INVESTIGATION OF CHD7 FUNCTION IN DEVELOPMENTAL MODELS OF

CHARGE SYNDROME

by

STEPHANIE ANN BALOW

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation advisor: Peter C. Scacheri, Ph.D.

Department of Genetics and Genome Sciences

CASE WESTERN RESERVE UNIVERSITY

May 2014

To my parents, for their constant love and support

! "! Table of Contents

List of tables!!!!!!!!!!!!!!!!!!!!!!!!!!!.... 4 List of figures!!!!!!!!!!!!!!!!!!!!!!!!!!!.. 5 Acknowledgements!!!!!!!!!!!!!!!!!!!!!!!!... 7 Abstract!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 9 Chapter 1: Introduction and Background!!!!!!!!!!!!!!.. 11 Overview of chromatin remodeling and development!!!!!!!!12 Chromodomain helicase DNA-binding (CHD) protein family!!!!!13 Subfamily I: CHD1 and CHD2!!!!!!!!!!!!!!...13 Subfamily II: CHD3, CHD4, and CHD5!!!!!!!!!!!15 Subfamily III: CHD6, CHD7, CHD8, and CHD9!!!!!!!. 17 CHD7!!!!!!!!!!!!!!!!!!!!!!!!!!!! 18 Molecular function of the CHD7 protein!!!!!!!!!!...18 Expression of CHD7 during development!!!!!!!!!. 22 CHARGE syndrome!!!!!!!!!!!!!!!!!!!!!!23 Overview!!!!!!!!!!!!!!!!!!!!!!!.. 23 Expansion of the CHARGE syndrome clinical presentation!! 23 Ocular coloboma!!!!!!!!!!!!!!!!!.24 Heart defects!!!!!!!!!!!!!!!!!!.. 24 Choanal atresia!!!!!!!!!!!!!!!!!.. 24 Growth retardation!!!!!!!!!!!!!!!!. 25 Genital abnormalities!!!!!!!!!!!!!!!. 25 Ear anomalies!!!!!!!!!!!!!!!!!!. 26 Other common phenotypes!!!!!!!!!!!!.. 26 Mutation spectrum!!!!!!!!!!!!!!!!!!!. 27 Clinical overlap with other syndromes!!!!!!!!!!! 28 Animal models of CHARGE syndrome!!!!!!!!!!...29 Regulation of rDNA expression!!!!!!!!!!!!!!!!....31 The nucleolus and building a ribosome!!!!!!!!!!...32 Structure and transcriptional regulation of rDNA!!!!!!!32 Epigenetic regulation of rDNA repeats!!!!!!!!!!....35 FBXL10 is an epigenetic modifier of rDNA expression!!!.....36 Dysregulation of ribosome biogenesis!!!!!!!!!!!!!.!39 The nucleolar stress response!!!!!!!!!!!!!!..39 Ribosomopathies!!!!!!!!!!!!!!!!!!!.!43 Summary and Research Aims!!!!!!!!!!!!!!!!!...46

Chapter 2: Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a zebrafish model of CHARGE syndrome!!!!!!...!..49 Abstract!!!!!!!!!!!!!!!!!!!!!!!!!...!...50 Introduction!!!!!!!!!!!!!!!!!!!!!!!!...!.52 Results!!!!!!!!!!!!!!!!!!!!!!!!!!...!.55 Organization of the zebrafish chd7 gene!!!!!!!!!...... 55 chd7-morpholino gene targeting recapitulates major features of CHARGE syndrome!!!!!!!!!!!!!!!!!!.55

! #! chd7 morphants develop defects in neural crest-derived craniofacial cartilage!!!!!!!!!!!!!!!!!!..63 Chd7 is required for normal cellular proliferation during zebrafish development!!!!!!!!!!!!!!!!!..68 Fbxl10 regulates rRNA levels during zebrafish embryogenesis!!!!!!!!!!!!!!!!!!!!...72 Rescue of chd7 morphant phenotype upon knockdown of fbxl10!!!!!!!!!!!!!!!!!!!!!!!!...76 Analysis of rRNA and cell cycle genes in fbxl10/chd7 double morphants!!!!!!!!!!!!!!!!!!!!!!...84 Discussion!!!!!!!!!!!!!!!!!!!!!!!!!...87 Materials and methods!!!!!!!!!!!!!!!!!!!!..91

