 2011a) and the MPD (Mouse Phenome Database) SNP collection (http://phenome.jax.org/SNP/) (Smith JA, 2010). The MGI database provides reference SNV and Indel information, as well as the corresponding genotyping assays. SNV queries can be based on strain, map position, marker range or associated genes. Currently, there are data from 86 mouse strains, but many holes exist in these databases and more assays are needed to fill in the blanks.

While conducting a positional cloning project to identify induced variants from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we detected several 129S6/SvEvTac polymorphisms in potential candidate genes. This ENU mutagenesis screen was performed on C57BL/6J males, which were bred to 129S6/SvEvTac animals carrying an inversion chromosome for the targeted region (Hentges et al., 2006a; Kile et al., 2003). The mouse reference sequence is based on C57BL/6J, which is one of the most widely used inbred mouse strains for the generation and analysis of transgenic mice and disease models (Al-Hasani et al., 2004; Tang et al., 2008a). 129S6/SvEvTac and 129S1/SvImJ have been used frequently for gene knock out studies (Hibma et al., 2007; Kuo et

24 al., 2010). 129S1/ImJ is one of the 17 additional mouse strains being sequenced by the Sanger Center (Turner et al., 2009). However, there is very little polymorphism data for 129S6/SvEvTac. As a result, it is important to develop SNV and other polymorphism detection assays to help identify 129S6/SvEvTac alleles to facilitate genotyping. Here we provide SNV and insertion/deletion (Indel) information for 7 genes located on chromosome 11 between these three commonly used laboratory mouse strains.

2.2 Methods We isolated genomic DNA from mouse tail biopsies from 129S6/SvEvTac and C57BL/6J animals; 129S1/SvImJ DNA was purchased from the Jackson Laboratory. Exons from each gene were sequenced individually. Each 600-800 bp PCR amplicon contained one or more exons plus upstream and downstream flanking sequence. Exons larger than 800 bp were sequenced by multiple overlapping sequencing reactions. All amplicons were sequenced in both directions using Big Dye v3.1 chemistry (Applied Biosystems, Foster City, CA). Primers are listed in Table 2.1.

We analyzed the data using Sequencher 4.9 (Gene Codes, Ann Arbor, MI) to identify polymorphisms among the various strains used in this study. The sequences from different samples were assembled automatically and then entered into a BLAT search against the UCSC genome database (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start) (Kent, 2002)(NCBI Build 37 (Mm11_95772_37)) to obtain the cDNA and genomic DNA sequences. All sequences (including cDNA and genomic regions) were assembled with our sequencing data into a contig that spanned one individual exon. The contig was scanned for any polymorphisms using features of the Sequencher program. Once a variation was identified, we checked the chromatogram manually to 25

distinguish true polymorphisms from sequencing errors. All SNVs and other polymorphisms were compared with SNV information from the MGI database (dbSNV Build 128) to pinpoint SNVs within the contig and identify unique polymorphisms. Exon or intron positions were determined using the UCSC genome database (http://genome.ucsc.edu/) (Fujita et al., 2011). We compiled all polymorphisms into tables containing gene location, nucleotide number, local sequence, existing SNV ID using the C57BL/6J as the reference strain.

Table 2.1 Primers used for this study T Gene-exon Forward sequence Reverse sequence Size m Mettl16-E1F TTCTCCATCCGCTAACCTAAGAAG TCAACAGATGTCTCCCACCTAGAC 60 624 Mettl16-E2F GGCACTAAATATGCTGGTTTCCTC AATCACACAAAGCACAGTCCTAGC 60 468 Mettl16-E3F GAACGCTGTACCATGGAGCTATAC ACTTCTCAATGATACCTGCAGCC 60 1081 Mettl16-E4F GTGTATCTGCTGTTGTGCTGAATG TTCTTCCTTCCACGCTTCTCTAAC 60 553 Mettl16- CTCTGAGTTATGTGTGAAATGGGC CCCAAATCTCAGGGCAATCTATAC 60 731 E5/6F Mettl16-E7F TCCAACTCTCCTTCATGGTTTATG CTGAACAACTTGTGCTAACACTGC 60 616 Mettl16- GATAAACTTGATGCGTCTCCATTG CTCCGGAAGCCTTAATAACTGAAC 60 504 E8/9F Mettl16-E10F ACCATGGGCTAATCCAAGTTAATG CAGAACTAGGTGGAGGTCTTCGAG 60 490 Mettl16-E11F CTCCCACTGGCTGTGTGTTC CTGAGATGGCTGGTAGTCTGTTTC 60 599 Mettl16-E12F GGAAATTTGAAGAGCTGGGTATTG TTCTAACCTATTTGGGAGGTGGC 60 577 Mettl16-E13F CCTATACTGCAGTTGGAAATACGC ACATGGTATACACACATATACACAGG 60 425 C Mettl16-E14F CTCTACATGCCTTCAGGGTCTATG AAGCTGCTATACCCAAATTTCGAC 60 928 Mettl16-E15F AAAGAGCAACAGGAGTTAGCAGC GGTCTACCGAAGCTTATCACACAA 60 353 Mettl16-E16F TCAATAAAGGCCTGAGATTCCTTC CAACAGCAGTGGGCTTAGTGTTAG 60 552 Evi2aE1F CACTAAAGATGCCATCATGTAATTT TCAAGACCTTCCTCATCCTGATTT 62 626 CC Evi2aE2aF CCCTGACTGTCTCAGCCTTCTAAT TTAACAGGCTGCACTTGAGATAGC 62 866 Evi2aE2bF CCATGTTCTCCTTTCCTTCAAGAT GGTGTTCTCCTCACAGACTTCCTT 62 825 Evi2aE2cF GCCTCAGGTTCCAGTAATCAGAAT ATTCAGACAGAGGTCATTCACTGC 62 845 Evi2aE2dF AGAGAAAGCACGAAGAAGGAACTG GTGTATGCCTAGTTCCCAAGGAAG 62 887 Pmsd11E1F ACGGGGCAGAGAGACTACAACT ACAGTGAGTCAGCAGCACCATAG 62 342 Pmsd11E2F TTCATTTTCTTTTCTTAGTGAAACGT AAAACAGGACTTTCCCTCACCTAT 62 320 G Pmsd11E3F CCATATGCTTGTGTGGAGCTTAAA CATCCTTCCACACACTACCACAGT 62 401 Pmsd11E4F CTATGGACTGTGTGAACAACATGG ACACTCCCATAACTGGAAGCACTA 62 314 Pmsd11E5F TCAGTTGTTGTGTTGAACTTGATGT AGCAGGACTTTACCGTGACAAAT 62 375 Pmsd11E6F GGGCTGGGTATGACAGCTCA GAGGTGAGCAGTAAGAAAGCATGTC 62 400 Pmsd11E7F TAGGAACTGGCTTGTGACTTGTG CTCTGACCACCACACTCTGTATCA 62 450 Pmsd11E8F TTTTGTTTTGCAGCCCAGAAG ATTAAGGGAGGAAAACAGGGATG 62 200 Pmsd11E9F TGATGGTGAGGACAGGCTGTTTAT CCTCGAGCTCAGGGATCTGC 62 311

Pmsd11E10F AGCTTGGTCATGAGAGAGTTTCCT GGCCTCTTACCAGACAGACATACC 62 430 26

Pmsd11E11/ AGGCAGAGCAGAGGACAGTAAGAT CATCTCTCTCCACTCCCTTACACA 62 574 12F Pmsd11E13F TAAATAGAAAGGCGTGCTGTGAAA CCAACTCTGCAAGAGAAGACAGAG 62 376 Pmsd11E14F AGAAGCTGACATAGGTGAGTGCTG CAATTCTGAAGAGGAGGAGAGGTG 62 500 Cct6bE1F TCAGTTCTTCCTGATTCACCACTC CGCACAAGGCTAACAGTTTCTATT 62 545 Cct6bE2F TAAGGTCATCCGGGAGAACTAATC GCACTGCATCTAATACCTGAATCC 62 415 Cct6bE3F GAAGCAAAGACTTTCATACAATGTT ACGGGAGCTTTCTCTTTAACCTTT 62 552 CA Cct6bE4F ACACATTTATGTTAGTGCCGTTGC TGAATTGTAAATGCTTGCCTTGTT 62 689 Cct6bE5F TATATTGGTGTTCCTCAGCTCTGC AGGGAGATCTTCAAGTCAGCATTT 62 587 Cct6bE6F TTTGAGCCGTGTAACTTAGAGAATA ACTCATGCACATACACACTCACAA 62 440 A Cct6bE7F TTACAAATACGAGCCATCATGTCC CTTACAAATGTCCACACAGTGCAA 62 549 Cct6bE8F GACTTTAGACCTCCCTTGCTTCAG CCCTGACTGTCCTAGAACTCACAA 62 552 Cct6bE9F ATAACCAAAGTGAGCATGGCATAA AAACTGTTCTTCATGGAAACACTCAC 62 158 Cct6bE10F TTGAACTGCCTTTGTAACTCAACC GGCAGCCTACCTTATCTCTCCTCT 60 621 Cct6bE11F GAAGAGGGCATTACATCCCATTAC CAAGTCCTTCATTTCCCACAAGTA 62 658 Cct6bE12F TAGGTTGGGAATGGGATTCTTCTA CTTGATGGTAATGGACTGAACCTG 62 469 Cct6bE13F GGAGAAAGGAGCCAATAGAGAAAG TACACAGAGGCTGCTGTTACTCCT 60 574

Table 2.1 Continued. Cct6bE14F GGTGAGGGTGCTTCCTTGATAATA AAGCTGGAATTGACTGGGATACTC 62 535 RfflE1F CCACACCTGTCCCATGAAAGT GGCCCTACTTCCCTCTGAGTCT 62 362 RfflE2F TGGTACTAACAGATTATCACTAACT AGGTGTGTGCTTTAGCTACTCG 62 393 TGC RfflE3F CGATATTTGGAACACTCACTCTGG GGGTCTATGCTGCTGTCTGAGTT 62 529 RfflE4F CTGTGTGTATCATCTCTGGCTGTG ACTGGACTCAAGGACTTCAGGACT 62 412 RfflE5F CTGTGTGTATCATCTCTGGCTGTG ACTGGACTCAAGGACTTCAGGACT 62 412 RfflE6F GTGTGTGGCTGTCTGCATAAAGTA AACTGTGTATTCAATGCCTGTTCC 62 484 RfflE7pcrF CTCCACATGACTTCTCTGAACACA GCAAGGGCAATACCTGACTCTACT 62 332 RfflE8aF TGATCTGAGAGTACCAGACTCAAA GGATCTTAGGACAGGAGAGACTGG 62 663 CC RfflE8bpcrF ACGCATGAACGAATGTCCTATCT GATCCAAACTCTGACCTGAGGAAT 62 586 RfflE8cpcrF CCAGTCTCTCCTGTCCTAAGATCC AGGACCAGAAGACAAAGACAAAGC 62 488 RfflE8dpcrF GGCCAATAGTATTCCTCAGGTCAG TTCTCAGCTGTGTCAGTGGTGTAA 62 569 Ap2b1-E1/2 GGGCTTCTTAACCTTACAAGCAAA ATTTACAGCCTTGCCCAAACTAGA 62 1144 Ap2b1-E3 CCTTGAAGCAAGTTGCAGTTCTTA CCTTAGTTTGTAAGCCCACACTCA 62 570 Ap2b1-E4 TAAAGTAGGGTCTGTGTGGCCTTT TCTCAAGACATTGGTGCTATGTTG 60 500 Ap2b1-E5 GGAAGCCAACACTGTCATCTGTAT ACAGCATGTCCACTCTCTATCAGC 62 527 Ap2b1-E6 AAAGAATGCCATGAGAGATTGGAT TACCCTGTAGTCGTGTCTCCAAGA 62 479 Ap2b1-E7 CTTGGAAGCTGTCTATGCTGTCAT AATACGCTAGGGATAGCAATGTCC 62 513 Ap2b1-E8F CAAGAAGGAAACTGGTGTGAGTGT AGGGCACCAGTCTATAAAGCAATC 62 590 Ap2b1- CAGGCAGAAAGACTGACTTGATTT CTTCTGGCCTACACAGGTAGTTGA 62 810 E9/10F Ap2b1-E11F TAGCAGAGTCAGTGCCTGTCTGA TTCAAAGTGCCTTACTTATACGTTTCC 62 292 Ap2b1-E12F CTTGGAATCCCTTGTGTATGGTTT TAAGCGCTGCAGCTTTGTTATCTA 62 429 Ap2b1- GCATGGTCAATGATTGCTTTGTA CTGCAACAGATCAAGAAGAGGTGT 62 950 E13AF Ap2b1- TTCTCAAGAAGCCATCAGAAACAC AGTCACCCACAGATACCAGATTCA 62 992 E13BF Ap2b1- GCCTCAAAGCAGATTCTGTTCAAT TTGAGATTTCAGGCCTGTGCTA 62 1000 E13CF Ap2b1- ATACTCTGACAAGCGACTTCAGGA TGAGAGCTCAGAACTGGCAAA 62 945 E13DF Ap2b1- CCTTTCATAACAGGTCACAACCAG TGAGAGCTCAGAACTGGCAAA 62 558 E13D2F Ap2b1-E14R CCCTGCCCATATCAATCTATGTTT AAAGAAGCTCTTCAGAGCAGCAAG 62 596 Ap2b1-E15R TCTCCTTCCATGTGTAGATTCTGG CGGCCAACATGATAAACCTATACA 62 564 27

Ap2b1-E16R TTCACTCCCACTGCTCAGTTC AGTCAAGACTGGAGGTTCCATTTC 62 507 Ap2b1-E17R GACAAAGCCTCTAAAGCACGTGTA ACGAGGCCCAAACTCAAATAGTTA 62 540 Ap2b1-E18R AAAGAAGCCAATTGCTCATAGACTT TTTGTCCTGTTCTCCAGAGTCAAG 62 541 T Ap2b1-E19R TAGATTGTGTTGGAGCAGGTCAA CTACCACAATTCTCCGCATGATAC 62 504 Ap2b1-E20R ACCTACAGACTGAGGATCATTGG TGAGATTTGAGGTGGCAGAGTAAG 62 527 Ap2b1-E21R CTCTTAACTGCATGCTTTCTTCCA TAGGGCTGTGCCTTAGAGAGATTT 62 427 Ap2b1-E22R CTGCCTGTATCAGAAGAAGCTCTG GGGAAACCTATAGGGTGGAGACTT 58 1187 Ap2b1- AGTCCTGTGTTGCAATCATGAAAT GAGGCAGGGTCTCAACTATGAAGT 62 287 E22AR Ap2b1- GCCACACAAGCTGTAGGTAGACAT AAGACTGTGTTTGATGAGGGTCAG 60 756 E23AR Ap2b1- TCTTGTGCTGACATTAGCATTCAC ACCAGTCTCAGCTCTGCTACTCAA 62 853 E23BR Ap2b1- GAACACTGAGTAGAAAGGGCGACT CAGTGAGCTGACAGACAGTGAGAA 62 833 E23CR Ap2b1- TGAGTAGCAGAGCTGAGACTGGTT GAACTTGGTCAACTTGTCTTTCCA 62 804 E23DR Ap2b1- TGTGAACACATTGGTGTGAGTCAT CCAGCTATGTATTGTCCTGGGAAC 62 1100 E23ER

2.3 Results We analyzed Nle1, Mettl16, Evi2a, Psmd11, Cct6d, Rffl and Ap2b1 on mouse chromosome 11. Chromatographs for representative SNVs within each locus are shown (Figure 2.1). We only detected differences between C57BL/6J and 129S6/SvEvTac in Mettl16 and Ap2b1. However, we summarized SNV information for sequenced genes.

We identified 21 new polymorphisms and compiled a list of all of the polymorphisms found to date within the Nle1 locus (MGI dbSNV Build 128) (Table 2.2) (Blake et al., 2011b). Nle1 is transcribed from the Crick strand, and variances are ordered in reference to Nle1, not the chromosome. The nucleotide position is noted in column one (NCBI Build 37) and the location within Nle1 in column two. Variances detected within exons are designated as synonymous (S) or non-synonymous (N). Variances reported in the MGI database, but not found in our sequencing studies are depicted by gray cells, and SNV IDs for previously identified changes are noted.

Figure 2.1 Represnetative chromatographs Mettl16 (A), Evi2a (B), Psmd11 (C), Cct6b (D), Rffl (E) and Ap2b1(F). The SNV in each sequence is marked in black box. We detected no polymorphisms among 129S6/SvEvTac, 129S1/SvImJ and C57BL/6J in Evi2a, Psmd11, Cct6b or Rffl.

These sites contain known polymorphisms within different inbred mouse strains. 29

We included known variants in the reference strain (C57BL/6J), as well as those found in the 129S6/SvImJ, C3H/HeJ, NOD and CzechII strains, and the strains that we sequenced (l11Jus1, l11Jus4, C57BL/6J, 129S6/SvEvTac, and C3HeB/FeJ). In total, there are 73 variances across the Nle1 locus, including 5’ and 3’ UTR sequences. We saw no discordance between the C57BL/6J sequences and the reference sequence. We discovered 9 new indels; 129S6/SvEvTac and C3HeB/FeJ had identical sequences across these regions, while C57BL/6J, l11Jus1 and l11Jus4 segregated together. cDNA comparison studies also indicate that the NOD allele (Genbank Accession # AK170853.1)

30 has a single nucleotide deletion in the 3’UTR sequence (Carninci and Hayashizaki, 1999). All polymorphisms identified in the 129S6/SvEvTac and C3HeB/FeJ strains are new. We identified five new expressed SNVs across the gene and one expressed indel in the 3’UTR. One previously reported expressed SNV (rs28209059) is a missense mutation (C 912 G; Table 2.2) in the 129S6/SvImJ and C3J/HeJ strains. This results in a neutral amino acid substitution (N 291 K) (Livingstone and Barton, 1993) within exon 8, which does not encode for any type of functional domain.

Table 2.2 Polymorphisms in the Nle1 locus A A A T T G C CzechII BC018399.1 T T A T A G C GenBankmRNA NOD AK170853.1 A A A A T T A G G G C G C G C T C G C3H /HeJ /HeJ A G A G A A G C C C A A G T C C T G C T C A G 129S1 /SvImJ G A T T C T G G G A A G G G C57BL /6J C57BL MGI dbSNP Build 128 dbSNP MGI SNP ID ID SNP rs28209039 rs28209040 rs28209041 rs28209042 rs29435903 rs29476130 rs29465874 rs29420143 rs28209043 rs28209044 rs29436903 rs29423644 rs29482604 rs28209045 rs28209046 rs29395234 rs28209047 rs28209048 rs28209049 rs29429431 rs28209050 rs28209051 rs28209052 rs28209053 rs28209054 rs28209055 rs29411088 rs28209056 rs28209057 rs28209058 A G A G T G A A A A G A A A G T C T C C T G G G G A /FeJ in10 C3HeB A A T A A A A A A A T T T T A G G G G G C C C G G G G in10 129S6 /SvEvTac Our study Our A T T A A A A T T A A A A G G C G G G G C C C G G G G del10 C57BL /6J C57BL ACATACATACCT 31 TCCCCTCGGGCC

GCAGCGTTAATC [C/T] TATGCTAAGGAC GCTACAGCGCCC TAGGACACTAAT GAGAACCGAGGC ACCAGCTGGACT TGGGGATGTTTC GGCACTACAAAC TAGAACGCCAGG TCCATGTCGCGC CTGAAGGACAGA GGTGACATAAGG CAAGAATCAGGA GCAAGGCACTGG TCTGCTTCGTTC GAATGCCCCTTA TATCGACGATTTTCT GGCACGTCGAAC GAGAGGTGAGGG GGGGATGTTTCC GGGAGAGATTAT CAGTGAGGGTCC ACCCCACGCGGA TGCTGTGGCCAG AGCCTGTGCCCA GTTCTGCGACCT [G/C] [A/G] GenomicSequence [A/C] [G/A] [C/T] [G/A] [G/C] [A/C] [A/T] [G/A] [A/T] [G/A] [A/G] [A/G] [T/G] [G/A] [T/C] [A/G] [A/G] [G/A] [C/T] [G/A] [T/A] [T/G] [::::::::::/ACAGAAATAT] [G/A] [C/G] CGGGCAACCTCC ACGGAGCGTTAA TCCCCACGTGGA TCGGGCCTCGG TGTCAGTCCCCCTCCC CGTGATGTCCAC AGCATCGTGGAC GGCTGGTAGATG CCCTGAGGAGAA ACTCAGTCCAGT AACCGAATCAAC CAGCAGGGTATG CACCTTTATCTC CTGGGGACCAGG CTCATAAAGTAA CCCACAGCAGAA CATTTGCTGTGG CCTGTGATCCAT GAGCAGGGGAGG CCGTTGCTGTCA GCCGTTGCTGTC CAGACTGGCTTC AAGTTTGCAAGC CTTCCGCAGAAG AGGTGACATAAG CCACCATTGGAC ACCTAGCATCAA GGTCACATATAC S S N AA AA L4 N Change I1 I1 I3 I3 I3 I3 I4 I4 I4 I4 I4 I4 I4 I4 I5 I6 I6 I6 I7 I7 I7 I7 I7 I7 I7 I7 I7 I7 US US US E2 E3 E3 E5 E6 E6 E6 Location Mutation AnalysisPerformed Using NCBIBuild 37 (Mm11_95772_37) Mutation 82721166 82718114 82721933 82721908 82721877 82721793 82721776 82721648 82721246 82720449 82720441 82720253 82720130 82719772 82719666 82719658 82719447 82719301 82719207 82719159 82719136 82719059 82718904 82718877 82718839 82718816 82718735 82718697 82718696 82718280 82718241 82718178 82718099 82718033 82717975 82717963 82717928 Nucleotideon Starts 82717908 Starts Chromosome11 Table 2.2 . Polymorphisms 2.2. in theNle1 locus Table

32 A T G G C G G in7 A T A A G C del1 del7 T T A T A T T A T C G C C C C C G T A T T C A T A T T C A T T G C A A C C G C C G G C G A rs3695207 rs3695151 rs3695110 rs3694664 rs3678304 rs28209059 rs28209060 rs28209061 rs28209062 rs28209063 rs28209064 rs28209065 rs29474275 rs28209066 rs28209067 rs28209068 rs28209069 rs29407135 rs29423161 rs28209070 G G C G G T A T A T A A A T G A T T G in6 in3 in2 in7 del2 in12 del2 del1 distinction between C57BL /6J and 129S6 /SvEvTac and/6J 129S6 /SvEvTac distinctionbetween C57BL distinctionbetween 129S6and 129S6/SvImJ /SvEvTac T A T A T A A A T A T T G G C G G G G in6 in3 in2 in7 del2 in12 del2 del1 A T A A A A G C C G C G G C C G C C G G CC AG del6 del3 del2 del7 del12 GCCAGGAAGTGC GAGAGAGAGAAAGAA TCCAAATTCAGT GGGGGGGAAAAAAGAGTAT AGCAAGCAGTGT TGTGTCAGAAGT 32 GTAGGCAGCCTG GGACACAAGCTT TTGAATCCGGGG GTGGTCAGATAC TGGGACGTGGTC CTCACCTTGGCT TCCATGCCAAAC AGGTTGTAGCGG TGGAGTTTCTGT CTTCATGTTGGT ACACAGAGCAAG GCCACGTGGCCA CAGAGCTGGGAC TAGACTTGTCGA CTAGGCTCCTTC TTCACCCAGTGG GTGTTCACCCAG GCCATAGTGTTC GAGAATCTGGCT CCCCACTCACAT [:/G]

[C/T] [C/G] [C/T] [::/GG] [A/T] [G/A] [C/T] [::::::/GAGAGA] [C/T] [G/A] [G/A] [AG/::] [G/A] [A/G] [:::::::/GCTGTTA] [::::::::::::/CAGGAGAACTGG] [::::::::::::/CAGGAGAACTGG] [C/A] [A/G] [A/G] [T/C] [C/:] [A/G] [CC/::] [G/C] [G/T] [:::/CAA] CTCCTTCCCGTA AAGTGCCATAGT GCTAAGTGCCAT GTCTGTGCTAAG CTAGGCTCCTTC CCACCCACTAGG ACACATCCACCC TGCAGGAATGGG ACTCACCCGCAC TCCAGAGCTGGG GATGAGAAGAGT AGGCCCAAGTGA TGAGGCCTGGCA CACCGCGTGGGA AAAGCACACCGC TCCCACAGCTTG AAGCAGTGTCGA CCAGTGCCACAC CCCAGCCAGTGC GGTGAGGGGTCT ATGCCATTTTAC CACAACAGTGGC GGTACCTGGGCC CTGGTACACGGC GCTTCTACCACA TCTACCTCCCAT GGGCAGGTGGGG GGCTTCCCCACT nodata available in current database N S S S S L1 N Bold=new SNP or indel or SNP Bold=new Blue=newinfo129S1/SvImJ for I8 I8 I8 I8 I9 I9 I9 I9 I9 I9 I9 I9 E8 E8 E8 E9 I10 I10 I10 I10 I10 I10 I10 I10 I10 I10 I10 I12 I12 I12 I12 E10 E11 3'UTR 3'UTR E = Exon; I = Intron; US = upstream;downstream;US= DS Intron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change 82717833 82717830 82717824 82717692 82717598 82717566 82717555 82717525 82717192 82717062 82716788 82716727 82716721 82716524 82716448 82716426 82716383 82716315 82715768 82715672 82715566 82715435 82715340 82715048 82714931 82714867 82714745 82714503 Starts 82717698 Starts 82717654 Starts 82716354 Starts 82715378 Starts 82714383 Starts Table 2.2 Continued. Table 82717705-82717706 82716431-82716432

The RefSeq annotated transcript for Methyltransferase like 16 (Mettl16), the most proximal gene in the group, is expressed from the Watson strand and contains 10 exons; 4 additional exons are predicted based on Ensembl annotations. We sequenced all 14 exons and identified 18 variants (Table 2.3). Although C57BL/6J is the mouse reference sequence, the dbSNV database lacks significant C57BL/6J information. Red lettering denotes the variant, grey boxes indicate new dbSNV information and bold lettering indicates novel sequence variants between C57BL/6J and 129S6/SvEvTac; all new variants have been deposited into the dbSNV database.