Chapter 3: Dissecting CHD7 functions in a human iPS cell model of CHARGE syndrome..!!!!!!!!!!!!!!!!!!!!!!!..98 Introduction!!!!!!!!!!!!!!!!!!!!!!!!!.99 Results!!!!!!!!!!!!!!!!!!!!!!!!!!...102 Early characterization and preliminary data in CHARGE syndrome patient-derived iPS cells!!!!!!!.102 Discussion!!!!!!!!!!!!!!!!!!!!!!!!!..111 Materials and methods!!!!!!!!!!!!!!!!!!!!.114

Chapter 4: Discussion and future directions!!!!!!!!!!!!..117 Summary!!!!!!!!!!!!!!!!!!!!!!!!!!118 Discussion and future directions!!!!!!!!!!!!!!!!120 Implications of zebrafish chd7 morphant model studies!!!120 Does chd7 regulate cellular proliferation in specific tissues?...... 120 What is the molecular mechanism of chd7 morphant rescue by fbxl10 knockdown!!!!!!!!!!!...121 Are these findings applicable to mammalian development?...... 125 Future experiments investigating the nucleolar and nuclear functions of CHD7 using iPS cells as a developmental model of CHARGE syndrome!!!!!!!..126 Summary remarks!!!!!!!!!!!!!!!!!!!!!....132

Bibliography!!!!!!!!!!!!!!!!!!!!!!!!!!....134

! $! List of tables

Chapter 2 Table 2-1. CHD7 mutant phenotypes comparison across multiple species!!!!!!!!!!!!!!!!!!!!!!!.62

Table 2-2. PCR primers designed to measure gene expression!!!!!97

! %! List of figures

Chapter 1 Figure 1-1. Members of the CHD family!!!!!!!!!!!!!!!..14

Figure 1-2. Simple schematic of a single mouse rDNA gene!!!!!!..34

Figure 1-3. Schematic of FBXL10 protein domain composition!!!!!..37

Figure 1-4. Overview of the nucleolar stress response!!!!!!!!!40

Figure 1-5. Disruption of ribosomal biogenesis alters nucleolar structure resulting in the stabilization of p53!!!!!!!!!!!..42

Chapter 2 Figure 2-1. Morpholino targeting of the zebrafish chd7 RNA induces an aberrant transcript!!!!!!!!!!!!!!!!!!..56

Figure 2-2. Zebrafish chd7 targeting results in CHARGE-like phenotypes!!!!!!!!!!!!!!!!!!!!!..60

Figure 2-3. chd7 morphants display variable defects in craniofacial cartilage!!!!!!!!!!!!!!!!!!!!!!...64

Figure 2-4. Sectioning of chd7 morphants reveals moderate to severe craniofacial cartilage abnormalities!!!!!!!!!!!.66

Figure 2-5. chd7 targeting impairs cellular proliferation!!!!!!!!...69

Figure 2-6. Targeting of zebrafish fbxl10 transcript modulates pre-rRNA expression!!!!!!!!!!!!!!!!!!!!!...73

Figure 2-7. Modulation of fbxl10 expression results in normal development of gross anatomical and craniofacial structures!!!!!!...... 75

Figure 2-8. Modulation of fbxl10 expression rescues CHARGE-like phenotypes and improves cellular proliferation defects.!!.77

Figure 2-9. Changes in cellular proliferation correlate with embryonic head size!!!!!!!!!!!!!!!!!!!!!!79

Figure 2-10. Sagittal sectioning of 4 dpf chd7/fbxl10 double morphants indicates variable restoration of ceratobranchial cartilage development!!!!!!!!!!!!!!!!!!!!..82

! &! Figure 2-11. Gene expression changes in cell-cycle regulators in chd7/fbxl10 double morphants!!!!!!!!!!!!!85

Chapter 3

Figure 3-1. The locations of CHD7 mutations in CHARGE syndrome patient-derived iPS cell lines!!!!!!!!!!!!!..103

Figure 3-2. Reprogrammed CHARGE syndrome patient cells express multiple markers of pluripotency!!!!!!!!!!!....105

Figure 3-3. CHD7 expression is significantly reduced in CHARGE syndrome patient-derived iPS cells!!!!!!!!!.!..106

Figure 3-4. CHD7 protein localizes to different cell sub-compartments in different cell types!!!!!!!!!!!!!!!!....109

Figure 3-5. Decreases in CHD7 expression do not affect 45S pre-rRNA expression in iPS cells!!!!!!!!!!!!!!!....110

Chapter 4 Figure 4-1. Model of interactions potentially involved in molecular mechanism of rescue in chd7/fbxl10 double morphants!....123

Figure 4-2. Multiple histone tail modifications demarcate enhancer activity and expression of target genes!!!!...... 130

! '! Acknowledgments

First and foremost, I would like to thank my advisor, Peter Scacheri, for his expertise, guidance, and his commitment to my success. My time in his laboratory has been invaluable and has helped me build strong skill sets both at the bench and in communication. It has been clear over these past 5 years that he has a vested interest in my well-being, and I appreciate this immensely. I am truly grateful for all his help, patience, and encouragement, which have helped mold me into a more confident and independent scientist.

I would also like to thank my committee members Ron Conlon, Peter

Harte, and Brian McDermott for their support and encouragement throughout my graduate career. They continuously provided vital outside perspective to my project by asking questions and providing suggestions. I am also indebted to our collaborator, Paul Tesar, for his help and advice. I would also like to extend my thank you to the entire departmental faculty and administrative staff. I truly believe that my interactions with the faculty over the years, whether in the classroom or casually in the hallway, have been vital in shaping my progress.

I would also like to thank the past and present members of the Scacheri laboratory, as without them, none of this would be possible. Although everyone has been a great source of camaraderie both personally and scientifically, there are three members that I would like to especially thank. I am particularly indebted to Cindy Bartels for her technical support and her overall willingness to drop everything to help me in times of need. I would also like to thank Olivia Corradin, who for the past several years, has not only been my baymate but also a good

! (! friend. She is always a constant source of positivity and has always been willing to listen and discuss. Lastly, I am beyond grateful for my friendship with my previous baymate and “lab brother”, Gabe Zentner. I owe him more than I can say for the scientific discussions that helped shaped the beginnings of this project and also for his mental support along the way.

Furthermore, I would like to thank my friends from both here and afar for all their support. I would like to especially thank my best friend, Meghana Gupta.

We met during the first week of graduate school orientation and I could not have been luckier to have met such a loyal and caring friend. She has been there for me in the best and worst of times over the years and, without her, I think I would have lost my sanity ages ago. I would also like to thank my dear friends Jason

Heaney, Lorrie Rice, Meetha Gould, Jackie Butler, Nicola Brynolf, and Stacie

Zurek.

Lastly, I would like to thank my family. My whole life they have been my pillars of encouragement and this time spent in graduate school was no exception. In times of uncertainly, they have firmly believed that everything happens for a reason and they constantly reminded me of our family motto –

“Work hard, you’ll be glad you did!” I cannot even begin to express my love and gratitude for all that they have done.

! )! Investigation of CHD7 Function in Developmental Models of CHARGE Syndrome

Abstract

by

STEPHANIE ANN BALOW

Epigenetic regulation of the genome is essential for regulating gene expression to ensure proper tissue differentiation and patterning during embryonic development. The importance of epigenetic regulation is best highlighted by the large number of disease that arises from its misregulation. One particular epigenetic regulator that signifies this importance is the chromatin remodeling protein, CHD7. Changes in CHD7 expression during development results in a congenital disorder known as CHARGE syndrome comprising a complex constellation of developmental abnormalities in humans affecting multiple organ systems such as the eyes, ears, and heart. The CHD7 protein functions to regulate both nucleoplasmic and nucleolar gene expression; however, the respective contributions of each of these functions to proper embryonic development and of their misregulation to CHARGE syndrome is not clear. To this end, we sought out to model changes in CHD7 using the zebrafish as a model for early embryonic development. Targeting of the zebrafish chd7 homolog resulted in developmental abnormalities in multiple organ systems overlapping with those affected in CHARGE syndrome patients. Further investigation in to the chd7 morphant zebrafish revealed global decreases in