We identified two new SNVs (10 and 18) and indels (11 and 14) within Mettl16. SNV 10 is a T to G transversion in intron 5, while SNV 18 is a G to C transversion located in intron 8. Indel 11 is a 3 bp insertion located within a string of Ts, and it is therefore impossible to determine the exact insertion site. Indel 14 is a 12 bp deletion in 129S6/SvEvTac. We confirmed the C57BL/6J genotype for polymorphisms 13, 16 and 17, and updated the dbSNV database for 13 additional C57BL/6J and all 16 129S6/SvEvTac alleles. Within the Mettl16 locus, 129S6/SvEvTac and 129S6/SvImJ are concordant for all variants tested with both strains. Notably, 6 SNVs (10, 13, and 15-18) and 2 indels (11 and 14) differ between 129S6/SvEvTac and C57BL/6J. 33

Table 2.3 Mettl16 polymorphisms A T A A G G G C A G T G C G C C ins3 ins12 129S1/SvImJ 129S6 /SvEvTac A A T MGI dbSNP Build 128 dbSNP MGI C57BL/6J SNP ID ID SNP rs26904580 rs26904579 rs26904578 rs26904560 rs26904559 rs26904553 rs26904552 rs26904551 rs26904530 rs26904514 rs26904513 rs29406771 rs29415056 rs26904512 A T A A A T G G G C G G C G C C ins3 ins12 129S6 /SvEvTac distinction between C57BL /6J and 129S6 /SvEvTac and/6J 129S6 /SvEvTac distinctionbetween C57BL Thisstudy A T A A A T T A A A T G G G C G Ref Ref C57BL /6J C57BL Blue=newinfo129S1/SvImJ for TTTATTTATTTA CTATTTAGAATA ATGCTTAGAGAG TTTTTTTTTTAG TAACTGCTTGAT TTCAACAAAAGA ATGGCCTTTCCC TATCAGAGCATG CCGCCGTCTCCA TCCCTTACTGGC TTGACCCAAAAA TTGCTGCTTTTT TGTACCTAGTGG TCCCTAAGCTCT CTTCTGTTCTCA GAACAAAAGGCA ATCCGGTTGCGC /TTT] [A/G] [G/T] [A/G] [T/C] [T/G] [T/G] [A/C] [A/G] [A/G] [C/T] [G/A] [G/C] [G/C] [T/C] [A/G] [A/G] … [ ../TTTATTTATTTA] 34 … [ GenomicSequence

TGTATTTCCCAG GGGTTTTTGTTT CAATATTTGTTT GTCTCCATTTGT TCAAAAATCTCT GCATTCTCAACT TGGCATAGTGCT TATCAGAGCATG TGGGTCCTTGGT AAGTGCTAGCTA GAGTTTCGTCAC ATTGCAGAGATA TTAAGGGCAAAC CTCGCCTAGGCC CTCATGATCAAC GGGTGAAAACTC nodata available in current database CTGAGCTGCTTA I1 I1 I2 I2 I2 I2 I3 I5 I5 I5 I6 I6 I6 I6 I6 I6 I8 5'UTR Location Genome Bold=new SNP or indel or SNP Bold=new 11 within within 74605915 74584265 74584508 74584686 74595996 74596009 74597473 74597505 74597800 74605879 74605904 74616406 74616488 74616975 74617278 74617316 74621916 Mettl16 Polymorphisms Anywhere 74605905~ Nucleotideon Chromosome Start74616809 E = Exon; I = Intron; US = upstream;downstream;US= DSIntron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Poly# Table 2.3 Table

Ecotropic viral integration site 2A (Evi2a) is expressed from the Crick strand and contains two exons (Table 2.4). Table 2.3 is organized the same as Table 2.2, with the following exceptions: amino acid changes are found in column 3 and the remaining column numbers are shifted. We designed 5 amplicons for sequencing Evi2a, as exon 2 spans 2,058 bp. We confirmed the 16 SNVs in the region and did not identify any additional alleles. However, we added genotyping information for C57BL/6J for all SNVs. Three SNVs (4 – 6) are located in exon 2; SNVs 4 and 5 cause missense changes (M), while SNV 6 is a silent variant (S).

Proteasome 26non-ATPase subunit 11 (Psmd11) codes from the Watson strand and contains 14 exons (Table 2.5). Table 2.4 has the same organization as Table 2.3. We designed 13 amplicons for sequencing. Exons 11 and 12 were amplified from one PCR product. We confirmed the 4 SNVs in the region and did not identify any additional alleles. However, we added genotyping information for C57BL/6J for all SNVs. SNV 2 causes a silent variant in exon 3.

Chaperonin containing Tcp1, subunit 6b (zeta) (Cct6b) is expressed from the Crick strand and contains 14 exons (Table 2.6). Table 5 has the same organization as Table 2. We sequenced each exon and confirmed 9 SNVs within this locus. SNV 1 is located downstream to Cct6b, while SNVs 2 and 3 are within 35

the 3’UTR and should be identified from mRNA. Although we did not identify any additional alleles, we added genotyping information for C57BL/6J for 9 SNVs.

Table 2.4 Evi2a polymorphisms Table 2.5 Psmd11 polymorphisms G T T G T C A C A C G T G G T C A T A G 129S1 /SvImJ 129S1 /SvImJ 129S6 /SvEvTac 129S6 /SvEvTac C57BL/6J MGI dbSNP Build 128 dbSNP MGI C57BL/6J MGI dbSNP Build 128 dbSNP MGI SNP ID ID SNP rs28228857 rs28228856 rs28228855 rs28228854 rs28228853 rs28228852 rs28228851 rs28228850 rs28228849 rs28228848 rs28228847 rs28228839 rs28228838 rs28228837 rs28228836 rs28228835 SNP ID ID SNP rs28212768 rs28197167 rs28197166 rs28197159 G T T G T C A C A C G T G G T C 129S6 /SvEvTac A T A G 129S6 Thisstudy G T T G T C A C A C G T G G T C /SvEvTac C57BL /6J C57BL Blue=newinfo129S1/SvImJ for Thisstudy A T A G C57BL /6J C57BL CTCCTTTGCTCT AAATGAGAATGT TCTTGTCTTACG TACATGTCACGG ACGTGGAGTAAT AGCCGCATGTGC TTTTAAAGAGGA ACTAACCTGAAC CTGGCTATGGCT TGCTCCGCGGCT GAGGCCCACACT CAGTGTCTGGCA CCCAGCCGTCAC CTACACATTGGG GCCTTCCTCATT ACAAGTGGCTTT [C/T] [C/T] [T/C] [G/A] [T/C] [G/A] [C/A] [T/C] [C/T] [G/A] [G/A] [T/C] [A/G] [G/A] [T/C] [A/C] TTGTTGTGTTGA CCCGGCTCTTTA GAAAGAAGTGTG CTGGTCCGGTCT GenomicSequence [G/A] [A/G] [T/C] [A/G] TATTTCTAACTC ACATAGTTGAAT TTTAAAAGATAT AATAGAAAAGCA GCAAGTGTGCAT TCTGTTGCTGTA GCCCTTCCTCTC AATCTGCAGGGT AACAGTGTCTGG GCTGCAACTCAA TGGAGCCTGTGA AGAAAGTCACCG TCCAGAAGACGA GTTTGTGGAAGA CCTTCAGAGCTC GGAGGAGTAAGA GenomicSequence 36

TAAAGCAGCTCG GGGCTGAGTTCA CAGCTCCCCGAG AACAGATGGTGT I1 I1 E2 E2 E2 3'UTR 3'UTR 3'UTR E25'UTR E25'UTR E25'UTR E25'UTR E25'UTR E15'UTR E15'UTR E15'UTR I2 I3 I4 E3 Locationbased on Location nodata available in current database nodata available in current database RefSeq:NM_001033711.1 S S AA Change AA M: L194F M: M: N171S M: AA Change AA Polymorphisms Polymorphisms Psmd11 Psmd11 80251677 80251820 80251912 80259283 Evi2a 79340133 79340257 79340568 79340705 79340773 79341225 79341344 79341355 79341781 79341847 79341961 79343815 79343851 79343882 79343973 79344056 Nucleotideon Nucleotideon E = Exon; I = Intron; US = upstream;downstream;US= DS Intron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change Chromosome11 E = Exon; I = Intron; US = upstream;downstream;US= DS Intron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change Chromosome11 1 2 3 4 1 2 3 4 5 6 7 8 9 11 10 12 13 14 15 16 Poly# Poly# Table 2.5 Table Table 2.4. Table

Table 2.6 Cct6b polymorphisms T T T A T C C G C 129S1/SvImJ 129S6 /SvEvTac G MGI dbSNP Build 128 dbSNP MGI /6J C57BL SNP ID SNP rs28225664 rs28225663 rs28225662 rs28225659 rs28225630 rs28225629 rs28225627 rs28225614 rs28225613 T T T T C C G C A 129S6 /SvEvTac Thisstudy T T T A T C C G C C57BL /6J C57BL CACACATTGTTT GGAGGAGCTTGC TCTAACAATATT ACTCACCAATGC GCTGCTGGGTAT ATCAGAGTGAGC TGTCTTCCAGAT GGGAAGCGAGTG AAGCTTGTGCCC [T/C] [T/C] [C/T] [G/A] [C/T] [A/C] [T/C] [T/C] [C/T] 37

GenomicSequence TTGTTGACTGAA TCTTCATGGAAA TGGGATACTCT ACGTATAAAATC GGATCAGCCAGG AAGGCCATGTGA CACACGCACCAC GATCAGCATCAG TCGGACTCCGGC nodata available in current database

I9 I5 I1 DS I12 I10 US 3'UTR 3'UTR Location Genome Polymorphisms 11 Cct6b 82532579 82532804 82532833 82533574 82549799 82550586 82555698 82577547 82577906 Chromosome Nucleotideon E = Exon; I = Intron; US = upstream;downstream;US= DS Intron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change 1 2 3 4 5 6 7 8 9 Poly# Table 2.6. Table

Ring finger and FYVE like domain containing (Rffl) is expressed from the Crick strand, contains 11 exons and produces 5 alternatively spliced products (Table 2.7). Table 2.7 is organized exactly the same as Table 2.3. We designed 13 amplicons for sequence analysis of Rffl; three span exon 11. To reduce complexity, we incorporated all splice variants into a single transcript. RefSeq NM_026097.3 contains exons 5-7 and 9-11. RefSeq NM_001164570.1 contains exons 2, 4, and 6 – 11. RefSeq NM_001007465.3 contains exons 1 and 6 – 11. RefSeq NM_001164569.1 contains exons 1, 2 and 6 – 11. RefSeq NM_001164571.1 contains exons 1, 6, 7 and 9 – 11. We confirmed 10 SNVs for this locus and found no variants between C56BL/6J and 129S6/SvEvTac. SNVs 9 and 10 are within the 5’UTR of exons 4 and 2, respectively. SNV 8 is a missense variant in exon 6, while SNVs 6 and 4 are silent changes in exons 11 and 8, respectively. SNVs 1-3 are located in the 3’UTR of Rffl. SNV 8 causes an Ala to Thr missense change at amino acid positions 90 (NM_001164569.1), 69 (NM_001164570.1) and 55 (NM_026097.3, NM_001007465.3 and NM_001164571.1) depending upon the RefSeq.

Adaptor-related protein complex 2, beta 1 (Ap2b1) is expressed from the Watson strand and contains 22 exons (RefSeq NM_001035854.2) (Table 2.8). Table 2.8 is organized exactly the same as Table 2.3. We designed 30 amplicons; 6 are 38

necessary to span exon 22, 5 are necessary to span exon 12, while exons 8 and 9 are amplified from a single PCR reaction. We identified 80 SNVs and 4 Indels; 19 of the SNVs (28 – 32, 36, 37, 41 – 48, 50, 65, 66 and 84) and all Indels (4, 12, 27 and 80) are novel. SNVs 1-3 are located upstream of the transcriptional start site of the two RefSeq genes (NM_001035854.2 and NM_027915.3), but within the 5’UTR of the predicted Ensembl transcripts (ENSMUST00000018875). Variant 4 (a 6 bp insertion) is located within the 5’UTR of exon 1. The remaining 3 indels (12, 27 and 80) are located in introns 3, 10 and the 3’UTR, respectively. SNV 32 causes a silent change in exon 11. The remaining novel SNVs are located within intron and the 3’UTR. Previously identified SNVs (33 and 56) lead

Table 2.7 Rffl polymorphisms T C G A C A C C C C SvImJ 129S1/ 129S6 /SvEvTac T C57BL/6J MGI dbSNP Build 128 dbSNP MGI SNP ID ID SNP rs6284732 rs28225526 rs28225525 rs28225524 rs28225509 rs28225508 rs28225507 rs28225470 rs28225441 rs28209144 T C G A C A C C C C 129S6 /SvEvTac Our study Our T C G A C A C C C C C57BL /6J C57BL Blue=newinfo129S1/SvImJ for CATTCGTTCATG GTAGTCATAGTC TCAGTTCACACG CAGGTTGCGGTA AGGCAAAGAAAC GAAATGGACCCC GGTACTCTGGCT GGTTTTCTGTCC AAGTGACTCAAC GGGAGGGCTGCA [C/T] [A/G] [C/T] [C/T] [C/T] [C/A] [C/A] [A/G] [T/C] [G/C] GenomicSequence 39

CTTTTCAGCAGC CAATACTAAACA TGCCTCTGTAGA CTCATCCTCAGT CCGGCAGATAGG GGTGCTTCTCCA TCCCAAGAAGAC CCATGGGCCGCT GGGTTGTGCTTG GTGTAAAGCCCC I8 I7 E8 E6 E11 3'UTR 3'UTR 3'UTR Location E25'UTR E45'UTR nodata available in current database S S M: A90T M: AA Change AA Polymorphism 82618486 82618575 82618921 82619561 82624485 82624548 82624681 82631935 82647709 82684226 Rffl Nucleotideon E = Exon; I = Intron; US = upstream;downstream;US= DS Intron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change Chromosome11 1 2 3 4 5 6 7 8 9 10 Poly# Table 2.7 Table

40 to missense variants (F490L and A602T) in exons 11 and 14, respectively. Additional expressed polymorphisms include exons 4, 7, 12, 13, 14, 17 and 20. We identified 5 variants between 129S6/SvEvTac and 129S1/SvImJ (blue boxes, 16, 33, 53, 60 and 82) and 46 differences between 129S6/SvEvTac and C57BL/6J (yellow boxes). Notably, SNV 33 leads to a missense variant between 129S6/SvEvTac and 129S1/SvImJ that could account for differences between these two distinct 129 strains.

The polymorphisms located in these 7 genes between 129S6/SvEvTac and C57BL/6J strain are summarized in Table 2.9. In total, we identified 15 Indels and 181 SNVs in all 7 genes; we are the first to report 44 of these SNVs and all Indels. Regarding our analyses, it becomes clear that the major distinctions between the 129S6/SvEvTac and C57BL/6J strains lie within Ap2b1, Nle1 and Mettl16, while the variations between 129S6/SvEvTac and 129S1/SvImJ lie in Ap2b1 and Nle1. This suggests that the remaining four genes might be more highly conserved among the 129S6/SvEvTac, 129S1/SvImJ and C57BL/6J strains.

We converted the current dbSNV information within this region into a heatmap that represents the polymorphism density across the region (Figure 2.2). Red 40

areas contain more variants than yellow segments. It is clear that the 7 target genes all map to regions that contain high or intermediate numbers of SNVs. Interestingly, there are two blocks with a low SNV density. The first lies between Mettl16 and Evi2a while the second falls between Evi2a and Psmd11. Based on the SNV density, we predict that these two regions contain elements under selective pressure in the two mouse strains or harbor important functional domains.

Table 2.8 Ap2b1 polymorphisms T A T A T A T T T T T T T A T C G C G C T G G C C A G G G C G C G C del6 ins1 del1 129S1 /SvImJ 129S6 /SvEvTac T G T T C C C T T C57BL /6J C57BL MGI dbSNP Build 128 dbSNP MGI SNP ID ID SNP rs6243183 rs28210348 rs28210347 rs28210346 rs28210329 rs28210328 rs28210304 rs28210288 rs28210286 rs28210279 rs29392050 rs28210260 rs28210259 rs28210258 rs29406029 rs29406086 rs28210254 rs28210253 rs28210244 rs28210243 rs28210242 rs29400459 rs28210303 rs28210303 rs28210287 rs28210285 rs28210284 C G T A C T A T G G C A A T T T A T T G G T T G A C T C G C C C G C del6 del1 ins1 129S6 /SvEvTac Our study Our C G T G T A T A G G T T A C C T A T T G G C C A G T T C C C C C G C Ref Ref Ref C57BL /6J C57BL ACCCCGCCCTTG TCCATTTGTTAC GGTTTCTTCGTC TGCTGTCATATT TTGACTTTTCTA CACATATGGGAT AAGGTGAAATGT CTTGTATTCATA TTTTTTTACTTT CAGTACGTCGCC TGCGTTTGCAGT GCTAGGATCTCC GACCTGGCCGTT CTAGTCCATTTG AAGGTAGTTTCC GTCTCCTGAGTG TTGTCGGTGGGT ACACTAGGGATA TATAGACTGGTG TCTCAAAATTTG CTGGAAGGTTTT TACACTAGGGAT GTTCCAAGGCAT TGTTAAATACAG GCTGAGAGCTTA TTCTACTGTTAA TGGACAGTCAGC TGAGGTAGCCGC TTCCTGGAAGGT ATAGACTGGTGC TTTGTGGTCAGT TTGTTCACGGGA ]AGCAGCGGGAGT GCTGCAGACCCC TCGTTGCTTGCT GTCTCAAAAGGG CCGTGCTCTAGA [:/T] [G/A] [T/C] [T/C] [C/T] [A/G] [C/T] [G/A] [T/A] [G/A] [C/:] [T/C] [T/C] [C/A] [A/G] [C/T] [G/A] [T/G] [G/A] [T/G [G/T] [T/C] [A/G] [G/A] [C/T] [C/G] [G/A] [C/T] [A/G] [T/C] [C/A] [T/A] [A/C] [C/T] [C/T] [C/G] [GCCGCC/::::::] GenomicSequence 41 CCTTGTGTATGG CCTTAGTAACTA GGCTTGTAATCA TTTTTACTTTAG GTCTCTTCCAT GTTTGTTTGTCA TAAATAGAGATG ACGTCTTTACAC TTCAGCATATTA TTTCACAAAAAT TCGATTTATCCC TGTAAGCATTCT GTAAGCATTCTG TCAGCATGGAGC CAGATGATTGCT CTTTAGCTTCTA TAGAGAGTGGAC AGATGATTGCTT CAACAGCTTTGT TAAGTTTGTGCC GGAGATGAGATC TTGGCCTTAGTA ACTAGAGAGTTT AGCTTTCCTAGA GGTGTTGGGGAT AACAAGAGCTGA TCATGGTTTCTT GTTGATGGGGTC TCTTGCCCCCCT TGAAAGTGAGCC ATTACTAGAGAG

AAAGCAACTGCG AGGCGACAGACA GGAGCCAGAAGT TGCGCTCCGGTG CGCTGCAGACCC GCCGCCGCCGCC I1 I2 I2 I2 I2 I3 I3 I3 I4 I4 I4 I4 I4 I5 I6 I6 I7 I7 I8 I9 US US US E4 E7 I10 I10 I10 I10 I10 I11 I11 I11 I11 E11 E11 E1UTR RefSeq: NM_001035854.2 Locationbased on S S S AA AA F469L Change Polymorphisms 83115978 83116045 83116118 83116563 83154911 83121650 83121867 83122050 83129752 83130051 83130082 83130094 83135573 83135730 83135731 83135857 83135884 83137966 83138305 83148637 83148754 83149060 83149173 83149174 83149996 83150389 83154700 83154714 83154720 83154754 83154758 83154908 83154957 83154991 83155035 83155044 Ap2b1 Nucleotideon start83116316 Chromosome11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Poly# Table 2.8. Table

42 T T T A T T A A T T A T A T T T T T C C C C C C G G G G C C G C G C G C G G G C C C A T G T T T A A G T C C rs3693555 rs3693644 rs3707795 rs3700640 rs6338647 rs4139523 rs6256784 rs6256883 rs6257414 rs6157128 rs28210238 rs28210233 rs28210226 rs29405321 rs28210225 rs28210224 rs28210193 rs28210192 rs28210191 rs28210189 rs28210188 rs28210187 rs28210181 rs28210179 rs28210178 rs28210093 rs29395887 rs13462751 rs13462753 T T A T T A C C C A T G A G G G T T C C G T C G T C G T C T T G G G A T C C C G C C C C C A T G T T G T A T A G G G T T C C G T A A C C G T T T A A G T C C G C CTCAACCCAGTA TTTAGTTCTGAA GTGGATCTTTTA TTAGTTCTGAAA CTTAGTATTTGT ACATTTCCCAAG TGTCAGAGCCAC CTGGTTGGCCTT TTTATCAGTGGT GTGTACGTTTAT GCCTTTTGCATT TTGTGGCTTCCT TCAATTGTATCT CACATATGGGAT GGACGTGATAGA TTAGGCACTTTG AGAGGTGGCTCA TCTCTGCCGCTC GAACTTAGCCAT TGCTTTGGCTTC AATCTTGGTGTT GATCGGGGTTAT CAGGTGATAGCC TGGTCCAACAGG GAGCTTGTGTCA GTCTCTGGAAAA CAGCCTCAGGTC GCCATAGTGAAG TGTGCCTAAGTC GCAGTGTTTTCT CTGAGTGTAGAG CCATTCACTGGG GTTTCCAATGAC CTTGCTGCTCTG ATACACACCTGT CTGGGGACAGAC GAGCTGCGGATC AAGTGGTCACAG AAAGAGCTGAAG ]TGGCCTGGAGGG [C/A] [G/A] [T/C] [T/A] [C/T] [T/C] [C/T] [G/C] [T/G] [G/A] [A/G] [A/C] [C/T] [A/C] [T/C] [C/T] [T/G] [C/T] [G/A] [C/T] [G/A] [T/G] [T/C] [C/A] [C/G] [G/A] [C/T] [A/G] [G/A] [A/C] [T/C] [G/A] [C/T] [A/T] [T/G] [G/A] [T/C [T/C] [C/T] [A/G] AAGCAGATTCTG AGTAGATTTCTA AATCTTGGTGTT GCCTCTAAAGCA AAATCCTGTAAC GCCAGGCTGATT TCAAATGGCATG CCTTCACACGCG TGTTTCTGACAT AAGCACGTGTAC CTTCTCACATGG AAAGACTTCTTA CACACTGCTCAC ATTTAAGAAAAT CTGGCATCTTGG AGTGACTTGCTG TGCATTCCACAC TTGGATTTTGGC AGCCCTGCTCCT TTTGGTGCTCCA GCCAACACAGGA CCCTGACCTTCG TGCAGCACTGAT GCAGATGGGAGC GAGCATTGACGT ACTCTGATGAGG ACAGGCTTGCTC ATCTGGGGCAGG AAGCTGGCAAGT GAAACACAGGAG TCTCAGACGACT AAAACTAGCCAG TCTGGGGCAGGG AACCAACCTGGA AAAGGGTAGCAT GACTGAGGATCA TCTCAGGGGGAC AGAACCCAGACC ACAAGGCATGGT TGACTGAGGCTG 42

I12 I12 I12 I12 I12 I12 I12 I12 I12 I12 I12 I12 I13 I13 I13 I14 I16 I16 I17 I18 I18 I18 I18 I21 I21 I15 I15 I15 I15 I15 E12 E12 E13 E14 E14 E14 E18 E21 3'UTR 3'UTR S S S S S S S A602T 83211375 83211619 83156122 83156168 83156221 83156391 83156465 83156506 83156567 83156628 83156662 83156694 83156695 83156739 83156788 83160098 83160173 83160451 83160512 83164338 83164488 83164532 83164640 83178192 83178405 83178577 83178974 83178981 83179047 83179406 83181262 83181627 83183224 83183468 83202971 83203016 83203129 83212053 83216679 83217239 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

43 T A A C G G ins1 rs28210091 rs28210090 rs28210089 rs13462752 rs28210088 T A C G G G ins1 distinction between C57BL /6J and 129S6 /SvEvTac and/6J 129S6 /SvEvTac distinctionbetween C57BL distinctionbetween 129S6and 129S6/SvImJ /SvEvTac T A C A G A Ref CTCAGTTTAACC CTCAGCAGGCCC ATTCCTGGAGTC TCAGAGTAGAAA GTGGAAAGACAA ACAGCCTGGCAC TCCCAGCTGCAA [T/G] [:/A] [A/G] [G/A] [A/C] [A/G] [C/T] 43 TAATCCTGTTTC GGAGGGAGATTC CTTGTCGATGCT GGTGGTATGTTG TGGCACTTGTCG AGGCTGAAAAAA TGGGAGAACTGG

3'UTR 3'UTR 3'UTR 3'UTR 3'UTR 3'UTR 3'UTR nodata available in current database Bold=new SNP or indel or SNP Bold=new Blue=newinfo129S1/SvImJ for 83217452 83217576 83217663 83217716 83217876 83217915 83218254 E = Exon; I = Intron; US = upstream;downstream;US= DS Intron; untranslated3' = 3'UTR= Exon;I = E region; untranslated5' 5'UTR = region silent= missenseS variant;= M change 78 79 80 81 82 83 84

Table 2.9 Summary of polymorphism analysis 4 5 0 0 0 0 72 23 44 C57BL/6Jand 129S1 /SvImJ Distinctionbetween 3 0 0 0 0 0 4 5 9 129S6/SvEvTac 129S6/SvEvTac Distinctionbetween and129S1 /SvImJ 2 5 0 0 0 0 91 40 46 C57BL/6J. Distinctionbetween 129S6/SvEvTac and 129S6/SvEvTac 2 0 0 0 0 9 4 15 Indel 1 1 0 0 2 0 5 11 19 UTR SNP 2 3 4 2 15 55 57 138 intron SNP in SNP 0 1 3 8 14 13 12 51 exon SNP in SNP 2 0 0 0 0 23 19 44 SNP Novel 4 9 16 16 10 46 80 181 SNP Identified Express Forward Forward Forward direction Reversed Reversed Reversed Reversed 2 8 10 14 14 13 22 exons Numberof 44 size 4049 7628 47830 43519 45057 66923

102337 Genomic Location 74584365-74632194 79340063-79344111 80242117-80285635 82532752-82577808 82617324-82684246 82900768-82908395 83116199-83218535 11 11 11 11 11 11 11 Chromosome beta 1 Name like16 6b(zeta) subunit11 containing non-ATPase non-ATPase ecotropicviral ringfinger and 1(Drosophila) adaptor-related proteasome26S The Distinction indicates the number of differences in SNPs or indel between 129S6/SvEvTac and C57BL/6J. and 129S6/SvEvTac between indel or SNPs in differences of number the indicates Distinction The 129S1/SvImJ. and 129S6/SvEvTac between indel or SNPs in differences of number the indicates Distinction The 129S1/SvImJ. and C57BL/6J between indel or SNPs in differences of number the indicates Distinction The UTR SNP indicates thenumber SNPswithinof theuntranslated regions UTRSNP methyltransferase FYVE like FYVE domain integrationsite2A proteincomplex 2, chaperoninsubunit Notchlesshomolog 2. 3. 4. 1. Rffl Nle1 Total Gene Evi2a Cct6b Ap2b1 Mettl16 Psmd11 Table 2.9Summerythepolymorphism of analysis Table

73 31

16 Nle1

7 7 4 4

Mettl16 Evi2a Psmd11 Cct6b Rffl Ap2b1

Chr11:74,584,365 Chr11:83,218,535

low high 1Mb Polymorphism concentration

Figure 2.2 Density map of polymorphisms located in the domain of study The heatmap represent current dbSNV information across this region. Red indicates highest amount of SNVs and yellow stands for lowest amount. The 7 genes in this study are marked on the heatmap based on their chromosome locations and gene sizes. Genetic variations including SNVs and Indel identified in this study are shown by black lines. Each black line correlates with the number of polymorphisms within a 10kb domain; longer lines signify increased polymorphism densities.

2.4 Discussion In order to identify the mutations generated from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we detected several 129S6/SvEvTac polymorphisms in the 7 genes discussed in the results. We found no non-synonymous mutations Ap2b1, Cct6b, Suz12, Rffl, Evi2a, and Psmd11. However, we identified a T 1184 G transversion (I 395 S missense mutation) in l11Jus1 heterozygotes in exon 10 of Nle1 (Table 2.2). This non-conservative substitution replaces an aliphatic, hydrophobic amino acid with a polar residue, which likely disrupts functionality (Livingstone and Barton, 1993). Subsequent mutation detection efforts resulted in the identification of a second missense mutation (T 484 C transition; S 162 P missense mutation) in exon 5 of Nle1 for the l11Jus4 allele (Table 2.2). This non- conservative amino acid substitution has a high probability to alter protein function, as serines easily form hydrogen bonds with polar substrates, while prolines are rarely found in active sites (Livingstone and Barton, 1993). In addition, we detected an endogenous C57BL/6J non-synonymous SNP (A 535 G transition; I 179 V) in exon 6 (Table 2.2). This well-documented SNP (rs2820949) leads to a very conservative amino acid substitution (Livingstone and Barton, 1993). Both mutants share the endogenous mutation, indicating that l11Jus1 and l11Jus4 homozygotes harbor two coding changes in Nle1–an ENU-induced allele and an endogenous C57BL/6J missense mutation. 46

The 7 genes in this study span a 6.8 Mb domain that contains a total of 154 genes, 9 miRNAs and 5 snoRNAs. We concentrated our efforts on genes that are expressed during pre-implantation development or in gametes. The Evi2a locus, which is adjacent to Evi2b, resides within intron 33 of the Nf1 gene. Although the function of Evi2a is unknown, the locus demarcates the boundary between the two conserved regions, as it contains a high number of polymorphisms in a relatively small genomic region. We identified 16 genetic variants within the 4kb Evi2a locus, while the average number of variants identified in this study is 6 within 10kb. Although C57BL/6J, 129S6/SvEvTac and

129S1/SvImJ are concordant for all variants of Evi2a, this gene could be crucial for the divergence of other mouse strains. Alternatively, polymorphisms within this intron could be important for Nf1 gene regulation. Consistent with this hypothesis, EVI2A is upregulated in malignant peripheral nerve sheath tumors in Neurofibramatosis patients harboring microdeletions within the NF1 locus (Pasmant et al., 2011). The dbSNV database indicates that high levels of allelic variants are present in the distal end of the 6.8Mb domain that overlaps with Cct6b, Rffl and Ap2b1. This is consistent with our results, as we sequenced 84 variants within this locus.

The 129 strain is commonly used for knockout and other genetic manipulation studies because of the efficiency of obtaining ES cells that colonize to the germline. (te Riele et al., 1992). This strain originated in 1928 and has diverged into more than 15 substrains. Different breeding strategies used in the past resulted in phenotypic and genetic diversity among these substrains (Simpson et al., 1997). In this report, we sequenced the 129S6/SvEvTac strain that has been maintained since 1992 at Taconic. According to Simpson et al., (1997), the origin of 129S6/SvEvTac can be traced back to the breeding of the “Steel” substrains, which resulted from the outcross of the 129/Sv strain to C3HeB/FeJ followed by 12-14 generations of backcrossing to the parental 129/Sv line. The resulting mice, 47

termed 129/SvEv, were distributed to Martin Evans and were maintained by selection for the SteelJ allele. The 129/SvEv strains underwent further genetic manipulations to produce 129/SvEvBrd and 129/SvEv-Gpilc. Taconic crossed 129/SvEvBrd and 129/SvEv-Gpilc to generate 129S6/SvEvTac. This line has been bred as a separate inbred line at Taconic since 1992.

129S1/SvImJ was developed as a control-inbred strain for many of the Steel- derived strains. (Petkov et al., 2004) Therefore, we included this strain as reference to compare the genetic variation between 129S1/SvImJ and 129S6/SvEvTac. Ap2b1, which has 84 polymorphisms across the locus, is the

48 only gene in this study to contain variants between the 129S1/SvImJ and 129S6/SvEvTac inbred strains. There are 46 variants between C57BL/6J and 129S6/SvEvTac, 44 alterations between C57BL/6J and 129S1/SvImJ and 5 SNVs between 129S6/SvEvTac and 129S1/SvImJ across the gene. Polymorphism #33 (rs28210244) is a C1571G transversion in exon 11 that causes an F469L amino acid substitution. The remaining variants between the two 129 strains lie within introns or in the 3’UTR.

Ap2b1 encodes one of the two large chain components of the clathrin assembly protein complex 2. Ap2b1 protein is found on the intracellular domain of transmembrane coated vesicles and is important for protein transport (Schmid et al., 2006). It is unclear why Nle1 and Ap2b1 contains so many polymorphisms and why there are differences between 129S6/SvEvTac and 129S1/SvImJ. It is possible that the genetic variations lead to alterations in gene regulation and/or protein structure changes that respond to strain-specific factors. However, it is equally likely that the high number of polymorphisms between 129S6/SvEvTac and 129S1/SvImJ is simply due to random silent mutations. Most of the variances within the Ap2b1 locus (82/84) lead to silent changes or are located within non-protein coding regions of the gene (i.e. introns or UTR), which is consistent with the latter explanation. 48

Although there are many variants between C57BL/6J and the 129-derived lines in this region of MMU 11, all of the polymorphisms between 129S6/SvEvTac and 129S1/SvImJ are located within a single gene. The domain proximal to Ap2b1 contains no variants between the two 129 strains. Regions of conservation suggest sequences critical for function, i.e. gene variation is not functionally tolerated. Phylogenetic studies of 9 different 129 strains indicates that the 129S6/SvEvTac and 129S1/SvImJ strains are highly related (Threadgill et al., 1997), Both were derived from a common ancestor bearing the Steel allele of the Kit ligand, although 129S1/SvImJ has been maintained at The Jackson

Laboratories and 129S6/SvEvTac is maintained by Taconic. Since 6 of the commonly used mouse embryonic stem (ES) cell lines have been derived from these two 129 strains, it is important to document the variants between these two 129 strains for future gene targeting and genotyping studies.

CHAPTER 3. ENU MUTAGENESIS REVEALS THAT NOTCHLESS HOMOLOG 1 (DROSOPHILA) AFFECTS CDKN1A AND SEVERAL MEMBERS OF THE WNT PATHWAY DURING MURINE PRE-IMPLANTATION DEVELOPMENT

The contents in this chapter have been published in BMC Genetics 2012, 13:106.

3.1 Introduction

Mouse chromosome (Mmu Chr) 11 shares significant synteny conservation with regions of six different human (Hsa) chromosomes: 22, 7, 2, 5, 1 and 17 (Boles et al., 2009). The largest domain of synteny conservation between mouse and human occurs on distal Mmu 11, which is entirely syntenic with Hsa chr 17 (Hentges et al., 2007). The gene-rich domain flanked by Trp53 and Wnt3 in this region of synteny conservation contains 2545 gene structures, including 1597 predicted protein-coding genes, 450 processed RNAs and 498 pseudogenes (Boles et al., 2009).

A large-scale, phenotype-driven ENU (N-ethyl-N-nitrosourea) mutagenesis screen targeted to this 34 Mb region of Mmu 11 demonstrated the wide functional diversity of this linkage group (Hentges et al., 2006b; Hentges et al., 2007; Kile et al., 2003). Functional analysis of 785 total pedigrees from this ENU mutagenesis screen resulted in the discovery of a variety of mutant phenotypes, including infertility, craniofacial abnormalities, neurological defects and lethality (Kile et al., 2003). Subsequent studies detailed the embryonic lethal phenotypes of 45 mutant lines that fell into 40 complementation groups (Hentges et al., 2006b; Kile et al., 2003). Resequencing efforts led to the identification of causative or putatively causative lesions in 31 genes in 17 lethal lines (Boles et al., 2009).

Although many mutations were identified in the sequencing study, the lesions in the l11Jus1 and l11Jus4 complementation group have yet to be identified. These two alleles survive through implantation but arrest prior to embryonic day (E) 6.5 (Hentges et al., 2006b; Kile et al., 2003). Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Since these two mutants fail during this critical window, we undertook a positional cloning strategy to identify the causative mutations in this complementation group. Here, we present evidence that both mutant alleles have non-conservative missense mutations in the Notchless homolog 1 (Drosophila) gene, Nle1. Moreover, targeted disruption of Nle1 in mice (Cormier et al., 2006b) results in an embryonic lethal phenotype that is remarkably similar to l11Jus1 and l11Jus4, providing further supporting evidence that Nle1 is disrupted in both mutant alleles.

NLE1, which is a member of the WD40 repeat protein family, was first identified as a suppressor of the notchoid phenotype in Drosophila (Royet et al., 1998a), and has been implicated in both positive and negative regulation of NOTCH signaling, depending upon developmental stage and species (Cormier et al., 2006b; Royet et al., 1998a). Studies in Drosophila and Xenopus demonstrate that NLE1 signals via the canonical NOTCH pathway (Cormier et al., 2006b;

Royet et al., 1998a). In invertebrates and lower vertebrates, the NOTCH pathway 51 is critical for directing cell fate prior to gastrulation, and also plays important, but varied roles in germ layer boundary formation. At the 4-cell stage in C. elegans, NOTCH signaling dictates an ectodermal cell fate in ABp daughter cells by repressing expression of TBX-37 and TBX-38 (Good et al., 2004). In sea urchins, the NOTCH pathway impacts the development and differentiation of the secondary mesenchymal cells, which are fated to produce mesodermal cells (Sherwood and McClay, 1999, 2001). In contrast, in X. laevis, induction of NOTCH signaling leads to an increase in endoderm-specific and a decrease in mesoderm-specific markers, while suppression of NOTCH signaling has the opposite consequence (Contakos et al., 2005).

The role of NOTCH signaling during the earliest stages of mammalian development is much less clear. Several lines of evidence demonstrate that NOTCH signaling is dispensable for gastrulation in mice. Single gene and compound knockout studies of the Notch receptors and ligands results in either viable animals or embryonic lethality at mid-gestation (Conlon et al., 1995; Dunwoodie et al., 2002; Hamada et al., 1999; Hrabe de Angelis et al., 1997; Jiang et al., 1998a; Jiang et al., 1998c; Krebs et al., 2004; Krebs et al., 2000; Krebs et al., 2003; McCright et al., 2001; Swiatek et al., 1994). Similarly, deletion of genes that block NOTCH signaling, such as Pofut1 and members of the γ- secretase complex, leads to embryonic failure after gastrulation and midline formation. POFUT1 adds O-fucose molecules to NOTCH receptors prior to their translocation to the cell surface, while Presenilin 1 and 2 are members of the γ- secretase complex (Kopan and Ilagan, 2009; Schwanbeck et al., 2011). This complex cleaves NOTCH at the cell membrane, releasing the NOTCH intracellular domain (NICD) into the cytoplasm. The NICD translocates to the nucleus and binds to RBPJ, thereby modulating transcription of downstream target genes.

Deletion of Pofut1, which effectively blocks NOTCH signaling through inhibition of post-translational modifications to NOTCH receptors (Shi and Stanley, 2006), 52 leads to embryonic lethality at E9.5 (Shi et al., 2005a; Shi and Stanley, 2003). Targeted disruption of Presenilin 2 in a Presenilin 1 null background leads to embryonic lethality at E9.5. Compound mutants exhibit cardiac, somite and neurological phenotypes (Donoviel et al., 1999). Finally, deletion of the co- repressor, Rbpj, causes somitogenesis defects, placental abnormalities and marked growth delay (Oka et al., 1995; Souilhol et al., 2006). These studies demonstrate that unlike lower vertebrates and invertebrates, and despite the fact that Notch receptors and ligands are expressed prior to and during gastrulation (Cormier et al., 2004a), NOTCH signaling is dispensable prior to gastrulation in mice.

Since Nle1l11Jus1 and Nle1l11Jus4 mutants have more severe phenotypes than mutations that disrupt NOTCH signaling in mice, we hypothesized that NLE1 interacts with NOTCH and other signaling pathways during pre-implantation development. To address this hypothesis, we conducted targeted gene expression studies in homozygous mutant embryos. Surprisingly, and in contrast to studies in Xenopus and Drosophila, our data indicate that canonical NOTCH signaling is not disrupted in Nle1 mutant embryos; instead, we discovered that Cdkn1a was upregulated, while several members of the Wnt cascade were downregulated in homozygous mutant embryos. These results highlight the differences in NOTCH signaling between mammals (where canonical NOTCH signaling is dispensable for gastrulation) and other species (where NOTCH signaling is required for gastrulation) and indicate that NLE1 could play divergent roles in development that depend upon other signal transduction cascades.

3.2 Methods 3.2.1 Mouse Strains, Meiotic Mapping and Generation of Mutants The l11Jus1 and l11Jus4 mutants were induced by ENU mutagenesis on a C57BL/6J background (Hentges et al., 2006b; Kile et al., 2003), and maintained in trans using a balancer chromosome (In(11Trp53;11Wnt3)8Brd) harboring a 35 Mb inversion between Trp53 and Wnt3 that expresses the agouti protein under the Keratin 14 promoter (Zheng et al., 1999). Thee inversion animals were on a 129S6/SvEvTac background. Animals carrying one copy of the balancer chromosome have light ears and tails due to ectopic agouti expression that reduces pigment (Hentges and Justice, 2004; Hentges et al., 2006b; Kile et al., 2003; Zheng et al., 1999). The l11Jus1 line has been continually maintained in our colony. The l11Jus4 line was resuscitated from cryopreserved spermatozoa of an l11Jus4/In(11Trp53;11Wnt3)8Brd male with a C3H/HeJ female (www.MMRRC.org, MMRRC:000074-UCD). Pups were genotyped at weaning and l11Jus4/C3H/HeJ males were backcrossed to In(11Trp53;11Wnt3)8Brd/Rex females on a 129S6/SvEvTAC genetic background. We genotyped the progeny of both lines at least every 10 generations by microsatellite analysis or direct sequencing of the mutations to ensure that we maintained the mutations.

To generate recombinant animals for meiotic mapping it is necessary to remove 54

the balancer chromosome. Animals heterozygous for the l11Jus1 mutation (l11Jus1/In(11Trp53;11Wnt3)8Brd) were mated to animals carrying one copy of the inversion and the dominant curly coat marker, Rex (In(11Trp53;11Wnt3)8Brd/Rex). We selected animals with a curly coat (i.e. inherited the Rex allele) and dark ears and tail (i.e. inherited the l11Jus1 mutation) for meiotic mapping. We intercrossed F1 animals to generate recombinant F2 animals, which were genotyped at several microsatellite markers (D11Mit4, 219, 245, 120, 39, 327, and 32) and single nucleotide polymorphisms (SNPs; Slc6a, Wsb1, Rad51l3 and Rasl10) along the 35Mb interval. Primers and PCR conditions are available upon request.

For the Notch PCR array studies, qRT-PCR analysis and Caspase 3 detection, we outcrossed heterozygous males to 129S6/SvEvTac females (Taconic, Hudson, New York) to eliminate genetic interactions with Wnt3. Heterozygotes, which had light ears and tails (Zheng et al., 1999) were mated, generating F2 blastocysts for analysis. Notch PCR array studies were conducted on N5F2 l11Jus1 embryos (Nle1 ). qRT-PCR studies were performed on N14F2 and N15F2 l11Jus1 l11Jus4 (Nle1 ) and N4F2 (Nle1 ) embryos dissected at E3.5. Caspase detection l11Jus1 assays were carried out on N15F2 (Nle1 ), as well as N14F2 or N15F2 (Nle1l11Jus4) embryos. All mouse studies were conducted in facilities approved by the American Association for the Accreditation of Laboratory Animals with the approval of the Baylor College of Medicine Animal Care and Use Committee or the Purdue University Animal Care and Use Committee.