! *! cellular proliferation accompanied by increased expression of cell-cycle regulator genes. By targeting the expression of fbxl10, a known negative regulator of both rRNA expression and cellular proliferation, we successfully modulated the levels of cellular proliferation and significantly restored the majority of morphological abnormalities. Collectively, these studies indicate that CHD7 plays a significant role in regulating cellular proliferation during embryonic growth and development and provides a novel explanation for the pathogenesis of CHARGE syndrome.

To further investigate this possibility, we next sought to test this hypothesis in a human model of embryonic development, or rather, human induced pluripotent stem (iPS) cells. Using CHARGE syndrome patient fibroblast lines, we successfully reprogrammed these cells to a pluripotent state that express several key genes including OCT4 and NANOG. In our initial characterization of these cells, protein expression assays revealed that CHD7 expression levels are severely reduced and are more consistent with a complete loss-of-function rather than a haploinsufficiency. Additionally, CHD7 protein subcellular localization is cell-type dependent. While these results are preliminary, our studies show that the use of CHARGE syndrome patient iPS cell technology can provide an invaluable model to study CHD7 function during development. Furthermore, through their differentiation potential, this model will allow for investigation into the relevance of cellular proliferation defects in the etiology of CHARGE syndrome.

! ! !

! "+! Chapter 1

Introduction and Background

! ""! Overview of chromatin remodeling and development

Eukaryotic genomes are packaged into chromatin, comprised of nucleosomes formed by the wrapping of ~147 base pairs (bp) of DNA around histone octamers. These units are then further packaged and compacted into progressively high-order structures to form chromosomes. Chromatin restricts

DNA access, and processes that require access to DNA, such as transcription, replication, and DNA repair, must contend with the inhibitory nature of chromatin.

For example, positioning of nucleosomes is critical for the action of cis-regulatory elements, such as gene promoters or enhancers, which must be exposed and bound by the transcriptional machinery (1). The importance of chromatin structure is illustrated best by the large number of diseases that result from its misregulation, such as Rett syndrome (2), CHARGE syndrome (3), alpha- thalassemia x-linked intellectual disability syndrome (4), and even cancer (5). It is due, in part, to this increased awareness of the link between chromatin structure and human disease that the topic of chromatin-level regulation of genetic processes has received renewed interest.

Regulation of chromatin structure and thus, gene expression, can occur through DNA methylation, covalent histone tail modifications, and ATP- dependent chromatin remodeling. ATP-dependent chromatin remodelers function by sliding nucleosomes to expose DNA segments or altering the composition of the histone octamer core (1). There are four major families of ATP-dependent chromatin remodelers: switching defective/sucrose nonfermenting (SWI/SNF), imitation switch (ISWI), inositol requiring 80 (INO80), and chromodomain

! "#! helicase DNA-binding (CHD). Together, these families are crucial for achieving normal development, cell differentiation, and maintenance of cellular identity (6-

8).

Chromodomain helicase DNA-binding (CHD) protein family

The CHD family is a group of highly conserved chromatin remodeling proteins that can be found in all eukaryotic organisms, with a single member in budding yeast to a complement of nine members in vertebrates (9). Members of this family contain several well-characterized protein domains including a set of tandem chromodomains at the amino terminus, a SWI2/SNF2 family ATPase domain, and a helicase domain. The tandem chromodomains recognize and bind nucleosomes and have a unique architecture that confers binding specificity for methylated H3K4 residues (9). The ATPase domain and its DEXDc sub-domain are both critical for ATP binding and hydrolysis (10). There are nine CHD family members in vertebrates and these can be further classified into sub-families based on the presence of additional functional domains (Figure 1-1).

Subfamily I: CHD1 and CHD2

The Subfamily I is defined by two CHD family members – CHD1 and

CHD2. Both of these proteins possess a defined DNA-binding domain and are known to bind A+T rich DNA sequences (9). CHD1 has been studied in multiple model organisms including yeast, Drosophila, and the mouse and has a general role in transcriptional regulation. It is known to associate with the open chromatin of actively expressed genes and can mediate binding of post- transcriptional

! "$!

Figure 1-1. Members of the CHD family. The CHD protein family has three protein domains in common amongst all members. These include tandem chromodomains (green), a SWI2/SNF2 family ATPase domain (purple) with a

DEXDc subdomain (yellow), and a helicase domain (red). Each subfamily has a well-defined domain distinguishing it from other subfamilies. Subfamily I has a well-defined DNA binding domain (orange), while tandem PHD domains (light blue) define members of the Subfamily II and BRK domains distinguish members of Subfamily III (navy blue).

! "%! factors and RNA splicing factors of the spliceosome (11-13). Studies in yeast also revealed that CHD1 is a component of the SAGA and SLIK chromatin remodeling complexes used to recognize sites of H3K4 tri-methylation and promote histone acetyltransferase activity (14). Furthermore, mutations in CHD1 and its Drosophila homolog have developmental consequences. In mouse embryonic stem cells, CHD1 expression is required for the maintenance of pluripotency. Loss of expression results in a decreased ability to differentiate cells to the endodermal lineage (11). Drosophila chd1 is also required for incorporation of the H3.3 histone variant for the fertilization of developing embryos (15).

While CHD2 is not as well characterized as CHD1, there is a clear requirement for this protein during development. Mutant mouse models of CHD2 display multiple systemic defects including abnormalities of the kidney, spleen, and liver, growth retardation, hematopoietic stem cell differentiation, and lordokyphosis (16-19). Homozygous null mutants are perinatal lethal (16). It was recently reported that human CHD2 mutations have been linked to epileptic encephalopathy (20). CHD2 has also been linked to abnormalities in the deposition and clearance of multiple histone variants. Loss of CHD2 expression resulted in abnormally slow removal of !H2AX after DNA damage and loss of

H3.3 in differentiating muscle cells (18, 21).

Subfamily II: CHD3, CHD4, and CHD5

Members of the second CHD subfamily are characterized by the presence of two tandem amino-terminus plant homeodomain (PHD) – zinc-finger-like

! "&! domains. These domains are implicated in transcriptional regulation through association with chromatin (22, 23). Both CHD3 and CHD4 are associated with the nucleosome remodeling and histone deacetylase (NuRD) complex. This complex is comprised of multiple transcriptional repressor proteins, such as

MTA1, MTA2, MTA3, RbBP7, RbBP4, and these interact with HDAC1 and

HDAC2 (6). The specific composition of the NuRD complex can vary between different cell types and alter its functions (24).

CHD4 has been implicated in several developmental and cellular processes. It was reported that CHD4 is required for T-cell development (25, 26) and also in the establishment of germ layer boundaries in the developing

Xenopus embryos (27). CHD4 is also a regulator of rDNA activity by regulating the expression of rDNA inhibitor, Tip5 (28). CHD3 has been implicated as a repressor of viral gene expression (29). CHD3 was demonstrated to repress the expression of the Herpes Simplex Virus genes through epigenetic modifying its genome to a heterochromatic state.

Mutations or deletions of the CHD5 gene are commonly associated with neuroblastoma (30). CHD5 expression is restricted mainly to neural tissue and is also reported to regulate gene expression through interactions with a separate

NuRD-like complex (31, 32). CHD5 is required for neuronal-specifc genes and regulates cell fate through the p19Arf/p53 pathway (32, 33). Furthermore, CHD5 is required for maintaining repression of several Polycomb target genes.

Misregulation of these genes results in aberrant expression and loss of cell potential to differentiation into mature neurons (30).

! "'! CHD subfamily III: CHD6, CHD7, CHD8, and CHD9

Members of the subfamily III are distinguished by the presence of a single or tandem BRK domain at the carboxy-terminus. While the in vivo function of these domains is yet to be tested, in vitro pull-down experiments suggest that the

BRK domains of both CHD7 and CHD8 can interact with the insulator protein,

CTCF (34, 35).