3.2.2 Embryo Analysis To determine time of death and perform phenotypic studies, we examined embryos after timed matings, with the day of the vaginal plug designated E0.5. We genotyped each one as described (Hentges et al., 2006b; Kile et al., 2003). DNA was isolated by incubating whole embryos (E6.5 to E9.5) in 1 X PCR buffer (Invitrogen, Carlsbad, CA) and 0.08 mg/ml Proteinase K (Invitrogen, Carlsbad, CA) at 55ºC for 2-3 hours. Proteinase K was inactivated by either heating to 95ºC 55

for 10 min or by phenol:chloroform:Isoamyl alcohol extraction followed by ethanol precipitation. Alternatively, embryos were incubated in 25 to 50 µl of 25 mM NaOH, 0.2 mM EDTA for 60 min. at 95ºC. Genomic DNA was neutralized by the addition of an equal amount of 40 mM Tris-HCl, pH 8.0 and stored at -20ºC. D11Mit327 was used to genotype the embryos in a 25 µl PCR reaction under the following conditions: 1 X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 250 pmoles of each primer and 0.625 U of Taq Polymerase (Invitrogen, Carlsbad, CA). After an initial denaturing step at 95°C for 5 min, D11Mit327 was amplified with the following cycling parameters: 30 cycles of 94°C for 30 s, 60°C for 30 s min followed by 72°C for 30 s, with a final 5 min incubation at 72°C. Products

56 were size fractionated on 5% Metaphor (Cambrex, Bio Science, Rockland, ME), 0.5 X TBE gels.

3.2.3 Histology Deciduas were dissected at E6.5–E8.5. Implantation sites were fixed for 3 hours in Bouin’s fixative, embedded in paraffin, sectioned in 5–7 µm slices and stained in hematoxylin and eosin as described (Noveroske et al., 2002). Stained sections were analyzed under light microscopy.

3.2.4 Candidate Gene interrogation Exons of candidate genes were bidirectionally sequenced directly from PCR amplicons using the Big Dye® Teminator v3.1 (Applied Biosystems, Foster City, CA) sequencing mix. Each amplicon contained at least one exon, plus ≥ 200 bp of flanking sequence. For the l11Jus1 mutation, genomic DNAs from 129S6/SvEvTAC, C57BL/6J and l11Jus1/In(11Trp53;11Wnt3)8Brd mice were sequenced as controls. For the l11Jus4 mutation, genomic DNAs from 129S6/SvEvTAC, C57BL/6J, In(11Trp53;11Wnt3)8Brd/C3H, and l11Jus4/C3H, and l11Jus4/In(11Trp53;11Wnt3)8Brd mice were sequenced as controls. Sequence data was analyzed (Sequencher; Gene Codes, Ann Arbor, MI) to identify mutations on the l11Jus1 and l11Jus4 alleles, as well as any additional 56 sequence variants.

3.2.5 Notch Pathway Expression of l11Jus1 mutants at E3.5 We analyzed Notch pathway gene expression in homozygous mutant and homozygous wild-type blastocysts using the SAB PCR Arrays (SABiosciences/Qiagen, Frederick, MD). Embryos were washed in EmbryoMax© M2 media (M2; EMD Milllipore, Billerica, MA) and transferred into 100 µl of RNAqueous lysis buffer (RNAqueous-Micro Kit, Applied Biosystems/Ambion, Austin, TX). After vortexing, we snap-froze each tube in liquid nitrogen and stored each sample at –80°. Total RNA was isolated following manufacturer’s

57 instructions, eluting with 20 µl of nuclease-free water. We used 5 µl of RNA to generate cDNA for genotyping in a half reaction of SuperScript One-Step RT- PCR with Platinum Taq (Invitrogen, Carlsbad, CA) with gene specific primers, oligo dT, and PCR primers (Table 3.1). We used nested PCR for sequencing (Figure 1E). We collected 45 mutant and 45 wild-type E3.5 embryos, and split embryos with the same genotype into 3 pools (i.e. biological replicates); each pool consisted of morulae, half-blastocysts, full blastocysts and hatched blastocysts. We then performed a linear amplification step on each pool using the RT2 Nano PreAMP cDNA synthesis kit (SABioscience, Frederick, MD). Each biological replicate was subdivided into 3 technical replicates. Data from each PCR plate were analyzed using an iCycler Real Time PCR detection system (Bio-Rad, Hercules, CA).

Lethal 1! +! Inversion! L4 Mutation L1 Mutation A. X! D. T484C T1184G B6 Variant Inversion! +! Rex! L4/C3H L1/L1 A535G

C3H/129 L1/Inv Light ears, non-curly! Light ears, curly! 129S6/SvEvTac Lethal 1! +! Lethal 1! +! 129S6/SvEvTac X! Rex! +! Rex! C57BL/6J C57BL/6J

2 3 4 5 6 7 8 9 10 11 12 13 Dark ears, curly! Dark ears, curly!

500bp Lethal 1! Rex! Lethal 1! Rex! +! +! +! +!

X L4!B6! L1! Lethal 1! + +! Rex! +! Rex! Lethal 1! +! E. Lethal Dark ears, curly Dark ears, curly Dark ears, non-curly 189! 487!538! 876/882! 1005! 1187! 1245! 100 bp!

F1! F2! R2! R1! GSP! Gene Speciﬁc RT Primer! B. Gene/Locus Forward & Reverse RT-PCR Primers! D11Mit245/Slc6a Forward & Reverse Internal Primers (R2 is the sequencing primer)! Wsb1 Mutation Sites! Single Nucleotide Polymorphism Site! Rad51l3 Rasl10b D11Mit120 F. L4/L4! B6! L1/L1! D11Mit39 S165P! I182V! I398V! Total: 487 139 284 0 20 23 5 12 2 2 L1/L1! L4/L4! Genotype 129S6/SvEvTac/129S6/SvEvTac C57BL/6J! 129S6/SvEvTac/C57BL/6J 129S6/SvEvTac! CzechII! C57BL/6J/C57BL/6J R.norvegicus! Not Genotyped H.sapiens! B.Taurus!

C. Slc6a Rad51l3 Mit120 Mit4 Trp53 Wsb1 Rasl10b Mit35 Mit39 Mit327 Wnt3 2 3 4 5 6 7 8 9 10 11 12 ! Missense Mutations! WD-40 Repeat-like Domain! Wsb1! Nle1! Mit120! NLE Domain!

Figure 3.1 Postional cloning of l11Jus1 and l11Jus4 A. Exclusion Mapping Breeding Scheme. Blue line: ENU-mutated allele (l11Jus1; C57BL/6J); Green line with the double arrow: 35 Mb inversion (In(11Trp53;11Wnt3)8Brd; 129S6/SvEvTac); yellow line: dominant Rex allele (129S6/SvEvTac), which confers a curly coat. Plus sign: wild type locus. All animals carrying two copies of the l11Jus1 allele will die in utero. Representative 58 phenotypes and crossover events are depicted in the F2 generation. B. Mapping the Nle1 mutation by haplotype analysis in the F2 generation. Each box represents a locus within Mmu Chr 11 with color indicating the genotype of the animals produced through heterozygous matings. Yellow boxes are non- informative, homozygous 129S6/SvEvTac genotypes; green boxes are heterozygous for 129S6/SvEvTac and C57BL/6J genotypes; while blue boxes indicate informative, homozygous C57BL/6J genotypes. Wsb1 & D11Mit120 define the boundaries of the critical region. C. Physical map of the non- recombinant interval. Nle1 lies ~700 kb centromeric to D11Mit120. D. Mutation analysis. Arrows indicate the location of each mutation. Stacked chromatographs show the sequence for each strain. E. The Nle1 cDNA and genotyping primers. The full-length, spliced mRNA product is shown with exons represented by empty boxes. Primer locations are depicted by arrows. The 5’ and 3’UTRs are

59 represented by a thin line on the mRNA transcript. F. NLE1 protein structure. Green oval :NLE specific domain; yellow boxes: WD40 Repeat-like domains; green diamonds: mutation sites.

Table 3.1 Primers used for this study

T Size Forward Sequence Reverse Sequence m (bp) Nle1 sequencing primers E1/2 CTTGACTCCTCCGAACACGAG AAACACAGCCTGTCTGTAGGTGAG 62 500 E3 GATTAAATTTGTCGCATGGTGGTA GTCTGTTACTTGCAACGTGAGTCC 62 475 E4 TATTTCTCCTCAGGGAATGGAGAG CCACACTCAGTCCAGTATCTGCTT 62 377 E5/6 CTGTGTTCTCCCTCACCTCTCC ATAGTAGGCCAAGCCGTTGCT 62 557 E7 ACAGCCTTGCTCTGCTGTTAGAA GGACCAGCTGGACTCTTGGTATAA 62 440 E8/9 TTCCTGATTCTTGCCTTATGTCAC AACCCTAACTAAGACAACCAAGAACAA 62 544 E10 TGGAGTTGCATGTAAGCTTGTGT GTCACTAGCCCTAAAGATGCCATT 62 488 E11/12 CCGGCCCAGGTACCTAGCTT ACCTACAGGTTCTCCCAGAGTCTCC 62 498 E13 ACTTGATACTTGGCAGTAGGCACA CTCCTGCTATCCAGTGCAAGG 62 570

Nle1 genotyping primers GSP GCTGTAATGTCCTGACTGT cDNA CTGTGTCGTACTCTTCAAGGTCAT CTGTGGAGTCATCTTCTCCATATC 60 637 cDNA 2nd TCAGACGACTTCACCTTATTCCTG CAGTCAACAGCATATACCTCATCG 62 351 gDNA geno TCTCCTTCAGCTCCTTCACTGT TCCAATGGTGGAGTATAGGGTATAA 60 341

cDNA amplification primers 1st ATATGGATCCGGCGCGCCGTCGACTT ATATCTCGAGGGCGCGCCGGATCCTT round TTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTT 67

2nd (NH2)ATATGGATCCGGCGCGCCGTCG (NH2)ATATCTCGAGGGCGCGCCGGAT round ACTTTTTTTTTTTTTTTTTTTTTTTT CCTTTTTTTTTTTTTTTTTTTTTTTT 67 Eed cDNA GTGTGACATTTGGTACATGAGGTT ACATTTATGATGGGTCAGTGTTGT 60 148 59

ABI TaqMan gene expression assay Assay ID Amplicon Size (bp) Trp53 Mm01731290_g1 119 Cdkn1a Mm04205640_g1 80 Gapdh Mm99999915_g1 107

3.2.6 PCR Array Data Analysis for Gene Expression Cycle threshold (Ct) values were calculated for all data obtained from 18 PCR plates. We calculated the optimal threshold values based on the value for each plate by selecting the auto calculate threshold position and the PCR base-line subtracted analysis mode from the iCycler Data Analysis Software (Bio-Rad, Hercules, CA). The highest threshold position was 1415 PCR base-line subtracted relative fluorescence units (RFU). We re-analyzed each plate by entering 1415 as the user defined threshold position. Therefore, we were able to compare replicates across multiple plates using Ct values generated from the common threshold position.

We used the SABiosciences RT2 Profiler Data Analysis Software to determine gene expression profiles (http://www.sabiosciences.com/pcr/arrayanalysis.php). This software calculated fold regulation values for each gene using the relative quantification 2-∆∆Ct method (Livak and Schmittgen, 2001). Each plate met the quality assurance criteria listed by the manufacturer for genomic DNA contamination, reverse transcription inhibition, and PCR cycling conditions. ∆Ct values were normalized using the mean values of three housekeeping genes: Gusb, Hsp90ab1, and Actb. All wells with a Ct value above 29.5 cycles were excluded from the analysis. This left 65 transcripts for analysis. 60

3.2.7 Caspase 3 Detection

Active Caspases were detected based on a fluorescent inhibitor of Caspases (FLICA) approach (Bedner et al., 2000; Slee et al., 1999). Zona-free embryos were placed on slides and incubated with FLICA caspase3 reagent (Image-iT™ LIVE Red Caspase-3 and -7 Detection Kit, Invitrogen, Grand Island, NY) in M2 medium at 37°C for one hour. FLICA was removed and the embryos were washed with M2 media, counterstained with Hoechst dye for 3 minutes and washed with buffer provided by the manufacturer. Embryos were fixed in 1% PFA for 10 min and mounted on cover slides. Each embryo was imaged with Zeiss

LSM510 microscope (20X objective) and the images were pseudo-colored using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). To genotype, we collected each embryo in 10 µl of 100 µg/ml proteinase K solution, incubated the embryos at 55°C for 10 minutes, and then heat inactivated at 95°C for 5 minutes. We used these lysates to genotype each embryo by two rounds of PCR using primers that flanked an insertion/deletion in exon 8.

3.2.8 Quantitative RT-PCR by Taqman RNA isolated from embryos separated by genotype (mutant vs. wild-type) and stage (morula, full blastocyst and hatched blastocyst) was reverse transcribed individually following the protocol by Tang and Colleagues (Tang et al., 2010). Following a 1:1 addition of 100% ethanol, RNA was concentrated with a SpeedVac for 15 mins, resuspended in 4.5 µl lysis buffer and reverse transcribed. We performed a two-step linear amplification process using barcoded primers as described (Tang et al., 2010). Products from the first and second rounds were purified using Zymo DNA concentration kits (Zymo Research, Irvine, CA) and eluted in 30 µl of 1 X T10E0.1. Eed expression was used to check the cDNA quality following the first linear amplification step (primers listed in Table 1). cDNA was quantified using a Bio-Rad SmartSpec™ Plus Spectrophotometer.

Ten ng of cDNA was used as a template for qRT-PCR in combination with 61 TaqMan® Gene Expression Master Mix (PN#4369514; ABI, Carlsbad, CA) and Taqman Gene-specific probes (ABI, Carlsbad, CA) on a Prism 7000HT Sequence Detection System (ABI, Carlsbad, CA). We assayed a minimum of three biological replicates for each group. Cycling reactions were performed in duplicate or triplicate. The relative expression of each gene was calculated based on the ΔΔCt value, where the results were normalized to the average Ct value of Gapdh. Samples that failed to generate a signal above threshold at the end of the reaction were given a Ct value of 40.

3.2.9 Statistical Methods SAB PCR Array Study. These data can be thought of as a complex nested block design: Plate is nested within Pool and Pool is nested within Genotype (Plate and Pool are both blocks). Transcript is nested within biological Role (each transcript was assigned one biological Role), and transcript and role are crossed with each level of nesting (i.e. each transcript is measured on each plate). The data were analyzed in a GLM, blocked by Plate nested within Pool, and Pool nested within Genotype. Transcript was nested within Role; and Role and Transcript crossed with the blocking factors Plate and Pool, and with the experimental factor Genotype. Each Plate and Pool acted as its own control. The relationship of Plates as technical replicates from the same Pools is recognized. Transcript describes the overall expression profile, while Role describes the overall Functional Profile, and their interactions with Genotype test (respectively) whether particular Transcripts differ from the average for the Role between Genotypes, and whether particular Roles differ as a whole between Genotypes. We partitioned out between-plate error and used this as the error term for analyses for two reasons: 1) the plate reader software controls within plate error and 2) the use of between-plate error is conceptually equivalent to (the source of error in a traditional ANOVA approach testing each gene independently. By using ∆Ct values, the analysis directly calculates ∆∆Ct. 62

qRT-PCR study by TaqMan analysis. We adopted a similar GLM approach to individually test and calculate the ∆∆Ct values from Cdkn1a and Trp53 gene expression studies. Since we used the –∆Ct for each individual as raw data, genotype interactions figures and tests a ∆∆Ct value. We also tested for common changes in gene expression in different stages of pre-implantation development and used the full pairwise comparisons table to generate the individual ∆∆Ct values and standard errors.

3.3 Results 3.3.1 Phenotypic Analysis of l11Jus1 and l11Jus4 We screened a cohort of 59 lethal mutants (45 of which were embryonic lethal) that were generated by ENU mutagenesis (Hentges et al., 2006b), and identified an allelic series of two mutants (l11Jus1 and l11Jus4) mapping to mouse chromosome 11 that failed to gastrulate. Histological sections performed at embryonic day (E) 7.5 show completely resorbing implantation sites compared to control littermates (Figure 3.2). In contrast, animals inheriting two copies of the 35 Mb inversion, In(11Trp53;11Wnt3)8Brd, are homozygous mutant for Wnt3 and display a distinct, much less severe phenotype during the gastrulation stage (Table 3) (Hentges et al., 2006b; Kile et al., 2003). Complementation studies revealed that the phenotype of the l11Jus1/l11Jus4 double heterozygotes is identical to either single homozygous mutant (data not shown), thereby placing l11Jus1 and l11Jus4 in the same complementation group.

A B C

Ex" 63

Em" D" D" D"

Figure 3.2 Mutant phenotypes H&E stained sections at E7.5 A. Wild type implantation site B. l11Jus1 implantation site. C. l11Jus4 implantation site. embryo (Em), extra-embryonic region (Ex), and maternal decidua (D).

Penetrance of l11Jus1 (L1) (Table 3.2A): We genotyped a total of 34 l11Jus1 (L1/L1) homozygotes (32 normal and 2 abnormal blastocysts), 117 heterozygotes (L1/Inv) and 49 animals homozygous for the inversion (Inv/Inv). We failed to genotype 98 embryos due to lack of DNA from normal (n=40) and abnormal (n=2) embryos; resorption sites (n=38) and lost embryos (n=18) accounted for the remainder of non-genotyped embryos. At E6.5, we detected 0 homozygous mutant embryos out of 62 total embryos. X2 analysis indicates that these numbers are statistically significant, with p<0.0001 (Table 3). At the blastocyst stage (E3.5), we detected normal Mendelian ratios, indicating that the time of death occurs between E3.5 and E6.5.

Penetrance of l11Jus4 (L4) (Table 3.2B). We genotyped a total of 28 l11Jus4 (L4/L4) homozygotes (all normal), 153 heterozygotes (L4/Inv) and 49 animals homozygous for the inversion (Inv/Inv). We failed to genotype 58 embryos due to lack of DNA from normal (n=9) and abnormal (n=2) embryos; resorption sites (n=46) and lost embryos (n=1) accounted for the remainder of non-genotyped embryos. At E6.5, we detected 0 homozygous mutant embryos out of 32 total embryos. X2 analysis indicates that these numbers are statistically significant, with a p-value of 0.004 (Table 3.2). At the blastocyst stage (E3.5), we detected all genotypes, but saw an unexpectedly high number of heterozygotes (p=0.014). 64

Together, these data indicate that l11Jus1 homozygotes and l11Jus4 homozygotes both die in utero prior to E6.5.

Table 3.2 Time of death for Nle1 mutants A. Time of Death for l11Jus1 Mutants

Abnormal Normal No Inv/ No Inv/ Day Resorb DNA L1/L1 L1/Inv Inv DNA L1/L1 L1/Inv Inv Lost Total

3.5 0 2 2 4 3 40 32 58 23 1 165

6.5 28 0 0 0 18 0 0 42 0 10 98

7.5 3 0 0 0 1 0 0 5 0 3 12

8.5 3 0 0 2 3 0 0 1 0 4 13

9.5 4 0 0 1 1 0 0 4 0 0 10

Total 38 2 2 7 26 40 32 110 23 18 298

B. Time of Death for l11Jus4 Mutants

Abnormal Normal No Inv/ No Inv/ Day Resorb DNA L4/L4 L4/Inv Inv DNA L4/L4 L4/Inv Inv Lost Total

3.5 0 2 0 8 3 6 28 86 27 1 161

6.5 16 0 0 4 9 0 0 19 0 0 48

7.5 7 0 0 0 4 0 0 19 0 0 30

8.5 13 0 0 5 5 0 0 11 0 0 34

9.5 10 0 0 0 1 3 0 1 0 0 15

Total 46 2 0 17 22 9 28 136 27 1 288 65

Table 3.3 X2 analysis of selected timed matings Total Genotype Observed Expected Stage p value Embryos L1/L1 34 50 L1/Inv 117 100 All 200 0.018 Inv/Inv 49 50

L1/L1 34 30.5 L1/Inv 62 61 E3.5 122 0.582 Inv/Inv 26 30.5

L1/L1 0 15 L1/Inv 42 30 E6.5 60 3.71703E-05 Inv/Inv 18 15

L4/L4 28 57.5 L4/Inv 153 115 All 230 5.17678E-07 Inv/Inv 49 57.5

L4/L4 28 38 L4/Inv 94 76 E3.5 152 0.014 Inv/Inv 30 38

L4/L4 0 8 E6.5 32 0.004 L4/Inv 23 16

Inv/Inv 9 8 66

3.3.2 Positional Cloning of l11Jus1 and l11Jus4 Since L1 and L4 homozygotes failed at the implantation stage, meiotic mapping would be difficult, at best, using traditional methods that rely on haplotype analysis in phenotypically mutant animals. To circumvent this obstacle, we narrowed the critical interval by exclusion mapping (Figure 3.1A). Exclusion mapping involves haplotype analysis of all progeny at weaning for several markers across the candidate interval (i.e. from Trp53 to Wnt3 on Mmu 11). Since homozygous mutants are embryonic lethal, any marker that is homozygous for the mutant allele (i.e. C57BL/6J) will effectively ‘exclude’ this

67 marker from the candidate interval. Parents were heterozygous for the l11Jus1 mutation and for the dominant coat color marker, Rex. Throughout the 35 Mb critical interval, l11Jus1 is on a C57BL/6J background, while Rex is on a 129S6/SvEvTac background. Since the balancer chromosome is not present in the F1 generation, it is possible to obtain animals that have recombination events on one or both parental alleles. These recombination events were visualized by haplotype analysis in the F2 generation (Figures 1, 4). We genotyped 487 progeny (974 individual meiotic events), and narrowed the critical region to a 4.4 Mb domain flanked by Wsb1 and D11Mit120 (Figure 3.1B, C).

Of the 75 genes in this interval, 16 top candidates were selected based on microarray expression data and mutant phenotype. We sequenced 8 of these genes in the process of identifying the l11Jus1 and l11Jus4 mutations: adaptor- related protein complex 2, beta1 subunit (Ap2b1); chaperonin containing Tcp1, subunit 6b (zeta) (Cct6b); suppressor of zest 12 homolog (Suz12); fringe isoform 1 (Rffl); ecotropic viral integration site 2a (Evi2a); proteasome (prosome, macropain) 26S subunit non-ATPase 11 (Psmd11); TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor (Taf15); and Notchless homolog 1 (Drosophila) (Nle1).

We found no non-synonymous mutations in our first 7 candidates (Ap2b1, Cct6b, Suz12, Rffl, Evi2a, Psmd11 or Taf15) (Lo et al., 2011). However, we identified a T 1184 G transversion (I 395 S missense mutation) in l11Jus1 heterozygotes in exon 10 of Nle1 (Figure 3.1D). This non-conservative substitution replaces an aliphatic, hydrophobic amino acid with a polar residue, which likely disrupts functionality (Livingstone and Barton, 1993). Subsequent mutation detection efforts resulted in the identification of a second missense mutation (T 484 C transition; S 162 P missense mutation) in exon 5 of Nle1 for the l11Jus4 allele (Figure 3.1D). This non-conservative amino acid substitution has a high probability to alter protein function, as serines easily form hydrogen bonds with

68 polar substrates, while prolines are rarely found in active sites (Livingstone and Barton, 1993). In addition, we detected an endogenous C57BL/6J non- synonymous SNP (A 535 G transition; I 179 V) in exon 6 (Figure 3.1D). This well- documented SNP (rs2820949) leads to a very conservative amino acid substitution (Livingstone and Barton, 1993). Both mutants share the endogenous mutation (Figures 2D, 7), indicating that l11Jus1 and l11Jus4 homozygotes harbor two coding changes in Nle1–an ENU-induced allele and an endogenous C57BL/6J missense mutation.