CHD6 and CHD9 are relatively understudied compared to the rest of the family. CHD6 is proposed to regulate transcriptional initiation and elongation as it colocalizes with both hypo- and hyper-methylated forms of RNA polymerase II

(36). It was also associated with multiple transcription factors including p300 and

SRC1 (36). Additionally, CHD6 was implicated in transcriptional repression of viral genes after infection with either the human papillomavirus and influenza virus (37, 38). No human diseases are conclusively associated with CHD6, but a balanced translocation was reported in one patient disrupting the CHD6 gene.

This patient presented with both severe mental retardation and brachydactylyl of the toes (39). Homozygous mutant mouse models lacking exon 12 of CHD6 are viable and morphologically indistinguishable from controls. However, these mutants do display differences in balance and coordination (40).

CHD9 or chromatin-related mesenchymal modulator (CReMM) is associated with skeletal tissue development and specificity. CHD9 is highly expressed in osteoprogenitor cells. It is reported to interact with skeletal tissue- specific at promoters of such genes as CBAF1, osteocalcin, myosin, and upon

TGF" stimulation also biglycan and BMP4 (41, 42). Marom et al also reported

! "(! that CHD9 interacts with the glucocorticoid receptor suggested that it plays a role in regulating osteoprogenitor cell transcription in response to hormonal cues (43).

The CHD8 protein is reported to interact with a number of other proteins or protein complexes to regulate gene expression. CHD8 has been shown to interact with CHD7 (44), elongating RNA Polymerase II (45), and CTCF (34). It was demonstrated that CHD8 interacts with CTCF at insulator sites to prevent heterochromatin spreading. Knockdown of CHD8 expression resulted in hyper- methylation of insulate sites near C-MYC and BRCA1 (34). CHD8 has also been reported to interact with the histone modifying WAR complex (WDR5, Ash2L,

RbBP5) to regulate expression of such genes as HOXA2 and "-catenin target genes (46, 47). Furthermore, CHD8 has been implicated in controlled cellular proliferation and cell survival (48, 49). While heterozygous CHD8 mutant mice are normal and fertile, homozygous mutants are embryonic lethal as they display both growth arrest and massive apoptosis by E7.5 (50).

CHD7 is the largest member of the CHD family and also one the most widely studied. It has a strong connection to the congenital disorder known as

CHARGE syndrome and, as such, many groups are dedicated to uncovering the molecular and developmental functions of this chromatin remodeler.

CHD7

Molecular functions of the CHD7 protein

Since the relationship between CHD7 and CHARGE syndrome was uncovered, there has been a steady interest in understanding the molecular

! ")! functions of this protein. Similarly to other CHD family members, CHD7 has been hypothesized to be a chromatin remodeler and functions to regulate gene expression. However, this function is far from straightforward, as CHD7 has been implicated in the regulation of tissue patterning, cellular differentiation, and cellular proliferation all in a cell-type specific context.

Confirming the role of CHD7 as a chromatin remodeler, CHD7 utilizes

ATP to catalyze nucleosome sliding (51). However, a series of studies undertaken by Schnetz et al was seminal to our understanding of CHD7 function on a genomic scale (52, 53). Through a series of ChIP-seq experiments, CHD7 was found to bind to thousands of loci across the genome in a cell-type specific manner. These loci had characteristics of classic enhancer elements including

DNaseI hypersensitivity, distal location relative to transcription start sites, correlation with H3K4me1/2, and ability to activate transcription in luciferase assays (52). In a follow-up study using mouse embryonic stem cells as a model, it was shown that CHD7 colocalizes with the pluripotency transcription factors

Oct4, Sox2, Smad1, and Stat3 (53). It was also found that there was an enrichment of CHD7 binding sites near embryonic stem cell-specific genes; however, loss of CHD7 did not affect self-renewal or pluripotency of these cells. It was hypothesized that CHD7 functioned as a transcriptional “rheostat” to fine- tune the expression of cell type-specific genes.

Since these initial studies, CHD7 has been reported to interact with a number of proteins to regulate gene expression in multiple tissue and cell types.