3.3.3 The Nle1 locus BLAT analysis of the mouse RefSeq cDNA (Accession # NM_15431) at the UCSC genome browser (Kent, 2002), reveals that the Nle1 locus contains 13 exons and spans 7628 bp of genomic DNA. cDNA and EST sequences indicate the potential for generating several alternatively-spliced transcripts. The RefSeq cDNA is predicted to encode a 485 amino acid protein that has an NLE1 domain at the N-terminus and 8 WD40-like repeats (Figure 1F). NLE1 is highly conserved, with orthologs in multiple species, even yeast and plants; it is over 91% identical among mouse, rat, human and cow (Figure 3.3). In addition, it is highly conserved in yeast (42%), drosophila (55%) and potato (58%) (data not shown). 68

L1 --MAAAVVEEAAAGDVQRLLVQFQDEGGQLLGSPFDVPVDITPDKLQLVCNALLAQEEPL 58 L4 --MAAAVVEEAAAGDVQRLLVQFQDEGGQLLGSPFDVPVDITPDKLQLVCNALLAQEEPL 58 C57BL/6 --MAAAVVEEAAAGDVQRLLVQFQDEGGQLLGSPFDVPVDITPDKLQLVCNALLAQEEPL 58 Mouse --MAAAVVEEAAAGDVQRLLVQFQDEGGQLLGSPFDVPVDITPDKLQLVCNALLAQEEPL 58 CZECHII --MAAAVVEEAAAGDVQRLLVQFQDEGGQLLGSPFDVPVDITPDKLQLVCNALLAQEEPL 58 Rat MAAAVEVSDEAAASDVQRLLVQFQDEGGQLLGSPFDVPVDITPDQLQLVCNALLAQDEPL 60 Human --MAAAVPDEAVARDVQRLLVQFQDEGGQLLGSPFDVPVDITPDRLQLVCNALLAQEDPL 58 Cow -MAAAAAADEAATRDVQRLLVQFQDEGGQLLGSPFDVPVDITPDKLQLVCNALLAQEDPL 59 *. . :**.: ******************************:***********::**

L1 PLAFYVHDAEIVSSLGKTLESQSVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVI 118 L4 PLAFYVHDAEIVSSLGKTLESQSVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVI 118 C57BL/6 PLAFYVHDAEIVSSLGKTLESQSVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVI 118 Mouse PLAFYVHDAEIVSSLGKTLESQSVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVI 118 CZECHII PLAFYVHDAEIVSSLGKTLESQSVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVI 118 Rat PLAFYVHDAEIVSSLGKTLESQSVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVI 120 Human PLAFFVHDAEIVSSLGKTLESQAVETEKVLDIIYQPQAIFRVRAVTRCTSSLEGHSEAVI 118 Cow PLAFYVHDAEIVSSLGRTLESQAVETEKVLDIIYQPQAIFRVRAVTRCTSSLEGHSEAVI 119 ****:***********:*****:*****::********:*********************

L1 SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSISWSPDGKKLASGCKNG 178 L4 SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSIPWSPDGKKLASGCKNG 178 C57BL/6 SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSISWSPDGKKLASGCKNG 178 Mouse SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSISWSPDGKKLASGCKNG 178 CZECHII SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSISWSPDGKKLASGCKNG 178 Rat SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSISWSPDGKKLASGCKNG 180 Human SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCKGHRHWVLSISWSPDGRKLASGCKNG 178 Cow SVAFSPTGKYLASGSGDTTVRFWDLSTETPHFTCQGHRHWVLSISWSPDGKKLASGCKNG 179 **********************************:*********.*****:*********

L1 QVLLWDPSTGLQVGRTLTGHSKWITGLSWEPLHMNPECRYVASSSKDGSVRVWDTTAGRC 238 L4 QVLLWDPSTGLQVGRTLTGHSKWITGLSWEPLHMNPECRYVASSSKDGSVRVWDTTAGRC 238 C57BL/6 QVLLWDPSTGLQVGRTLTGHSKWITGLSWEPLHMNPECRYVASSSKDGSVRVWDTTAGRC 238 Mouse QILLWDPSTGLQVGRTLTGHSKWITGLSWEPLHMNPECRYVASSSKDGSVRVWDTTAGRC 238 CZECHII QILLWDPSTGLQVGRTLTGHSKWITGLSWEPLHMNPECRYVASSSKDGSVRVWDTTAGRC 238 Rat QILLWDPSTGTQVGRTLTGHSKWITGLSWEPLHMNPECRYVASSSKDGSVRVWDTTAGRC 240 Human QILLWDPSTGKQVGRTLAGHSKWITGLSWEPLHANPECRYVASSSKDGSVRIWDTTAGRC 238 Cow QILLWDPSTGKQVGRALTGHSKWITALSWEPLHANPECRYVASSSKDGSVRVWDTTAGRC 239 *:******** ****:*:*******.******* *****************:********

L1 ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 298 L4 ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 298 C57BL/6 ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 298 Mouse ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 298 CZECHII ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 298 Rat ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 300 Human ERILTGHTQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 298 Cow ERTLTGHAQSVTCLRWGGDGLLYSASQDRTIKVWRAHDGVLCRTLQGHGHWVNTMALSTD 299 ** ****:****************************************************

L1 YALRTGAFEPAEATVNAQDLQGSLKELKERASSRYNLVRGQGPERLVSGSDDFTLFLWSP 358 L4 YALRTGAFEPAEATVNAQDLQGSLKELKERASSRYNLVRGQGPERLVSGSDDFTLFLWSP 358 C57BL/6 YALRTGAFEPAEATVNAQDLQGSLKELKERASSRYNLVRGQGPERLVSGSDDFTLFLWSP 358 Mouse YALRTGAFEPAEATVNAQDLQGSLKELKERASSRYNLVRGQGPERLVSGSDDFTLFLWSP 358 CZECHII YALRTGAFEPAEATVNAQDLQGSLKELKERASSRYNLVRGQGPERLVSGSDDFTLFLWSP 358 Rat YALRTGAFEPAEATVNAQDLQGSLKELKERASSRYNLVRGQGPERLVSGSDDFTLFLWSP 360 Human YALRTGAFEPAEASVNPQDLQGSLQELKERALSRYNLVRGQGPERLVSGSDDFTLFLWSP 358 Cow YALRTGAFEPAEASVNAQDLRGSLQELKERALSRYNLVRGQGPERLVSGSDDFTLFLWSP 359 *************:**.***:***:****** ****************************

L1 AEDKKPLARMTGHQALINQVLFSPDSRIVASASFDKSVKLWDGRTGKYLASLRGHVAAVY 418 L4 AEDKKPLARMTGHQALINQVLFSPDSRIVASASFDKSIKLWDGRTGKYLASLRGHVAAVY 418 C57BL/6 AEDKKPLARMTGHQALINQVLFSPDSRIVASASFDKSIKLWDGRTGKYLASLRGHVAAVY 418

Mouse AEDKKPLARMTGHQALINQVLFSPDSRIVASASFDKSIKLWDGRTGKYLASLRGHVAAVY 418 69 CZECHII AEDKKPLARMTGHQALINQVLFSPDSRIVASASFDKSIKLWDGRTGKYLASLRGHVAAVY 418

Rat AEDKKPLARMTGHQALINQVLFSPDSRIVASASFDKSIKLWDGRTGKYLASLRGHVAAVY 420 Human AEDKKPLTRMTGHQALINQVLFSPDSRIVASASFDKSIKLWDGRTGKYLASLRGHVAAVY 418 Cow AEDKKPLARMTGHQALINQVVFSPDSRVIASASFDKSIKLWDGRTGKYLASLRGHVAAVY 419 *******:************:******::********:**********************

L1 QIAWSADSRLLVSGSSDSTLKVWDVKAQKLATDLPGHADEVYAVDWSPDGQRVASGGKDK 478 L4 QIAWSADSRLLVSGSSDSTLKVWDVKAQKLATDLPGHADEVYAVDWSPDGQRVASGGKDK 478 C57BL/6 QIAWSADSRLLVSGSSDSTLKVWDVKAQKLATDLPGHADEVYAVDWSPDGQRVASGGKDK 478 Mouse QIAWSADSRLLVSGSSDSTLKVWDVKAQKLATDLPGHADEVYAVDWSPDGQRVASGGKDK 478 CZECHII QIAWSADSRLLVSGSSDSTLKVWDVKAQKLATDLPGHADEVYAVDWSPDGQRVASGGKDK 478 Rat QIAWSADSRLLVSGSSDSTLKVWDVKAQKLTTDLPGHADEVYAVDWSPDGQRVASGGKDK 480 Human QIAWSADSRLLVSGSSDSTLKVWDVKAQKLAMDLPGHADEVYAVDWSPDGQRVASGGKDK 478 Cow QIAWSADSRLLVSGSSDSTLKVWDVKAQKLSTDLPGHADEVYAVDWSPDGQRVASGGKDK 479 ******************************: ****************************

L1 CLRIWRR 485 L4 CLRIWRR 485 C57BL/6 CLRIWRR 485 Mouse CLRIWRR 485 CZECHII CLRIWRR 485 Rat CLRIWRR 487 Human CLRIWRR 485 Cow CLRIWRR 486 ******* Figure 3.3 NLE1 protein sequence alignment NLE1 is highly conserved among mouse, rat, cow and human.

3.3.4 Expression Analysis of the Notch Pathway in Mutant Embryos Since Nle1l11Jus1/l11Jus1 embryos die shortly after implantation, while disruption of NOTCH signaling by multiple methods (i.e. targeted deletion of Notch receptors and ligands, (Conlon et al., 1995; Dunwoodie et al., 2002; Hamada et al., 1999; Hrabe de Angelis et al., 1997; Jiang et al., 1998a; Jiang et al., 1998c; Krebs et al., 2004; Krebs et al., 2000; Krebs et al., 2003; McCright et al., 2001; Swiatek et al., 1994), γ-secretase (Donoviel et al., 1999) or Pofut1 (Shi et al., 2005a; Shi and Stanley, 2003)) in mice leads to embryonic lethality after mid-gestation, we hypothesized that Nle1l11Jus1/l11Jus1 and Nle1l11Ju41/l11Jus4 mutants had defects in multiple signaling pathways. To test for defects in NOTCH signaling, we analyzed Notch pathway gene expression in homozygous mutant (i.e. Nle1l11Jus1/l11Jus1) blastocysts using the PAMM-059 Notch Pathway SAB PCR Array (SABiosciences/Qiagen, Frederick, MD). We compared expression levels of 84 Notch pathway genes in Nle1l11Jus1/l11Jus1 pre-implantation embryos to Nle+/+ control littermates.

We eliminated 19 genes, including 8 Notch downstream targets (Cflar, Ifng, Il2ra, Pparγ, Cd44, Dtx1, Krt1 and Ptcra) and the Notch ligand, Dll1, due to lack of expression (i.e. had a Ct value ≥ 29.5). This left 65 genes for statistical analysis, including the Notch receptors (Notch1-4), Jagged ligands (Jag1-2) and receptor 70 processing and modifying enzymes (i.e. the γ-secretase complex and protein O- fucosyltransferase genes: Adam10, Adam17, Psen1, Psen2, Psenen and Pofut1). Notch target genes include Cdkn1a (a marker of cell cycle arrest), Hes1, Hey1, Stat6, Nr4ar, Nfkb1 and Pparg (transcriptional regulators), Ccnd1 (cell cycle), as well as Chuk, Il17b and Krt1 (downstream targets with unspecified functions in the NOTCH pathway). In addition, the PCR array contains several members of the Wnt (Aes, Axin1, Lrp5, Fzd1-7 and Wnt11) and Hedgehog signaling pathways (Gli1, Gsk3, Shh, Smo and Sufu). To ensure biological significance, genes with less than a 1.5 fold change were included in the statistical analyses, but not considered differentially expressed.

We analyzed the data using a GLM blocked by plate (technical replicate) nested within pool (biological replicate) and pool nested within genotype. Transcript levels were nested within biological function/pathway (role). Role and transcript were crossed with the blocking factors plate and pool, and with the experimental factor genotype. Therefore, testing for genotype X transcript interactions will identify single genes that are statistically upregulated or downregulated in mutant embryos, while testing for genotype X role interactions identifies groups of genes with similar biological functions (i.e. Wnt pathway, transcriptional regulation, etc.) that are as a whole misregulated in mutant embryos compared to wild-type controls. The Role (GLM: F7,84=248.3; P<0.0001) and Transcript (F7,84=224.0; P<0.0001) effects were significant, indicating the presence of consistent functional and expression profiles in both genotypes.

The Genotype*Transcript interaction was significant (F57,684=1.5490; P=0.0073), indicating that at least one transcript differed from the overall mean of transcripts within the same Role. Of the 16 Notch target genes detected in this study, 6 were overexpressed by at least 1.5 fold (Cdkn1a, Nfkb1, Hes1, Erb2, Il17b, Mapk27). However only Cdkn1a, which was upregulated by 4.7 fold in mutant samples (Figure 3.4) (p=1.94X10-8), was statistically significant using post hoc tests corrected for multiple comparisons (accepting p<0.000769); none of the other 71

genes approached significance, even at an uncorrected threshold of p<0.05.

Seven genes demonstrated a more than 1.5 fold reduction in expression in Nle1l11Jus1/l11Jus1 embryos: Lrp5, Fzd7, Fzd4 and Wnt11, which are members of the Wnt signaling pathway, and Chuk, Nr4a2 and Stat6. However, none of these met the rigorous criteria (p<0.000769) that accounts for the multiple comparison analysis. Setting aside Bonferroni corrections for false positive detection rate, and accepting a false discovery rate (FDR) of 5% leads to the inclusion of Fzd7 (p=0.00168), Nr4a2 (p=0.00422), Fzd4 (p=0.00461) and Chuk (p=0.02492); using an FDR of 10% leads to the inclusion of Wnt11 (p=0.02679) (Figure 3.4).

Fold change in expression Mutant / Wildtype 0.25 0.50 1.00 2.00 4.00 8.00

Cdkn1a Cflar Gsk3b Nfkb1 Apoptosis

Ccnd1 Ccne1 Cdc16 Cell Cycle Ctnnb1 Fos Lrp5 Numb Runx1 Zic2 Differentiation

Adam10 Adam17 Jag1 Jag2 Notch1 Notch2

Notch3 Notch Notch4 Psen1 Psen2 Psenen

Smo

Sufu Shh

Ep300 Fosl1 Gli1 Hes1 Hey1 Hoxb4 Transcript Kat2b Mycl1 Nfkb2 FunctionalRole Nr4a2

Pofut1 Transcription Rfng Stat6 Tead1 Tle1

Cbl Chuk 72 Erbb2 Il17b Il6st Lmo2 Lor Map2k7 Mtap1b Neurl1a

Pdpk1 Undetermined Sel1l Snw1 Stil Supt6h

Aes Axin1 Fzd1 Fzd3 Fzd4 Fzd5 Wnt Fzd6 Fzd7 Wnt11

2 1 0 -1 -2 -3 DCT(Mutant) - DCT(Wildtype)

Figure 3.4 Expression of Nle1l11Jus1 embryos using a Notch-specific PCR array Each transcript is listed (left), with fold change indicated (top). The mean expression level for each transcript is indicated. The least-squares mean ΔΔCT ±SE is shown for each transcript, with equivalent fold-change. The dashed line indicates that Cdkn1a is the only gene with significant differences in expression following a Bonferonni correction for multiple comparisons. The dotted line represents transcripts where p < 0.05. Biological roles were taken directly from the SAB website.

This analysis indicates that multiple members of the Wnt pathway are downregulated in Nle1 mutant embryos.

3.3.5 Confirmation of Cdkn1a in Different Stages in Pre-Implantation The SAB PCR array study was performed on three pools (n=15) of E3.5 embryos at different stages (i.e. a mix of morula, blastocysts and hatched blastocysts). This could have introduced biased expression or masked subtle alterations in stage-specific gene expression due to pooling of embryos from different stages. To control for these possibilities and confirm the PCR array results, we analyzed expression of Cdkn1a between wild type and mutant embryos at multiple embryonic stages (morula, full blastocyst and hatched blastocyst) in single embryos using TaqMan assays. Expression of each gene was normalized relative to expression of Gapdh and compared to the stage-matched wild type 73 controls. Wild type expression was set at a value of one.

We used a multivariate GLM model to calculate ΔΔCt and fold change and to properly control and test for differences between the mutant alleles and stages of development. Using a least squares mean, which corrected for all of the variables in the analysis (line, genotype, stage of development), we did not detect differences in Cdkn1a expression between mutants compared to control embryos as a function of stage (morula, blastocyst and hatchet blastocyst; l11Jus1 l11Jus4 F2,32=0.2701; P=0.7650), line (Nle1 and Nle1 ; F1,32=1.293; P=0.2640) or stage and line (F2,32=0.0574; P=0.9444), indicating that we did not detect

74 differences in expression due to developmental stage or mutant line. However, overall, Cdkn1a was expressed at 4.69 fold (95% CI: 1.02 - 21.5 fold) higher levels both in Nle1l11Jus1/l11Jus1 and Nle1l11Jus4/l11Jus4 mutant embryos compared to wild-type controls at all stages (GLM: F1,32=4.2561; P=0.0473). These data are consistent with our PCR array findings for l11Jus1 mutant embryos, show that there are no significant differences in expression of Cdkn1a at the different stages tested in the PCR array, and demonstrate that the phenotypes associated with l11Jus1 and l11Jus4 do not differ at the molecular level.

3.3.6 Apoptosis Occurs at E4.5 in Nle1l11Jus1/l11Jus1 and Nle1l11Jus4/l11Jus4 Mutants These Cdkn1a expression findings are intriguing, as targeted disruption of Nle1 in mice indicated that mutant embryos started to undergo apoptosis at E3.5 plus 1 day in culture, which is approximately equivalent to E4.5 embryos (i.e. hatched blastocysts) (Cormier et al., 2004a). If our mutant embryos were also undergoing apoptosis, we would expect that they would not show high levels of Cdkn1a, as Cdkn1a expression is most often an indication that cells have exited the cell cycle following a DNA damage response or other type of cellular stress event (Brugarolas et al., 1995; Deng et al., 1995). However the function of CDKN1A in cell cycle arrest and apoptosis is still unclear, as other studies have shown that Cdkn1a expression levels can be upregulated in cells undergoing apoptosis 74

(Gartel, 2005). Given that Cdkn1a was expressed at much higher levels in Nle1 mutants compared to wild-type embryos, we hypothesized that animals expressing high levels of Cdkn1a would be protected from apoptosis at E3.5, but would ultimately become apoptotic by E4.5, which would be consistent with previous studies (Cormier et al., 2004a).

Therefore, we analyzed Caspase 3 activity at E3.5 and E4.5 (Figure 3.5; Table 3.4). We tested these data in a single logistic multiple regression. We detected Caspase 3 activity solely in homozygous mutants (Likelihood Ratio χ2 = 21.75; p<0.0001) at E4.5 (LR χ2 = 15.11; p=0.0001) p<0.0001. Strain had no effect (LR

χ2 < 0.0001; p>0.9999). We did not find any evidence for an interaction between genotype and stage (LR χ2 < 0.0001; p>0.9999). Overall, these data indicate that apoptosis appears at E4.5, but not at E3.5; that apoptosis is only seen in homozygous mutants, and that these two effects are independent and additive in both alleles.

Wildtype Mutant

E3.5

10µm 10µm

E4.5 75

10µm 10µm

Figure 3.5 Caspase 3 detection in Nle1l11Jus1 mutant embryos Caspase 3 detection in mutant and wild type embryos at E3.5 (200X) and E4.5 (200X). Positive Caspase 3 staining is only detected in E4.5 mutants. Red staining indicates the presence of Caspase 3 signal and blue is Hoechst staining. White arrows point out Caspase staining. Merged images are shown.

Table 3.4 Logistic multiple regression analysis of caspase 3 detection Homozygous Genotype/stage Homozygous WT Heterozygous Total Mutant Caspase 3 + – + – + –

l11Jus1 E3.5 0 1 0 5 0 2 8 morula/blastocyst

l11Jus1 E4.5 0 2 0 5 2 0 9 hatched blastocyst

l11Jus4 E3.5 0 4 0 5 0 3 12 morula/blastocyst

l11Jus4 E4.5 0 3 0 4 3 0 10 hatched blastocyst

3.3.7 qRT-PCR Analysis of Trp53 in Mutant Embryos We then asked whether the apoptotic phenotype is Trp53-dependent. Taqman assays were conducted to compare Trp53 expression between wild-type and mutant embryos. Identical GLM models were used to calculate fold change and test significance, with additional planned contrasts performed to test differences at different embryonic stages. Trp53 expression did not differ between control and mutant embryos overall (GLM: F1,33=0.5316; P=0.4711), by line

(F1,33=0.0057; P=0.9404), by embryonic stage (F2,33=0.0994; P=0.9056), or the interaction of line and stage (F2,33=0.1782; P=0.8376).

Together, the gene expression and Caspase 3 data indicate that Nle1l11Jus1/l11Jus1 and Nle1l11Jus4/l11Jus4 homozygotes undergo Caspase 3-mediated apoptosis at the hatched blastocyst stage. This is not mediated by alterations in mRNA levels of Trp53, and apoptosis does not correlate with upregulation of Cdkn1a expression in homozygous mutant embryos, as upregulation of Cdnk1a occurs from the morula through the hatched blastocyst stage, even though apoptosis only is observed at the latest stages. Furthermore, we demonstrate that several members of the Wnt pathway are downregulated in mutant embryos, suggesting that NLE1 interacts with the WNT pathway during pre-implantation development.

3.4 Discussion We present evidence that the l11Jus1 and l11Jus4 mutant phenotypes are caused by non-conservative missense mutations in the Nle1 gene. Gene targeting studies indicate that l11Jus1 and l11Jus4 phenocopy the null allele (Cormier et al., 2006b). These ENU mutants were created on a C57BL/6J background, which also contains a conservative missense mutation in Nle1. Therefore, l11Jus1 and l11Jus4 homozygotes harbor two mutations within predicted functional domains of NLE1. Previous studies in Drosophila and Xenopus indicate that NLE1 is a member of the NOTCH pathway (Cormier et al., 2006b; Royet et al., 1998a). NOTCH signaling facilitates short-range cell-cell communication during diverse cellular processes, in multiple tissues and at a multitude of developmental stages. Loss of function or gain of function mutations in various factors that are fundamental to canonical NOTCH signaling are often associated with developmental disorders, adult-onset diseases and a variety of cancers in humans (Kopan and Ilagan, 2009).

However, the function of NLE1 in NOTCH signaling remains elusive. Initial studies in Drosophila show that NLE1 function is context and dosage dependent. NLE1 was identified as a dominant suppressor of the viable mutant allele, notchoid (Royet et al., 1998a). Notchoid mutants have characteristic wing 77

notches; in the notchoid mutant background, Nle1 heterozygosity (i.e. Nle1/+) rescued the notchoid phenotype, while simultaneously causing shortened and thickened wing veins (Royet et al., 1998a). Interestingly, overexpression of wingless, the Drosophila Wnt ortholog, in the notchoid background also rescues the notchoid phenotype (Couso and Martinez Arias, 1994; Hing et al., 1994), while overexpression of Notch leads to shortened, thickened wing veins (Matsuno et al., 1995). Since NLE1 can bind the NOTCH intracellular domain (NICD), these experiments suggest that NLE1 is a negative regulator of NOTCH, and that NLE1 functions by blocking the ability of the NICD to regulate expression of downstream targets. Studies in Xenopus come to the opposite

78 conclusion, as overexpression of Murine (Cormier et al., 2006b) or a combination of Xenopus and Drosophila (Royet et al., 1998a) Nle1 mRNAs into single blastomeres at the 4-cell or 2-cell stage, respectively, lead to decreased numbers of primary neurons at the early neurula stage. These results indicate that NOTCH activity was upregulated following injection of Nle1 mRNAs, suggesting that NLE1 positively regulates NOTCH signaling.

These studies indicate that NLE1 acts as both a positive and negative regulator of NOTCH signaling. If NLE1 acts as a general positive regulator of NOTCH signaling during murine pre-implantation development, then elimination of NLE1 could lead to compensatory over-expression of the Notch receptors, ligands and protease family members, but reduced expression of downstream target genes. In contrast, if NLE1 functions as a negative regulator of NOTCH signaling, we would predict that disruption of NLE1 would lead to increased expression of Notch target genes. To our surprise, we saw no generalized misregulation of Notch target genes in the PCR array study. In addition, the Notch receptors, ligands and other family members were not significantly altered. At a false discovery rate of 5%, the only genes that are misregulated in the PCR array study were: Cdkn1a, Nr4a2, Fzd7, Fzd4 and Chuk. Cdkn1a, Nr4a2 and Chuk are downstream targets of NOTCH signaling, while Fzd7 and Fzd4 encode 78

receptors in the WNT pathway. To tease out the gene expression changes that occurred during specific pre-implantation stages, we analyzed expression of Cdkn1a on individual staged embryos (morulae, full blastocysts and hatched blastocysts). Cdkn1a was significantly upregulated in Nle1l11Jus1/l11Jus1 and Nle1l11Jus4/l11Jus4 animals at all three stages. These studies indicate that mutations in Nle1 do not significantly affect the NOTCH pathway during pre-implantation development.

CDKN1A is a powerful cyclin-dependent kinase inhibitor that functions in several developmental pathways and negatively regulates the cell cycle at G1 via a

TRP53-mediated response to DNA damage (Abbas and Dutta, 2009). This can happen directly by competing with DNA polymerase δ for PCNA binding sites at the replication fork, leading to decreased DNA synthesis (Moldovan et al., 2007). Alternatively, CDKN1A can inhibit CDK2, which leads to suppression of E2f- dependent transcripts, downregulation of components of the DNA synthesis machinery and reduced firing at origins of replication (Zhu et al., 2005). In addition, CDKN1A can act as a negative regulator of Caspase-mediated apoptosis (Abbas and Dutta, 2009). Gene targeting studies demonstrated that the inner cell mass of Nle1-/- embryos was undergoing apoptosis via a caspase- dependent mechanism in E3.5 blastocysts that were cultured for 24 hours (Cormier et al., 2006b). We analyzed Caspase 3 activity in blastocysts and hatched blastocysts at E3.5 and E4.5. Consistent with the results of Cormier and colleagues (2006), we demonstrate that l11Jus1 and l11Jus4 show evidence of apoptosis only in hatched blastocysts.

Although we show upregulation of Cdkn1a and downregulation of several members of the Wnt pathway, how these two networks work together to regulate pre-implantation development is still unknown. One attractive possibility is via the TRP53-mediated stress response pathway, which is upstream of CDKN1A. We predicted that if our Nle1 mutations were causing severe cellular damage, the 79 cell would not be able to recover during cell cycle arrest (at E3.5), which would then force the cell to undergo apoptosis (at E4.5). If this were true, we would expect to see increased expression of Trp53, as the cells proceeded through apoptosis. However, we did not detect altered expression of Trp53 by qRT-PCR studies in our mutants at any stage. In retrospect, this result is not that surprising, as TRP53-mediated apoptosis (via Cdkn1a upregulation) is not necessarily correlated with mRNA expression of Trp53, but is instead associated with upregulation of the active, acetylated form of the TRP53 protein (Yamakuchi and Lowenstein, 2009). Alternately, it is possible that the expression changes in

Trp53 were too subtle to detect, or apoptosis could be occurring via non-TRP53 mediated pathways (Gurley et al., 2009). 80

CHAPTER 4. NLE1 MISSENSE MUTATIONS TIGGER TRP53 ACTIVATION AND DISRUPTION IN RIBOSOMAL BIOGENESIS DURING MURINE PRE- IMPLANTATION DEVELOPMENT

4.1 Introduction

Previously, we reported that missense mutations in Notchless (Nle1) result in embryonic lethality at peri-implantation (Lossie et al., 2012). Mutant embryos implant and elongate, but fail to establish and/or maintain maternal/fetal contacts, ultimately failing prior to gastrulation. Nle1 mutant embryos display alter expression of Cdkn1a and several members of the Wnt pathway (e.g. Fzd4, Fzd7, and Wnt11). The Wnt pathway is critical for establishing and maintaining pregnancies (Pey et al., 1998; Wang et al., 2004; Xie et al., 2008). For example, targeted disruption of Wnt3 results in embryonic lethality prior to primitive streak formation (Barrow et al., 2007). Wnt pathway genes are highly expressed in blastocysts (Kemp et al., 2005; Kemp et al., 2007; Lloyd et al., 2003; Mohamed et al., 2004), as well as in the uterus during peri-implantation (Hayashi et al., 81

2007; Hayashi et al., 2009; Miller and Sassoon, 1998).

CDKN1A is thought to play important roles in cell cycle arrest and/or apoptosis through activation of TRP53-mediated programmed cell death (Abbas and Dutta, 2009). Activation of TRP53 can be induced by cellular stress or DNA damage (Yuan et al., 2005). In our case, we believe the cellular stress is resulted from the missense mutation in NLE1. Therefore, it is crucial to tease out the functional role of NLE1 in mouse early development. Nle1 was first identified as a suppressor of the notchoid phenotype in Drosophila, and has been determined to interact with

82 the intracellular domain of Notch (Royet et al., 1998). NLE1 acts both as a positive and a negative regulator of NOTCH signaling, depending upon developmental stage and species (Cormier et al., 2006b; Royet et al., 1998a). Studies in Drosophila and Xenopus demonstrate that NLE1 signals via the canonical NOTCH pathway (Cormier et al., 2006b; Royet et al., 1998a). In invertebrates and lower vertebrates, the NOTCH pathway is critical for directing cell fate prior to gastrulation, and also plays important, but varied roles in germ layer boundary formation (Good et al., 2004). However, our findings refute the possibility of NLE1 in the NOTCH pathway in mouse early development, as mutation of Nle1 does not lead to altered expression of Notch downstream genes (Lossie et al., 2012). Furthermore, several lines of evidence demonstrate that NOTCH signaling is dispensable for gastrulation in mice. Single gene and compound knockout studies of the Notch receptors and ligands results in either viable animals or embryonic lethality at mid-gestation (Conlon et al., 1995; Dunwoodie et al., 2002; Hamada et al., 1999; Hrabe de Angelis et al., 1997; Jiang et al., 1998a; Jiang et al., 1998c; Krebs et al., 2004; Krebs et al., 2000; Krebs et al., 2003; McCright et al., 2001; Swiatek et al., 1994). Similarly, deletion of genes that block NOTCH signaling, such as Pofut1 and members of the g- secretase complex, leads to embryonic failure after gastrulation and midline formation (Shi et al., 2005a; Shi and Stanley, 2003). These studies demonstrate 82

that unlike lower vertebrates and invertebrates, and despite the fact that Notch receptors and ligands are expressed prior to and during gastrulation (Cormier et al., 2004a), NOTCH signaling is dispensable prior to gastrulation in mice.

Several studies in yeast and plants (which lack NOTCH signaling) demonstrate a role for NLE1 in ribosomal biogenesis (Chantha et al., 2007a; Chantha et al., 2006; Chantha and Matton, 2007; de la Cruz et al., 2005b; Strain et al., 2001). Ribosomal biogenesis is necessary during preimplantation development, as genetic ablation of proteins involved in rRNA processing usually causes growth arrest prior to implantation (Gallenberger et al., 2011; Lerch-Gaggl et al., 2002;

Romanova et al., 2006; Zhang et al., 2008). For instance, in mice with mutations in the proteins involved in ribosomal rRNA synthesis and processing, such as pescadillo-1 (PES1) (Lerch-Gaggl et al., 2002), RNA polymerase 1 (Rpol1) (Yamamura et al., 2008), surfeit gene 6 (SURF6) (Romanova et al., 2006), RNA binding motif protein 19 (RBM19) (Zhang et al., 2008), EMG1 nucleolar protein homolog (S. cerevisiae) (EMG1) (Ding et al., 2010), WD repeat domain 36 (WDR36) (Gallenberger et al., 2011), and lin-28 homolog A (C. elegans) LIN28a (Vogt et al., 2012), mutant embryos are arrested at the morula stage, leading us to hypothesize that NLE1 is involved in murine ribosomal biogenesis pre- implantation development.

The general paradigm for ribosomal biogenesis is the coordinated assembly of approximately 80 ribosomal proteins and four ribosomal RNAs to generate the 60S and 40S ribosome precursors(Lempiainen and Shore, 2009). This process requires all of the RNA polymerases. RNA polymerase I transcribes the 47S precursor rRNA, which is subsequently processed into 28S, 18S and 5.8S rRNAs. 5S rRNA is transcribed by RNA polymerase III, translocated to the cytoplasm and then imported back into the nucleolus where it incorporates into the large ribosomal subunit (Venema and Tollervey, 1999). Ribosomal proteins and other factors that assist rRNA processing and assembly, rRNA export and final 83

maturation are transcribed by RNA polymerase II (Kressler et al., 1999). Mis- regulation of ribosomal biogenesis (e.g. defects in rRNA transcription and disruptions in rRNA processing) triggers ribosomal stress, and in most case, causes the breakdown of nucleolar structure which leads to activation of the RP- MDM2-p53 pathway and p53-dependent cell cycle arrest and/or apoptosis (Deisenroth and Zhang, 2010) .

Several lines of studies have shown that the appropriate amount of ribosome is required for allowing G1-S transition and further regulates cell cycle progression through the regulation of p53. For example, conditional deletion of the 40S

84 ribosomal protein S6 in mouse hypatocytes leads to defects in 40S ribosome production and a block of entry into S phase (Volarevic et al., 2000). Drug induced inhibition of rRNA synthesis caused a delay in the G1-S transition due to the reduction of 18S and 28S rRNA produced during the G1 phase (Derenzini et al., 2005). These pieces of evidence point out that there appears to be a threshold level of ribosomes necessary for progression in the cell cycle, and that disruptions in rRNA processing could seriously disrupt important cellular checkpoints during pre-implantation development.

P53 is part of a feedback loop that includes the E3 ubiquitin ligase MDM2, as MDM2 catalyzes ubiquitination of P53, leading to protein degradation (Deisenroth and Zhang, 2011). Many of the p53 regulators function through binding to and inhibiting the function of MDM2 or induce posttranslational modifications that block the MDM2-P53 interaction. Upon induction, the nucleolar protein ARF (Alternative Reading Frame), a product of the INK4A/ARF locus, binds to MDM2 to inhibit degradation of P53 (Sharpless, 2005). In addition, protein modifications to MDM2 (e.g. ubiquitination, sumoylation and phosphorylation) inhibit the MDM2-p53 interaction, thereby stabilizing p53 activity (Meek and Knippschild, 2003). Like ARF, a number of ribosomal protein (RPs) have been reported to interact with MDM2 to block p53 ubiquitination, and specifically ‘lock in’ the active 84

form of p53 (Deisenroth and Zhang, 2011). For instance, the ribosomal proteins RPL5, PRL11, RPL23 and S7 bind MDM2 and therefore stabilize p53 activity. Furthermore, inhibition of rRNA transcription by drugs (5-fluorouracil, actinomycin) or by deleting the Pol I complex also causes similar outcomes through RP- dependent inactivation of MDM2. The active p53 thereby induces the expression of Cdkn1a, inhibits cyclin E/Cdk2 complexes and leads to pRB-dependent cell cycle arrest (Zhao et al., 2003).

In yeast, NLE1 (Rsa4) mutations block processing of the 35S/27S precursor rRNA to the mature 25S and 5.8S rRNAs (Bassler et al., 2010). The

85 corresponding mammalian transcripts are the 36S/32S pre-rRNA, and the mature 28S and 5.8S rRNAs). Given that mutations in Nle1 induce Cdkn1a and ultimately lead to apoptosis (Lossie et al., 2012), we hypothesize that the primary cellular insult to the mutant embryos was a disruption in rRNA processing of the 36S/32S pre-rRNA transcript that activates the RP-MDM2-p53 pathway and leads to cell cycle arrest and/or apoptosis in mutant embryos. To test this hypothesis, we determined that TRP53 is activated in the mutants and several TRP53 regulated genes involved cell cycle and apoptosis pathways are mis- expressed. Next, we performed RNASeq using the MiSeq platform to sequence and quantify total cellular rRNA. We also examined nucleologenesis by detecting the expression pattern of the nucleolar protein, Fibrillarin during pre-implantation development.

4.2 Methods 4.2.1 Immunofluorescence Incubation of the embryos in acid Tyrode’s solution (Sigma-Aldrich, Cat# T1788- 100ML, St. Louis, MO) denuded the embryos of the zona pellucida. Embryos were fixed in 1% paraformaldehyde for 10 minutes, wash in 1X PBS, and then directly stored in 70% Ethanol at 4°C for later use. Fixed embryos were washed three times in PBST (0.1% Triton X-100 in 1X PBS), permeabilized for 30 minutes in 0.5% Triton in PBS at room temperature, and blocked for two hours in 3% BSA, 0.1% Triton X-100 in PBS at room temperature. Embryos were then incubated with the primary antibody in blocking buffer at 4°C overnight. The primary antibodies used in this study are rabbit polyclonal anti-Fibrillarin antibody (1:100 dilution) (Abcam, Cat# ab5821, Cambridge, MA) and rabbit polyclonal anti-p53 (acetyl K378) (1:750 dilution) (Abcam, Cat# ab61241, Cambridge, MA). We incubated the embryos in Chromeo 642 Goat Anti-Rabbit secondary antibody (Active Motif, Cat# 15044, Carlsbad, CA) using a 1: 1000 dilution in PBST for one hour at room temperature. Embryos were washed in 1 X PBST in 0.5 ul eppendorf tubes for 3 X 10 minutes between each step and stirred with pipette tips. Embryos were counterstained with Hoechst dye for 15 minutes and mounted on slides for imaging. Each embryo was imaged using a Zeiss LSM510 86

microscope (20 X objective); images were pseudo-colored using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). We genotyped each embryo following immunofluorescence analysis by collecting them separately in 10 µl of a 100 µg/ml proteinase K solution prepared in PCR water. Embryos were lysed by incubation at 55°C for 10 minutes, followed by heat inactivated at 95°C for 5 minutes to the eliminate proteinase K. We used these lysates to genotype each embryo by two rounds of PCR using primers that flanked an insertion/deletion in exon 8 (Lossie et al., 2012) (primers listed in Table 4.1).

TableTable 4.1. 4 .Primers1 Primers used for used this study for this study Amplicon Forward Sequence Reverse Sequence Tm Size (bp) Nle1 genotyping primers Nle1 GSP GCTGTAATGTCCTGACTGT cDNA geno CTGTGTCGTACTCTTCAAGGTCAT CTGTGGAGTCATCTTCTCCATATC 60 637 cDNA geno 2nd TCAGACGACTTCACCTTATTCCTG CAGTCAACAGCATATACCTCATCG 62 351 Nle1 gDNA geno TCTCCTTCAGCTCCTTCACTGT TCCAATGGTGGAGTATAGGGTATAA 60 341

cDNA amplification primers ATATGGATCCGGCGCGCCGTCGACT ATATCTCGAGGGCGCGCCGGATCCTT 1st round TTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTT 67

(NH2)ATATGGATCCGGCGCGCCGTC (NH2)ATATCTCGAGGGCGCGCCGGAT 2nd round GACTTTTTTTTTTTTTTTTTTTTTTTT CCTTTTTTTTTTTTTTTTTTTTTTTT 67 Eed cDNA GTGTGACATTTGGTACATGAGGTT ACATTTATGATGGGTCAGTGTTGT 60 148

ABI TaqMan gene expression assay Assay ID Amplicon Size (bp) Ccne1 Mm00432367_m1 63 Ccng1 Mm00438084_m1 89 Cdk9 Mm01731275_m1 150 Mad2I Mm00786984_s1 144 Plk1 Mm00440924_g1 92 Bax Mm00432051_m1 84 Mdm2 Mm01233136_m1 113 Gapdh Mm99999915_g1 107

4.2.2 Quantitative RT-PCR Individual total RNA from each embryo was segregated according to genotype (mutant vs. wild-type) and stage (morula, full blastocyst and hatched blastocyst). cDNA from individual embryos was produced from reverse transcription following the protocol by Tang and Colleagues (Tang et al., 2010). RNA was precipitated by addition of 100% ethanol (1:1; V:V) in a 15 minute SpeedVac run and 87 resuspended in 4.5 µl of lysis buffer prior to reverse transcription using a two- step linear amplification process with barcoded primers (Tang et al., 2010). Products from the first and second rounds were purified using Zymo DNA concentration kits (Zymo Research, Irvine, CA) and eluted in 30 µl of 1 X T10E0.1. Eed expression was used to check the cDNA quality following the first linear amplification step. cDNA was quantified using a Bio-Rad SmartSpec™ Plus Spectrophotometer, and 10 ng of cDNA was used as a template for qRT-PCR in combination with TaqMan® Gene Expression Master Mix (PN#4369514; ABI, Carlsbad, CA) and Taqman Gene-specific probes (Life Technologies, Grand Island, NY) (Table 4.1) on a StepOnePlus™ Real-Time PCR System (Life

Technologies, Grand Island, NY). We assayed a minimum of three biological replicates for each group; cycling reactions were performed in duplicate. The relative expression of each gene was calculated based on the ΔΔCt value, where the results were normalized to the average Ct value of Gapdh. Samples that failed to generate a signal above threshold at the end of the reaction were given a Ct value of 40, as described previously (Lossie et al., 2012) .

4.2.3 Sample Preparation for rRNASeq We staged E3.5 embryos and collected four mutants and four wild-type morulae for this study. To examine the repertoire of rRNA in these samples, we extracted total RNA from single embryos; we genotyped each embryo using 25% of the total RNA, as described (Lossie et al., 2012). Total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Cat#170-8890, Hercules, CA) according to manufacturer’s instructions. The iScript system contains both oligo(d)T and random hexamers, to ensure accurate representation of rRNA transcripts. The entire 20ul solution was concentrated using SpeedVac for 10 minutes to obtain the appropriate sample volume (5 µl) for RNASeq. The entire cDNA sample was used for the Illumina Nextera XT sample preparation (Illumina, Cat# FC-131-1024, San Diego, CA) following the manufacturer’s instruction. Prior to normalization, we analyzed 5 µl from each sample using the High 88

Sensitivity DNA Assay (Agilent Technologies, Santa Clara, CA) for quality control purposes. After sample preparation, each sample was given a unique DNA barcoded index during Tagmentation and library preparation, according to standard protocols (Illumina, Cat# FC-131-1002, San Diego, CA). Pooled libraries were loaded into the MiSeqTM platform (Illumina, San Diego, CA) using a MiSeq™ Reagent Kit v2 (500 Cycles).

4.2.4 Data analysis The filtered Fastq files generated from the MiSeq run were trimmed by the Purdue Genomics Core Facility to remove the index and adapter sequences. The Fastq files were uploaded into CLC Genomics Workbench (CLC Bio, Cambridge, MA) for analysis. We downloaded individual rRNA sequences from Mus musculus [45S rRNA (NR_046233.1); 28S rRNA 5’end (M19226.1); 28S rRNA 3’end (K01366.1); 18S RNA (NR_003278.3); and 5.8S rRNA (J01871.1)] from NCBI (http://www.ncbi.nlm.nih.gov/nuccore/) (Pruitt et al., 2009) and assembled them into one contig that represented the entire, unprocessed 45S rRNA using Sequencher 5.0 (Gene Codes, Ann Arbor, MI). See figure 4.1 for an explanation of rRNA processing.

18S 5.8S 28S 45S rRNA 5’ETS ITS1 ITS2

41S rRNA

18S 5.8S 28S 5’ETS ITS1 ITS2

21S 12S 89

18S 5.8S 28S Figure 4.1 Mammalian rRNA prcessing The 45S pre-rRNA is transcribed by RNA Polymerase I. It is processed into mature 18S, 5.8S, and 28S rRNAs by endonucleases and exonucleases, which cleave the external (ETS) and internal transcribed spacers (ITS). This figure is modified from Burger and Eick, 2013

We delineated the 5’ETS, ITS1 and ITS2 domains to the full-length, in silico- assembled unprocessed rRNA transcript and then mapped the MiSeq reads from each sample to the 45S rRNA sequence under the following parameters: similarity fraction 0.9, length fraction 0.5, nonspecific map-ignored. The remaining parameters were set to default values. We used this data to generate the coverage table that shows the coverage at each base pair (bp). To quantitate the levels of 28S, 18S, 5’ETS, and ITS2 transcripts, determined the range of 28S, 18S, 5’ETS, and ITS2 in our reference contig, and then averaged the coverage within the range of 28S, 18S, 5’ETS, and ITS2. For example, 5’ETS starts from bp 1 to bp 4008 in the 45S contig, we then averaged the coverage between bp 1 to bp 4008 to denote the average coverage of 5’ETS. To reduce amplification bias, we normalized the 28S coverage to 18S, and normalized ITS2 to 5’ETS. Since ITS2 is cleaved when the 32S transcript is processed into the 28S rRNA, we are able to quantify the levels of 32S rRNA and 28S RNA by comparing the coverage between ITS2 and 28S sequence. We computed the ratio between normalized ITS2 and normalized 28S, and then compared this ration between WT and mutant. (See formula in Table 4.3). A Student’s t test was used to determine significance.

4.3 Results 4.3.1 TRP53 Is Activated in Mutant Embryos Previously, our data indicates that apoptosis occurs at E4.5 in mutant embryos (Lossie et al., 2012). Although we failed to detect altered Trp53 expression in mutant embyros (Lossie et al., 2012) , we hypothesized that TRP53 is activated in our mutants. To test that hypothesis, we performed immunocytochemistry to detect expression of acetyl-K378 TRP53 (equivalent to K381 in human) in Nle1l11Jus1/l11Jus1 embryos at E3.5 and E4.5 (Fig. 4.2, Table 4.2). Acetylation of lysine 378 by p300/CBP has been shown to activate TRP53, which induces transcription of downstream genes, eventually leading to apoptosis (Kim et al., 2012; Xu et al., 2011). Using single logistic multiple regression, we determined that acetyl-K378 TRP53 is found solely in homozygous mutants (Likelihood Ratio χ2 = 31.69, P<0.0001) at E3.5 (LR χ2 = 16.57, P<0.0001) and E4.5 (LR χ2 = 15.01, P = 0.0001). We did not find any evidence for an interaction between genotype and stage (LR χ2 < 0.0001; p>0.9999), suggesting no difference was found between E3.5 and E4.5 with the same genotype. Overall, these data indicate that TRP53 is activated at both E3.5 and E4.5, suggesting that apoptotic phenotype could be mediated by acetylation of TRP53. 91

Wildtype Mutant

E3.5

10 µm 10 µm

E4.5

10 µm 10 µm 92

Figure 4.2 Acety-K378 TRP53 expression in Nle1 embryos Acety-K378 TPR53 detection in wild-type and mutant embryos at E3.5 (200X) and E4.5 (200X). Active TRP53 is only detected in the mutants. Red staining indicates the presence of acetyl TRP53 signal and green is the Hoechst staining. Merged images are shown.