These proteins include BRG1 and PBAF (54, 55), R-Smads (56), Sox2 (57), and

! "*! CHD8 (44). In a study by Bajpai et al, CHD7 was described as interacting with the BRG1-containing complex PBAF, and this interaction was crucial for the expression of several tissue-specific genes, such as TWIST and SOX9, and was required for viability of migratory neural crest cells (54). In another study, it was found that CHD7 interacts with several R-Smads (Smad 1, 5, and 8) upon BMP stimulation in the mouse embryonic heart. CHD7 specifically localized to the enhancer and promoter of Nkx2.5. Changes in Chd7 expression reduced the expression of several critical cardiogenic BMP target genes including Nkx2.5 resulting in reduced cellular proliferation and a loss of atrioventricular valve cushion development (56). Furthermore, CHD7 has been shown to interact with

Sox2 in neural stem cells to regulate gene targets of the Notch and Sonic

Hedgehog signaling pathways such as the genes Gli2 and Jag1 (57). Loss of

SOX2 expression results in anophthalmia, which is sometimes found in CHARGE syndrome. Approximately 58% of SOX2 binding sites colocalized with CHD7 sites in neural stem cells indicating that perhaps expression changes in these common regulatory targets of SOX2 and CHD7 may help explain the disease similarities.

Additionally, CHD7 is reported to regulate the expression of key transcription factors required for developmental tissue patterning and differentiation in the heart (58), the developing neural tube (59), and the central nervous system (60-63). Common to many CHARGE syndrome patients are neural abnormalities including mental retardation and olfactory bulb defects, but the pathology of these defects is not yet clear (60). Potential explanations for

! #+! these defects were uncovered in mouse models. CHD7 was found to interact with a conserved BMP4 regulatory element in the developing telencephalon.

Heterozygous mutations of Chd7 resulted in expanded Bmp4 expression resulting in altered cell identities and ultimately incomplete invagination of the developing telencephalon midline (63). Furthermore, it was validated by two separate groups that changes in Chd7 expression altered the proliferation and differentiation potential of neural stem cells in the subventricular zone of the lateral ventricle (61, 62). In these cells, CHD7 expression altered the expression of key neurogenesis genes Sox4 and Sox11 (62) and the retinoic acid signaling pathway (61).

Studies in the developing neural tube concluded that CHD7 has a complex relationship regulating key homeobox gene expression. Changes in expression of Gbx2 and Otx2 due to CHD7 loss resulted in abnormal patterning of cerebellar identity in rhombomere 1 (59). Furthermore, Randall et al demonstrated that

CHD7 and TBX1 have an epistatic relationship in the developing pharyngeal ectoderm. Decreases in TBX1 levels had no detectable effect on Chd7 expression levels. However, targeting of both genes resulted in a synergistic interaction with developmental consequences for the 4th pharyngeal aortic arch, thymus, and ear. However, the full interactions between these two genes are yet to be elucidated (58).

In addition to a role in regulating nucleoplasmic gene expression, it was also found recently that CHD7 localizes to the nucleolus of the cell to regulate the transcription of rDNA (64). In this study, it was reported that CHD7 binds to rDNA

! #"! repeats, but it specifically localizes to transcriptionally active repeats demarcated by high H3K4me2 histone levels and low DNA methylation. Knockdown of CHD7 in this study resulted in reduced rRNA expression levels accompanied by lower cellular proliferation and protein synthesis. Interestingly, levels of rRNA transcription were also significantly reduced in several developmentally affected tissues of Chd7 mutant mouse embryos. The targeting of CHD7 to the rDNA was also validated in two independent studies focusing on the expression patterns of the long and short CHD7 isoforms and the roles of CHD7 in mouse sub- ventricular zone neurons (61, 65).

Expression of CHD7 during development

CHD7 expression has been studied during development using in situ hybridization across a multitude of organisms including humans, mice, and zebrafish (66-69). These studies show that the general patterns of CHD7 expression are conserved across species, and also that CHD7 expression is highly dynamic, suggesting strong spatiotemporal regulation throughout development. In these organisms, CHD7 is expressed highly and ubiquitously during early embryonic development. However, these expression patterns are gradually restricted during later stages to tissues affected in CHARGE syndrome.