Table 4.2 Logistic multiple regression analysis of active TRP53 detection Stage 129/129 L1/129 L1/L1 Total Acetyl-K378 + – + – + – TRP53 E3.5 morula/ 0 7 0 9 3 0 19 blastocyst E4.5 hatched 0 9 0 3 3 0 15 blastocyst

4.3.2 Altered Gene Expression in the Cell Cycle and Apoptosis Pathways Acetylation of Lysine 378 by p300/CBP activates TRP53, which activates transcription of downstream genes, eventually leading to apoptosis (Kim et al., 2012; Xu et al., 2011). Previously, we showed that Cdkn1a is upregulated in the mutants at all stages (morula, blastocyst and hatched blastocyst), and several members of the Wnt pathway are down-regulated (Lossie et al., 2012). Since Cdkn1a is a transcriptional target of p53 that controls cell cycle regulation, we wanted to determine if mutant embryos display misregulation of additional p53 target genes and/or genes involved in the cell cycle. Therefore, we analyzed expression of CyclinE1 (Ccne1), CyclinG1(Ccng1), Cdk9, Mad21, Plk1, Bax and Mdm2 between wild-type and Nle1l11Jus1/l11Jus1 mutant embryos at both the morula 93

and E3.5 hatched blastocyst stages using Taqman assays. Expression of each gene was normalized relative to expression of Gapdh and compared to stage- matched wild-type controls. Wild-type expression was set at a value of one.

Ccne1, Ccng1 Mad2l and Plk1 are involved in cell cycle regulation (Derenzini et al., 2005; Mandal et al., 2010; Ren et al., 2002; van de Weerdt and Medema, 2006; Zhao et al., 2003), while Bax is a regulator of the caspase-mediated apoptotic pathway (Basu and Haldar, 1998; De Angelis et al., 1998; Salakou et al., 2007). Mdm2 is a transcription target of p53 that functions in a feedback loop, as the MDM2 protein inhibits TRP53 (Ard et al., 2002; Deisenroth and Zhang,

2011). We used a Generalized Linear Model (GLM) to calculate DDCt and fold change, as well as to properly control and test for differences among the mutant alleles and stages of development. Our data showed that Ccne1 was expressed at 5.56 fold lower levels in mutant morulae compared to wild-type controls (P=0.021) (Fig 4.3A). However, we did not detect differences in Ccne1 expression between control embryos and mutants at the hatched blastocyst stage (P=0.29). In contrast, Cdk9 expression was 3.22 fold decreased in the mutant hatched blastocysts (P=0.0066) (Fig 4.3A), but was not significantly altered at the morula stage (P=0.68). Ccng1, Mad2I and Plk1 expression did not differ between control and mutant embryos at either stage. Mdm2 and Bax were highly up-regulated in hatched blastocysts (Mdm2: P=0.047; Bax: P=0.041) (Fig 4.3B), but were not significantly different at the morula stage.

In addition, our NOTCH PCR array data revealed that several genes in the Wnt pathway were down-regulated in mutant embryos (Lossie et al., 2012). To determine if the canonical Wnt pathway is altered, we analyzed β-catenin expression. We confirmed that β-catenin was expressed at 2.63 fold lower levels in mutant hatched blastocysts (P=0.041), which suggests that the canonical Wnt pathway could be disrupted in our mutants.

Overall, we found stage-specific downregulation of Ccne1, Cdk9, and β-catenin in Nle1 mutants, while Mdm2 and Bax were highly up-regulated. CCNE1 (Cyclin E1) is required for progression through the cell cycle at the G1/S transition (Mandal et al., 2010), and CDK9 is involved in the replication stress response in S phase (Zhang et al., 2013). Therefore, down-regulation of Ccne1 and Cdk9 indicates that mutant embryos display a major defect at the G1/S transition, which is consistent with previous studies showing that the G1/S transition is blocked under ribosomal stress (Derenzini et al., 2005; Volarevic et al., 2000). Therefore, increased levels of Mdm2 and Bax are expected, as Mdm2 and Bax are transcriptional targets of activated TRP53, and BAX can induce activation of

95 caspases-dependent apoptosis (Ard et al., 2002; Basu and Haldar, 1998; Salakou et al., 2007). This result supported our previous finding that showed the Nle1 mutants were undergoing caspase-dependent apoptosis.

A Down regulated genes 2

1.5

1 WT 0.38 Mutant

Fold Change 0.5 0.18 0.31

0 Ccne1 Cdk9 β-catenin

B Up-regulated genes 100 43.53

60 WT 40 12.86 Mutant 96 Fold Change 20

0 Mdm2 Bax

Figure 4.3 Altered gene expressions in Nle1 mutants Genes that were mis-regulated in the mutants were displayed in fold changes by comparing wilt-type (WT) and mutant expression. Fold change of the wild-type is set to one. (A) Down-regulated genes included Ccne1 in mutant morulae (P=0.021), Cdk9 in mutant hatched blastocysts (P=0.0066), and β-catenin in mutant hatched blastocysts (P=0.041). (B) Up-regulated genes are Mdm2 (P=0.047) and Bax (P=0.041) both in mutant hatched blastocysts.

4.3.3 rRNA Processing Is Disrupted in the Nle1 Mutants Studies in yeast demonstrated that NLE1 (called Rsa4 in yeast) functions in the sequential processing of the 32S pre-rRNA to the mature 28S rRNA (Figure 4.1) (Bassler et al., 2010; de la Cruz et al., 2005). Cleavage of internal transcribed spacers 2 (ITS2) produces the mature 28S rRNA transcript. If NLE1 participates in murine rRNA processing, we expect to observe a disruption of 32S to 28S rRNA processing in our mutants as we predict the function of NLE1 to be conserved across species. To test this, we quantified all potential rRNA products using RNASeq. Because it is difficult to get enough amount of RNA from embryos to perform Northern blot for quantifying rRNA, RNAseq circumvents the technical limitations (i.e. obtaining nanogram quantities of starting material) encountered with pre-implantation embryos and allows us to directly sequence and quantify all of the rRNA products in individual embryos.

We compared the rRNA expression in four wild-type E3.5 morulae and four mutant morulae. The reason for choosing morulae is that we hypothesize that disruption in rRNA processing is the primary cellular insult to the mutant embryos. Since we observed alteration in gene expression as early as the morula stage, we expect to see disruption in rRNA processing at or before the morula stage. If mutation in NLE1 leads to a reduction of 28S rRNA and an accumulation of 32S 97

rRNA, we would expect to see increased levels of internal transcribed spacers 2 (ITS2) in the mutants. Based on the rRNASeq results, we detected a 2.65 fold higher coverage of ITS2/28S in the mutants (P=0.022, t-test) compared to wild type embryos (Fig. 4.4, Table 4.3), while the 18S, 5.8S and total 28S coverage show no differences (p≥0.82), indicating that only the 32S/28S processing is being disrupted.

A 5’ETS 18S 5.8S 28S 45S rRNA ITS1 ITS2 a d B b c Mutant

Overall coverage: 0-730 Counts <5 5-100 101-200201-300 301-400 401-500 >501 C 3.50 2.65 3.00 2.50 2.00 1 1.50

ITS2/28S rRNAITS2/28S 1.00 Relative amount of Relativeof amount 0.50 0.00 mutant WT Figure 4.4 RNASeq analysis of rRNA processing (A) Gene structure of 45S rRNA. Black boxes indicate the mature 18S, 5.8S and 28S region, and black lines are the external or internal transcribed spacers (5’ETS, ITS1 and ITS2) a, b, c, and d denote the coverage regions that are corresponding to the formula in Table 4.3 (B) Heat map of rRNASeq coverage in 45S rRNA. The average coverage from 4 biological replicates was used to generate this heat map. The overall coverage ranges from 0 to 730 counts. (C) Quantification on the relative amount of ITS2/28S rRNA, which is based on the

average coverage on the ITS2 and 28S regions. Student t-test was performed to 98 determine significance (P=0.022).

TableTable 4.3 4 .Average3 Average coverage coverage at each rRNA of subunitseach rRNA subunits Average Average Average Average Average Relative Average Genotype ITS2 28S 5' ETS 18S 5.8S ITS2/ETS 28S/18S Ratio ITS/28S ITS2/28S coverage coverage coverage coverage coverage mutant 7.86 259.128 3.564 197.516 156.482 2.205 1.312 1.681 mutant 1.778 97.609 1.046 80.091 68.154 1.7 1.219 1.395 1.953 2.65 mutant 12.616 721.356 4.908 730.794 540.121 2.572 0.987 2.606 mutant 3.21 71.953 2.179 104.02 34.454 1.473 0.692 2.13

WT 3.034 219.827 3.127 324.31 201.421 0.97 0.678 1.431 WT 0.501 19.741 1.019 32.006 24.211 0.492 0.617 0.798 0.737 1 WT 3.076 391.521 3.53 321.034 308.852 0.871 1.22 0.715 WT 0.002 352.162 0.263 291.704 241.369 0.007 1.207 0.006 Formula a b c d =a/c =b/d =(a/c)/(b/d) P value= 0.022 by Student t-test Mutants have relaitve 2.648 folds higher coverage of ITS2/28s than WT

Abnormal ribosomal biogenesis is usually coupled with disrupted nucleolar structure (Gallenberger et al., 2011; Zhang et al., 2008). Therefore, we examined nucleolar structure by detecting the expression pattern of fibrillarin, a nucleolar protein. In the E3.5 mutants, we observed circular stainings of fibrillarin (Fig. 4.5), which is similar to the structure of immature nucleolus precursor body in E2.5. In mice, the inactive nucleolus precursor body is gradually organized into a functional nucleolus during four- to eight-cell stages and is disappeared in morula stage. For the E3.5 wild-type embryos, fibrillarin staining shows clear punctate structure. 99

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A E2.5 8-cell embryo

10 µm

E3.5 Mutant Blastocyst 1 10 µm

E3.5 Mutant Hatched blastocyst 2 10 µm

3 E3.5 WT Hatched blastocyst

10 µm Hoechst Fibrillarin Merge B 100

1 10 µm 2 10 µm 3 10 µm Mutant blastocyst Mutant hatched WT hatched blastocyst blastocyst Figure 4.5 Immunofluorescent images of nucleolar structure (A) Embryos at indicated stages were stained for fibrillarin (red), showing in the middle panel. Nucleus is labeled using Hoechst fluorescent blue dye (left panel) (200X). Fibrillarin staining reveals the morphology of nucleolar precursor body in E2.5 wild-type embryo. At E3.5, the nucleoli are smaller than E2.5 stage. In the

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E3.5 mutant blastocyst and hatched blastocyst, fibrillarin staining show a circular structure (yellow arrows in (B)) that resembles nucleolar precursor body in E2.5, while the E3.5 wild-type embryo displays normal punctate structure (blue arrows in (B)). (B) Enlarged images from (A).

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4.4 Discussion The I11Jus1 and l11Jus4 alleles harbor missense mutations in the Nle1 gene (Lossie et al., 2012). Studies in Drosophila and Xenopus indicate that NLE1 is a member of the NOTCH pathway that functions as a negative regulator of Notch signaling (Cormier et al., 2006a; Royet et al., 1998b). However, our previous data refuted the possibility that NLE1 functions as a negative regulator of NOTCH signaling during mammalian pre-implantation development, as mutation of Nle1 does not lead to increased expression of key Notch downstream target genes. On the contrary, reports in yeast and plants (which lack NOTCH signaling) demonstrate a role for NLE1 in ribosomal biogenesis (Chantha et al., 2007a; Chantha et al., 2006; Chantha and Matton, 2007; de la Cruz et al., 2005b; Strain et al., 2001).

Here, we present evidence that support a role of NLE1 in ribosomal biogenesis in mammalian pre-implantation development, as l11Jus1 and l11Jus4 mutants show disruption in 32S/28S rRNA processing and abnormal nucleolar structures. Furthermore, Nle1 mutants displayed abnormal activation of TRP53 at both E3.5 and E4.5, disruptions in the cell cycle and eventual apoptosis. The big question is what is the initial insult that leads to activation of TRP53, and initiation of the

apoptotic mechanism. We provided evidence here that the causative insult is 102

likely induction of ribosomal stress that occurs due to mutations in Nle1. Activation of TRP53 induces expression of TRP53 target genes, including Cdkn1a (Lossie et al., 2012), Bax and Mdm2. Activation of Cdkn1a can cause cell cycle arrest, which is also supported by our data, since Ccne1 (Cyclin E1) (which controls GS/S transition) and Cdk9 (which regulates replication) are down-regulated in the E3.5 mutants, implicating a disruption in G1/S transition. Previously, we determined that E4.5 mutants were undergoing caspase- dependent apoptosis (Lossie et al., 2012), which can be initiated by the overexpression of Bax, which is consistent with our data.

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It is not surprising that mouse embryos with defects in genes that encode for proteins involved in ribosomal biogenesis fail during pre-implantation development. For example, mutations in multiple proteins involved in ribosomal rRNA synthesis and processing (e.g. pescadillo-1 (PES-1) (Lerch-Gaggl et al., 2002), RNA polymerase 1 (Yamamura et al., 2008), SURF6 (Romanova et al., 2006), RBM19 (Zhang et al., 2008), EMG1 (Ding et al., 2010), WDR36 (Gallenberger et al., 2011), LIN28 (Vogt et al., 2012)) cause embryonic lethality at the morula stage. However, at a gross level, our mutant phenotype appears around E4.5-E5.5 as they implant but fail to outgrow. This delayed phenotype is probably due to compensation by other protein.

Other WD40 proteins play important roles in rRNA biogenesis. WDR12, which contains an NLE1 domain and several WD40 repeats (Nal et al., 2002), shares 34.8% identity to NLE1 on the protein level, as determined by the SIM program (http://web.expasy.org/sim/) (Huang and Miller, 1991) and on the cDNA level (Kolmogorov-Smirnov P-value=0.0467) (Pearson, 1996). The yeast WDR12 homolog, Ytm1, and the NLE1 homolog, Rsa4, both interact with the AAA+- ATPase Rea1 in directing rRNA processing, (Bassler et al., 2010; Bottcher et al., 2009). Disruptions of Ytm1 and Rsa4 block processing of the 35/27S precursor rRNA to the mature 25S and 5.8S rRNAs (Bassler et al., 2010) (In mammals, 103 these correspond to 36/32S pre-rRNA, 28S rRNA and 5.8S rRNA). Based on the findings described above, it is possible that the function of NLE1 and WDR12 overlap, therefore mutation in NLE1 causes a slightly later phenotype. We can test this hypothesis by disrupting Wdr12 in the Nle1 mutants to see if the embryos arrest earlier.

The association between ribosomal stress and TRP53 activation still needs to be determined in our mutants. Several studies reported that ribosomal stress induces the RP(Ribosomal protein)-MDM2-p53 pathway (Daftuar et al., 2013; Dai and Lu, 2004; Gilkes et al., 2006). That is, disruption of rRNA biogenesis releases ribosomal proteins from the nucleoli to nucleoplasm where they bind to

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MDM2 and inhibit p53 degradation, thereby activating p53 the apoptotic pathway. To test if the RP-MDM2-p53 pathway is activated in our mutants, our next step will be to investigate the possible interaction between ribosomal proteins and MDM2 in our system.

Together, our data implicate NLE1 in ribosomal biogenesis that mediates cell cycle arrest and apoptosis via TRP53 signaling. Intriguingly, ribosomal biogenesis is critical for pre-implantation development in mice as described before. These pathways could provide novel targets for the design of therapeutic interventions for infertility.

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CHAPTER 5. DISCUSSION

5.1 NLE1 and Its Potential Biological Function in Ribosomal Biogenesis Through conducting positional cloning and sequencing analysis, we determined that the l11Jus1 (L1) and l11Jus4 (L4) phenotypes are caused by non- conservative missense mutations in the Nle1 gene. l11Jus1 and l11Jus4 were generated by N-ethyl-N-nitrosourea (ENU) mutagenesis on the C57BL/6J (B6) background, which also contains a non-conservative missense mutation in Nle1. Therefore, l11Jus1 and l11Jus4 homozygotes harbor two mutations within the predicted functional domain of NLE1. Although the endogenous C57BL/6J mutation is not lethal, it potentially interacts with the L1 and L4 mutations, as there is significant loss in viability of l11Jus1 and l11Jus4 on the C57BL/6J background (Lossie et al., personal observation).

NLE1 belongs to the WD40 repeat family of proteins; it contains eight WD40 105 repeat-like domains and a NLE domain at the N terminus. The L4 mutation and

B6 mutation are located in the second WD40 domain, while L1 is in the sixth WD40 domain. Despite the differences in L1 and L4 alleles, the phenotype is indistinguishable and the gene expression data between l11Jus1 and l11Jus4 are not significantly different (Lossie et al., 2012). Gene targeting studies also indicate that the mutant phenotypes of l11Jus1 and l11Jus4 phenocopy the null allele (Cormier et al., 2006a). These data suggest that these two single point mutations in the coding region are each sufficient to disrupt protein function. ENU mutagenesis has demonstrated that single point mutations can recapitulate the null phenotype. For example, Favor et al. (2001) identified nine independent

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Pax6 mutations generated by ENU mutagenesis, out of those, seven mutations expressed phenotypes characteristic of null alleles (Favor et al., 2001). A mouse model of the Human Fragile X Syndrome that harbors a missense mutation in Fmr1I304N (created by targeted knock-in approach) also displayed Fmr1 null-like phenotypes, including increased audiogenic seizure rates, decreased acoustic startle responses, and greater exploratory behavior (Zang et al., 2009).

The fact that L1 and L4 phenocopy the null allele suggests that the WD40 repeat domains are critical for the function of NLE1. The eight WD40 repeats in NLE1 are predicted to form a ‘β-propeller’ structure serves as a role for protein-protein interaction. The L1 and L4 mutations may cause structural change to the β- propeller in NLE1 that disrupts the interaction between NLE1 and its binding partner, leading to a malfunctioned protein complex. So far, no protein in the WD40 family has been found to display enzymatic activity (Xu and Min, 2011). WD40-repeat proteins usually comprise components within higher order molecular complexes that are responsible for sequential or simultaneous interactions. In addition to the interaction through the β-propeller motif, WD- repeat proteins may have other functions provided by the N- or C-terminus (Nal et al., 2002; Steimle et al., 2001).

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NLE1 and WDR12 are structurally similar; they both contain an NLE domain and WD40 repeats. WDR12 is a critical component of the PeBoW complex in mammalian cell lines. The PeBoW complex contains PES1 (pescadillo), BOP1 (block of proliferation) and WDR12. It is required for maturation of the 60S large ribosomal subunit and for cell proliferation in rat fibroblasts(Holzel et al., 2005; Rohrmoser et al., 2007). Knocking down Pes1 and Bop1 by dominant negative mutations or siRNA technology blocks processing of the 36/32S precursor rRNA into the mature 28S rRNA and 5.8S rRNA, and subsequently disrupts maturation of the 60S large ribosomal subunit. Maturation of the 40S small subunit is not affected. Moreover, p53 activity is induced after knockdown, followed by a block

107 of cell cycle in G1 phase and growth arrest (Eick et al., 2006; Holzel et al., 2005; Pestov et al., 2001b).

The PeBoW complex is conserved throughout evolution (Rohrmoser et al., 2007; Woolford et al., 2008). The yeast PeBoW ortholog (Nop7, Erb1 and Ytm1 are orthologs of Pes1, Bop1, and WDR12 respectively) is also involved in ribosome biogenesis and cell cycle progression (Miles et al., 2005). Similar to the mammalian systems, ablation of the proteins leads to disruption in large ribosomal subunit maturation (Miles et al., 2005; Oeffinger et al., 2002). Woolford et al. (2008) further identified how these three proteins interacting with each and what are the interacting domains in each of the three proteins. Specifically, Erb1 and Nop7 form a heterodimer, and then Ytm1 is recruited to assemble into a preribosome (Miles et al., 2005). The recruitment of Ytm1 stabilizes the association between Erb1 and Nop7. Erb1 serves as a bridge that connects Ytm1 and Nop7 via its N-terminus (Pestov et al., 2001a; Woolford et al., 2008). Ytm1 binds to the N-terminus of Erb1 through its C-terminal WD40 domain, while Nop7 interacts with Erb1 via the central region (Woolford et al., 2008).

Since NLE1 and WDR12 are the closest known family members, I predict that

NLE1 interacts with analogous proteins through the WD40 repeats to form a 107

protein complex that is similar to PeBow. By searching the Saccharomyces Genome Database (SGD) (http://www.yeastgenome.org/) (Cherry et al., 2012), I determined 44 proteins that interact with Rsa4. Out of them, 33 proteins are involved in ribosomal biogenesis and the rest are involved the other processes such as DNA replication and ubiquitination. Interestingly, Erb1 and Nop7 are a subset of these 44 proteins reported by a very recent study (Ohmayer et al., 2013). It is likely that NLE1 forms a complex with Erb1 and Nop7 to exhibit the similar function as PeBoW.

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The functional relationship between NLE1 and WDR12 has been identified in yeast. The orthologs for WDR12 (Ytm1) and NLE1 (Rsa4) both interact with the ATPase, Rea1 (Bassler et al., 2010; Bottcher et al., 2009). Rea1 is a conserved pre-ribosomal factor found in the nucleoplasm that is a component of the pre-60S ribosomal subunit (Galani et al., 2004; Nissan et al., 2004). Rea1 is required for the formation of 60S. It is necessary for processing of ITS2 from the 32S rRNA to 28S rRNA and is specifically involved in a late step that generates the mature 5.8S rRNA from the 7S pre-rRNA (Fig. 1.4) (Galani et al., 2004). Electron microscopy demonstrated that Real1 contains two major domains: an ATPases Associated with cellular Activities (AAA) motor domain that binds to the pre-60S particles, and a long flexible tail harboring a metal ion-dependent adhesion site (MIDAS) (Bottcher et al., 2009). The Rea1 MIDAS domain contacts the N- terminal NLE domain of Ytm1 and Rsa4 directly and sequentially during 60S subunit biogenesis. Therefore, the NLE domain can also be called the MIDAS interacting domain (MIDO) (Bassler et al., 2010; Bottcher et al., 2009).

A conserved glutamate (E114 in Rsa4 and E80 in Ytm1) in the MIDO is essential for interacting with MIDAS (Bassler et al., 2010; Bottcher et al., 2009). Both Ytm1 (WDR12) and Rsa4 (NLE1) share this conserved amino acid. This site is

necessary for proper, sequential exportation of the 60S subunit from the 108

nucleolus to the cytoplasm. Mutations in this glutamate residue in Ytm1 cause an early block at the transition of 60S from the nucleolus to the nucleoplasm (Bassler et al., 2010). The analogous glutamate mutation in Rsa4 inhibits exportation of the pre-60S particles from the nucleoplasm to the cytoplasm (Bottcher et al., 2009). Since Ytm1 is recruited first to the pre-ribosomal particle in the nucleolus, it is possible that Ytm1 and Rsa4 share the same binding site on the pre-60S subunit and the yeast PeBoW complex blocks the association of Rsa4 to the nucleolar pre-60S transcript until it is ready to be exported to the cytoplasm. How Rea1 mediates the replacement of Rsa4 from Ytm1 is still unknown. Since all of these proteins are conserved across species, it is possible

109 that in mammalian, Rea1 homolog (MDN1) drives a similar interaction between NLE1 and WDR12 during 60S ribosomal biogenesis. I speculate that MDN1- mediated export of the 60S transcript from the nucleolus to the nucleoplasm, and from the nucleoplasm to the cytoplasm occurs through interactions with WDR12 and NLE1, respectively (Fig. 5.1). To test that, future studies could be conducted to determine the interaction of WDR12 and NLE1 with MDN1 by co- immunoprecipitation and immune-FLIM-FRET detection to identify the intracellular localization of these proteins and protein complexes. This could be conducted in embryonic stem cells from Wdr12 and Nle1 mutants. Furthermore, these studies could be designed to examine the distribution of pre-60S subunits after mutation of the MIDO domain of within NLE1 and WDR12, and immunostaining of RPL27 to track the 60S ribosomal subunits.

109

110

Nucleolus Nucleus Cytoplasm

pre-60S pre-60S

WDR12 NLE1

WDR12 MIDAS NLE1

MDN1

Figure 5.1 Proposed model for the biological function of NLE1 The ATPase MDN1 acts at distinct, successive 60S biogenesis steps. The MDN1 MIDAS domain interacts directly with the N-terminal MIDO domain of WDR12. MDN1 then releases WDR12 from pre-60S particles in an ATP-dependent manner. The released WDR12 is replaced with NLE1 to direct the nuclear exit of pre-60S particles (modified from Baßler et al, 2010).

110

5.2 The rRNASeq study

To the best of my knowledge, we are the first to use RNASeq to analyze rRNA processing. The most common ways to study rRNA processing are by Northern blot and pulse-chase labeling of rRNA by 32P-orthophosphate. These methods require significantly more RNA than can be obtained from a single blastocyst. For example, northern blot require a minimum of 20 µg of total RNA or 4 µg of mRNA (Bergeron et al., 2001). However, a 64-cell embryo produces approximately 1 ng of total RNA. It is prohibitive to collect and genotype enough embryos to conduct this type of assay. Although quantitative RT-PCR could be used to address this

111 question, there are significant potential biases with this technique. First, there are several repetitive sequences in the rDNA, which means that each set of primers must be thoroughly validated prior, and although polymorphisms exist in the repetitive sequences (James et al., 2009), this experiment requires that primers be designed for specific regions of the transcript, which may or may not span enough polymorphisms to ensure accurate amplification, which can lead to nonspecific binding. Second, it is difficult to determine the appropriate control for these experiments. 18S rRNA is often used as a control because it is less variant in expression level under normal condition. However, in our case we are uncertain if 18S rRNA proceesing is also disrupted. Third, in order to quantify different rRNA subunits, one needs to design and validate multiple primer sets. To overcome these limitations and difficulties, we took an advantage of the Next Generation Sequencing (NGS) technology. We used the Illumina MiSeq platform and Nextera Sample Preparation kit to sequence total RNA in individual embryos. Nextera system only requires 1 nanogram of input material and uses a limited number of cycles of PCR amplification for library preparation. As we expect 1 nanogram of total from one blastocyst, the Nextera system allows us to perform rRNASeq in single embryo. RNASeq overcomes many of the limitations of northern blot and reduces the amplification bias of qRT-PCR. RNASeq also

allows us to quantify all of the rRNA products in one single experiment. 111

We are able to assemble the raw reads from MiSeq to the 45S pre-rRNA. By comparing the coverage of different rRNA subunits between the mutants and the wildtype, we detected a 2.65 fold higher coverage of ITS2 in the mutants. This indicates a block of processing from the 32S rRNA to the 28S rRNA because ITS2 is being cleaved during this process under normal condition. This result is consistent with the study in yeast (Bassler et al., 2010; de la Cruz et al., 2005).

112

5.3 Mutations in NLE1 Affect Wnt Signaling

NLE1 was first identified in Drosophila by a suppressor screen of the notchoid phenotype (Royet et al., 1998b). The same research group also determined that NLE1 is a regulator in the Notch pathway. Based on this evidence, we hypothesized initially that NLE1 is also involved in Notch signaling during mouse pre-implantation development. However, our Notch PCR array data refuted this possibility as none of the Notch receptor, ligands, or downstream targets were misregulated in the l11Jus1 mutants. To our surprise, several genes in the Wnt pathway were downregulated. Using Taqman qRT-PCR assays, we determined that β-catenin is expressed at lower levels in the mutants in comparison to wild- type embryso, indicating that the canonical Wnt pathway is potentially downregulated, at least at the transcriptional level.

The mechanisms underlying disruption of Wnt signaling in the Nle1 mutant needs to be elucidated. As we demonstrated that TPR53 is activated in the mutant embryos, it is possible that Wnt is regulated by TRP53. Since TRP53 inhibits self-renewal and promotes differentiation of human ES cells (Jain et al., 2012), the mutants could be inducing Trp53 in an attempt to differentiate cells stuck in a pluripotent state that was brought on by abnormal Wnt signaling. Interestingly, this study links upregulation of Trp53 with induction of microRNA-34 (miR-34). 112 This miRNA cluster is a link between the TRP53 and Wnt pathways (Kim et al., 2011); miR-34a, miR-34b and miR-34c are direct transcriptional targets of TRP53, as activation of TRP53 mediated by DNA damage or oncogenic stress induces miR-34 expression. Chromatin-immunoprecipitation and luciferase assays further showed that TRP53 binds to and activates transcription of the miR-34 genes (He et al., 2007). MiR-34a also regulates TRP53 through post-transcriptional binding to SIRT1, which inhibits translation. SIRT1 deacetylates and reduces activity of TRP53, thereby increasing the active form of TRP53 and inducing expression of TRP53 targets such as Cdkn1a and Puma (Yamakuchi et al., 2008). As a result, Trp53 and miR-34a form a positive feedback loop.

113

Kim and colleagues (2011) further demonstrate that TRP53 and miR-34 are negative regulators of canonical Wnt signaling. Loss of TRP53 function by conditional knock out, siRNA, or introduction of mutant p53 in human cell lines all lead to increased expression of b-Catenin, WNT1 and LRP6. Loss of miR-34a by addition of anti-miR-34a RNAs also induced Wnt activity by preventing binding of miR-34a to its cognant sequences on Wnt1, Wnt3, Lrp6 and β-catenin (Kim et al., 2011). These studies uncover the interaction of TRP53 and Wnt pathways by the intermediate of miR-34a.

Therefore, we expected miR-34a to be overexpressed in the Nle1 mutants. However, we did not find significant upregulation of miR-34a in mutant embryos from the 16-cell stage through the hatched-blastocyst. The expression levels of miR-34a are quite variable in this study; some cells show marked upregulation, while others do not. It is possible that the variable expressivity we found resulted from the inability to precisely stage each embryo. In addition, determining differential expression of microRNAs is difficult, as they often display subtle changes in expression (Stadler et al., 2010), which increases the difficulty of detecting differences in expression. Furthermore, since we showed that the

transcript levels, but not the protein levels of β-catenin were downreguated, and 113

miR-34a most often act to inhibit translation, other mechanisms could account for regulating the transcription of β-catenin.

Another inhibitory effect of TRP53 on β-catenin is through the ubiquitin- proteasome system, which requires an active glycogen synthase kinase 3β (GSK3β) (Sadot et al., 2001). GSK3β has been shown to phosphorylate β- catenin, thus targeting it for proteosome degradation (Wu and Pan, 2010). Induction of wild-type TRP53 by transfection or DNA damage down-regulates β- catenin. The reduction in β-catenin levels was accompanied by abatement in the protein amount and transcriptional activity of β-catenin due to GSK3β targeted

114 proteasome degradation. Mutating the N terminus of β-catenin prevented this effect, where the N terminus compromises degradation of β-catenin by the proteasomes. Blocking GSK3β activity or overexpressing the dominant-negative ΔF-β-TrCP also prevents β-catenin degradation (Sadot et al., 2001).

DNA damage (induced by camptothecin) activates the nuclear pool of GSK3β with no activation of the cytosolic pools (Bijur and Jope, 2001). This research group determined that TRP53 interacts with GSK3β in the nucleus following DNA damage. Furthermore, they found that binding of TRP53 to GSK3β increases the activity of GSK3β, and that activated GSK3β induces transcriptional activity of Trp53 (Bijur and Jope, 2001). Under normal conditions, GSK3β regulates TRP53 levels through phosphorylation of the central domain of MDM2. For efficient degradation of TRP53, MDM2 required this phosphorylation within the central domain to maintain the low basal levels of TRP53 (Kulikov et al., 2005).

As previously discussed, ribosomal stress induces the release of ribosomal proteins (RPs) from the nucleolus, allowing RPs bind to MDM2, thereby TRP53- MDM2 complexes are disrupted, stabilizing TRP53 (Deisenroth and Zhang, 2011). Under these conditions, TRP53 is increased, which allows TRP53 to bind

to GSK3β, thereby resulting in increased levels of active GSK3β. When GSK3β 114

is activated, it phosphorylates β-catenin, leading to proteasome degradation of β- catenin. This can further mediate down-regulation of β-catenin and Wnt signaling. To test if this model is true in our mutants, we can determine if GSK3β is expressed at higher levels and bound to TRP53 in the nucleus of the mutants.

5.4 Altered Nucleolar Structure and p53 Activation in the Mutant

Using immunostaining with antibodies targeting Fibrillarin, we determined the Nle1 mutant blastocysts and hatched blastocysts show nucleolar structures that are similar to the immature nucleolar precursor bodies (NPB). The nucleolus precursor bodies are derived from the parental nucleolar remnant and are

115 established in the pronuclei after fertilization. The NPB appear as a clear sphere delineated from the surrounding nucleoplasm (Flechon and Kopecny, 1998). The sphere is composed of a dense meshwork of thin fibrils. In mice, the inactive nucleolus precursor body is gradually organized into a functional nucleolus during four- to eight-cell stages. The zygotic nucleolus is developed mainly limited to the surface of NPB, while the core of the original NPB remains compact and is still detectable up to the morula stage (Flechon and Kopecny, 1998; Maddox-Hyttel et al., 2007). The fact that we detected an NPB-like structure in mutant blastocysts indicates a potential delay in mature nucleoli formation.

In our mutant blastocysts, not all of the blastomeres display NPB-like structures. If the L1 and L4 mutations in Nle1 can mediate abnormal nucleolar structure, and NLE1 is expressed throughout the embryo, we would expect all of the blastomeres to show altered nucleolar structure. However, only some of the cells in the inner cell mass show this defect, indicating that NLE1 may not be expressed equally in every blastomere. In addition, activated TRP53 is not expressed uniformly throughout the mutant embryos; it is detected mainly in the inner cell mass. This leads us speculate if abnormal nucleoli and active TPR53 are co-localized in the same blastomere.

115

Recently, several studies have reported that mouse blastocysts display a salt- and-pepper distribution of lineage-specific transcription factors (Chazaud et al., 2006; Kang et al., 2013; Plusa et al., 2008; Xenopoulos et al., 2012). The emergence of the epiblast and primitive endoderm within the inner cell mass (ICM) of a mouse blastocyst required initial co-expression of epiblast- and primitive endoderm- specific markers in all cells of the inner cell mass. This is followed by a mutually exclusive pattern, where cells expressing epiblast- and primitive endoderm- specific markers are distributed in a salt-and-pepper fashion independent of their location within the ICM. For example, the epiblast lineage markers (NANOG, SOX2 and OCT4) and the primitive endoderm lineage

116 markers (GATA4, GATA6, SOX7 and SOX17) are mutually exclusive; cells express either epiblast markers or primitive endomerm markers, not both. (Artus et al., 2011; Chazaud et al., 2006; Meilhac et al., 2009; Plusa et al., 2008; Yamanaka et al., 2010). Future studies can be aimed at determining if mutations in Nle1 disrupt detect expression of these markers in the Nle1 mutant to determine if cell lineage decision is disrupted.

5.5 Model for Signaling Regulation in Nle1 Mutants

Based on our findings and other published studies, we developed a model to explain how Nle1 interacts in multiple pathways that are imperative for embryonic development (Fig. 5.2). We demonstrated that NLE1 is involved in ribosomal biogenesis, as the Nle1 mutants show a defect in 32S/28S rRNA processing and display abnormal nucleolar structure. Disruption in ribosomal biogenesis leads to ribosomal stress that mediates activation of TRP53 through binding of ribosomal proteins to MDM2 (Daftuar et al., 2013; Dai and Lu, 2004; Deisenroth and Zhang, 2011; Gilkes et al., 2006). We confirmed that TRP53 is activated in mutant embryos, as acetyl-K378 TRP53 is expressed at higher levels in the mutants. This acetylation can be accomplished by histone acetyltransferases, CBP and p300 (Feng et al., 2005; Krummel et al., 2005). Activation of TRP53 induces two mutually exclusive pathways–cell cycle arrest or apoptosis. First, we found 116 upregulation of Cdkn1a and down-regulation of cell cycle regulators, such as CyclinE1 and Cdk9 at E3.5. CCNE1 (Cyclin E1) is required for the G1/S transition, while CDK9 regulates replication at S phase. Therefore, downregulation of CyclinE1 and Cdk9 suggests that mutant embryos are arrested at the G1/S cell cycle transition at E3.5. This finding is consistent with previous reports that the amount of ribosomes produced can determine G1/S transition (Derenzini et al., 2005; Volarevic et al., 2000). When the damage accumulates we predict that the embryo cannot recover from cell cycle arrest. Therefore, the mutants undergo caspase-dependent apoptosis at E4.5 via overexpression of Bax. We found that Wnt signaling is also downregulated in the

117 mutants. How WNT being misregulated is still unclear. As previously discussed, it could be regulated by activation of TRP53, which triggers GSK3β activity and proteasome degradation of β-catenin, leading to inhibition of the WNT signaling (Sadot et al., 2001).

To further support our model, future studies will need to be conducted. First, we want to determine if ribosomal proteins bind to MDM2 to activate TRP53. Second, we want to investigate if GSK3β is the link between TRP53 and Wnt signaling in our model. Last, we want to identify the biological function of NLE1 in ribosomal biogenesis. We propose that NLE1 has the same function as determined in yeast that it is responsible for the transportation of 60S ribosomal subunit from nucleoplasm to cytoplasm via binding to MDN1 (Bassler et al., 2010). Biochemical studies such as co-immunoprecitiation and FRET can be used to determine this interaction.

117

118

phosphorylation acetylation p53

Ribosomal stress RPs p53 CBP/p300 ? SIRT1 MDM2 NLE1 p53 ? GSK3β BAX CDKN1A β-catenin TCF/LEF BCL2 Cyclin E1 CDK9 caspase WNT signaling Cell Cycle Apoptosis Arrest

Figure 5.2 Proposed model for the regulatory network in Nle1 mutants I propose mutations in NLE1 lead to ribosomal stress and activation of the RPs- MDM2-p53 pathway. Activation of p53 mediates upregulation of Cdkn1a and cell 118 cycle arrest. When the mutants cannot recover from cell cycle arrest, they undergo caspase-dependent apoptosis. Downregulation of the Wnt signaling may result from activation of GSK3β via TRP53. GSK3β phosphorylates β- catenin, thus targeting β-catenin for proteosome degradation.

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VITA

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VITA

EDUCATION Purdue University, West Lafayette, IN Dec 2013 Ph.D., Animal Sciences, Interdisciplinary Life Science (PULSe) Emphasis Areas: Genetics, epigenetics, mouse embryology Advisor: Dr. Amy Lossie GPA: 3.70/4.00 Certificate: Applied Management Principles (AMP), May 2013 Krannert School of Management

National Taiwan University, Taipei, Taiwan Jun 2009 Bachelor of Science., Animal Science and Technology, GPA: 3.84/4.00

University of Illinois at Urbana Champaign, Champaign, IL Spring Exchange study, Animal Sciences, GPA: 3.73/4.00 2009

RESEARCH EXPERIENCE Aug 2009- Graduate Research, Purdue University Dec 2013 • Discover NOTCH, WNT and TRP53 pathways depend upon

each other during mouse pre-implantation development 141 (Publication 3)

• Identify polymorphisms in a commonly used mouse strain (Publication 4) Collaborative Research • Contribute molecular technique and analytical skill to support the findings at IUSOM that examine DNA methylation during neuronal maturation (Publication 2) • Involve in a collaborative epigenetic technology development project ! Identify specific regions of the genome for single cell epigenetics studies ! Present updates and contribute to group discussion in biweekly meetings ! Teach students in collaborative groups challenging skills

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Technology Development • Establish and conduct a new protocol for assessing DNA

methylation using Illumina Nextera and MiSeq system

(Publication 1)

Undergrad Research Reproductive and Development Biology Lab, Jan-May UIUC, Champaign, IL 2009 • Facilitated the study on polycistic ovarian syndrome using Ossabaw pigs and acquired swine In vitro fertilization (IVF) technique

Genetics and Epigenetic Lab, Jul-Aug Purdue University, West Lafayette, IN 2008

• Conducted genotyping and gene expression studies in mutant mice

Animal Reproduction lab, National Taiwan University, Taiwan Sep 2007- • Improved the isolation and differentiation of mouse/swine Dec 2008 mesenchymal stem cells (MSCs) into neurons. Obtained microinjection techniques for gene transfering

LEADHERSHIP EXPERIENCE Student mentor, Purdue University Jul 2010- • Supervise undergrads and rotating grad students to help them Dec 2013 complete their works • Plan daily schedule for new comers in the lab and check their progress 142

• Provide support and advices to mentees in choosing courses and labs

Teaching assistant: Teach labs and recitations at BIOL111 Jan 2013- Fundamentals of Biology II May 2013 • Introduced concept and requirement to students and assisted them in their exercise • Reviewed lecture materials, led group discussion and graded assignments

Resident assistant, National Taiwan University Sep 2006- • Coordinated orientation and social activities for residences Jun 2007

• Handled challenging issues and emergency in the doom

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CAREER DEVELOPMENT • Data Collector Interns of Hunger in America Study, Food Apr 2013- Finders Food Banks, Inc present ! Interact with clients at local food pantries and help clients take surveys ! Information collected will help the public better understand food insecurity in our local area and help Food Finders provide better services • ABE 691 Life of a Faculty Entrepreneur: Discover, Spring Development, Translation 2012 ! Presented a business plan for a biotech startup company ! Learned technology development and commercialization strategies • Associate staff at the Center for International Agricultural Nov 2007- Education and Academic Exchange, National Taiwan University Dec 2008 (NTU) ! Interacted with colleges in China and in the US to establish exchange agreements ! Introduced Taiwanese culture and biodiversity to international scholars • Web manager at Department of Animal Science and Sep 2006- Technology, NTU Jun 2007 ! Created and maintained the website for prospective students

SKILLS

• Fluent in English and Mandarin writing, speaking and listening

• Certificate in Good Clinical Practices (GCP) and Good Document 143

• Molecular Techniques ! DNA, RNA isolation from single blastocysts ! Library preparation and data analysis for next-generation sequencing ! Bisulfite sequencing and methylation-specific PCR ! qRT-PCR (Taqman, SYBR green) • In situ hybridization, Immunocytochemstry, cloning and genotyping • Rodent Husbandry Techniques ! Dissection for mouse embryo collection from E0.5 to E13.5 ! Maintaining, breeding and weaning of mutant mouse lines ! Euthanasia by carbon dioxide inhalation and cervical dislocation • Data Analysis ! CLC workbench, Sequencher, SAS, HTML web design, Photoshop, MS Office

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SERVICES/ACTIVITIES • Judge at Lafayette Regional Science and Engineering Fair Mar 2013 • Raised fund to Purdue Badminton Club for equipment Aug 2011- maintenance and traveling May 2012 • Taught science to K6-12 students at Burnett Creek Elementary Feb 2013, school, West Lafayette, IN, Sunnyside Middle School, Lafayette Oct 2011 • Coordinated orientation and social activities for incoming May 2010- Taiwanese student Apr 2011 • Decreased the usage of disposable tableware and printer paper Sep 2007- at National Taiwan University when joining the Roots & Shoots Jun 2009 program in the Jane Goodall Institute

AWARDS/GRANTS • Student Scholarship to IMGC, International Mammalian Genome 2010-2012 Society • LOUJA Graduate Travel Award. Purdue Animal Sciences May 2012 st • 1 Place of Graduate Student Poster Competition, Purdue Animal Apr 2012 Sciences • Travel award, Purdue PULSe Graduate Office 2010-2013 • Presidential Award, National Taiwan University 2007-2008 • Scholarship from the Alumni association, Animal Science and Dec 2007 Technology, NTU

PROFESSIONAL AFFILIATIONS • The Genetic Society of America (GSA) • International Mammalian Genome Society (IMGS) • American Society of Animal Science (ASAS) 144 • Chinese Society of Animal Science (CSAS)

PUBLICATIONS 1. Lo, CL., Bai Y., Shen F., Lelievre, SA., Lossie, AC., (2013). MiSeq Uncovers Altered BRCA1 Methylation in 3D Breast Cancer Cultures (In preparation) 2. Zhou FC., Resendiz, M., Lossie, AC., Chen, Y., Lo, CL. (2013). Cell-wide DNA Demethylation in the Maturing Brain. (Submitted to Cell, Jan 25, 2013) 3. Lossie, AC., Lo, CL., Baumgarner, KM., Cramer, MJ., Garner, JP., Justice, MJ. (2012). ENU Mutagenesis Reveals that Notchless Affects Cdkn1a and Several Members of the Wnt Pathway During Murine Pre-implantation Development. BMC Genet, 12;13(1):106 4. Lo, CL., Shen, F., Baumgarner K, Cramer, MJ, Lossie, AC. (2012). Identification of 129S6/SvEvTac-Specific Polymorphisms on Mouse Chromosome 11. DNA and Cell Bio, 31(3): 402-414.

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PRESENTATIONS • Lo, CL., Sherill JB. and Lossie, AC., Notchless Impacts Multiple Signaling Pathways During Pre-implantation, American Society of Human Genetics (ASHG) 2013 Annual Meeting, Oct 22-26, 2013, Boston, MA, USA • Lo, CL., Sherill JB. and Lossie, AC., Notchless Impacts Multiple Signaling Pathways During Pre-implantation, 2013 Sigma Xi Graduate Student Research Awards Competition, Feb 13, 2013, Purdue University • Lo, CL., Sherill JB. and Lossie, AC., Notchless Impacts Multiple Signaling Pathways During Pre-implantation Development, 26th International Mammalian Genome Conference (IMGC), Oct 21-24, 2012, St. Pete Beach, Florida, USA (Selected talk in the main conference) • Lo, CL., Chun H. and Lossie, AC., Optimization of RNA-seq techniques to Capture Transcriptome from Single Blastocyst, PULSe Retreat and Poster Session, Aug 16, 2011, West Lafayette, IN • Lo, CL., Chun H. and Lossie, AC., Optimization of RNA-seq techniques to Capture Transcriptome from Single Blastocyst, Mouse Genetics Conference, Jun 22-25, 2011, Washington DC • Lo, CL., Buit T., Shen F. and Lossie, AC., Gene Expression Studies of ncRNAs within The Odz4 Locus, 24th International Mammalian Genome Conference (IMGC), Oct 17-20, 2010, Crete, Greece • Lo. CL., Cramer, MJ., Klein J. and Lossie, AC., Identification of Allele-specific ncRNAs Expression in Mouse Odz4, Celebration of Science Graduate Student Award Banquet, May 14, 2010, West Lafayette, IN • Lo, CL., Hsiao SH., Wu CY., Peng SY., Cheng CC., and Wu SC., Neuronal Differentiation Potency of EGFP Mice Bone Marrow-Derived Mesenchymal Stem Cells, Chinese Society of Animal Science Conference, Dec 07, 2008, Taipei, Taiwan

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