These tissues include the developing retina, inner ear, cranial nerves, kidneys, olfactory epithelium, and brain. Interestingly, there are some discrepancies in tissue expression between models. For example, one would expect to see high

CHD7 expression in the heart, an organ severely affected with decreases of

! ##! CHD7. However, CHD7 expression in the heart was not detected in the human in situ experiments and not all mouse models (56, 66-68).

CHARGE syndrome

Overview

Two separate clinicians, Hall and Hittner, first identified the collective spectrum of clinical features that we now know as CHARGE syndrome in 1979

(70, 71), but it was not until two years later that the acronym “CHARGE” was coined and used as the basis for the first diagnostic critieria (72). CHARGE is an acronym for the cardinal features of the syndrome including ocular coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear anomalies. In general, the incidence rate of CHARGE syndrome is approximately

1:10,000 (73). Although several of the developmental abnormalities of CHARGE syndrome can result in neonatal mortality, with corrective surgery and symptom management patient survival rate is improved to about 70% at 5 years of age

(74).

Expansion of the CHARGE syndrome clinical presentation

Clinically, the most commonly seen features include external ear abnormalities, cranial nerve dysfunction, semicircular canal hypoplasia, and delayed attainment of motor milestones, but the developmental changes in patients are far more complex and diverse than the simple acronym CHARGE implies (75). Two separate clinicians, Verloes and Blake, have proposed separate sets of criteria for a clinical diagnosis of CHARGE syndrome (76, 77).

! #$! Both of these criteria sets are based on separate “major” and “minor” abnormalities; however, both are in agreement that an ocular coloboma and/or choanal atresia are required for the diagnosis of classic CHARGE syndrome. The different clinical features used to diagnose CHARGE syndrome are discussed below.

Ocular coloboma

Malformations of the eye can be found in approximately 86% of all

CHARGE syndrome patients (78). Malformations can present as either unilateral or bilaterial and affect development of either the anterior or posterior eye. Iris colobomas do not affect vision in the patient, however, it can predispose them to light sensitivity (79). Malformations of the posterior eye, or uveo-retinal colobomas, however do typically disrupt the macula and ocular nerve, diminishing vision (79).

Heart defects

Heart defects are typically very complex and present in 76-85% of patients

(75). These structural defects can include conotruncal anomalies such as

Tetralogy of Fallot, AV canal defects, and aortic arch anomalies (79).

Choanal atresia

Choanal atresia, or the blocking of nasal passages, is present in 38-61% of newborn CHARGE syndrome patients (75). This malformation often causes breathing problems in the infant, while if bilateral, causes immediate distress in the newborn and can be fatal if not corrected (79, 80). Patients can also present

! #%! with a tracheoesophageal fistula, or an abnormal connection of the trachea to the esophagus, creating both breathing and swallowing difficulties (80).

Growth retardation and developmental delays

This cardinal CHARGE syndrome phenotype refers to both stunted physical and developmental growth of CHARGE syndrome patient. CHARGE syndrome infants are typically born with both normal weight and length. However, as children progress, patients are both short and underweight for their age. It is currently unclear if this is a result of a CHD7 mutation or a side effect of their other developmental abnormalities (i.e. feeding difficulties, frequent hospitalizations for surgeries) that play a role in the physical growth changes (80,

81). CHARGE syndrome children also have delays in reaching motor milestones, such as head control and motility, and also mental retardation (79). Although, it is still unclear on to what extent delays in cognitive development are due to central nervous system dysfunction and how much is due to deficits in sensory stimulation caused by the combination of hearing and vision changes in children

(82).

Genital Abnormalities

CHARGE syndrome patients often present with structural abnormalities and hypogonadotropic hypogonadism. In 50-60% of patients, males present with micropenis or cryptorchidism while females have hypoplasia of the uterus and labia (79). In patients with hypogonadotropic hypogonadism, it has been reported that they have decreased levels of luteinizing hormone, follicle-stimulating

! #&! hormone, testosterone, and estradiol often affecting the development of secondary sexual characteristics (80).

Ear anomalies

Changes in external and internal ear development are one of the most common phenotypes seen in CHARGE syndrome patients with an occurrence frequency of 94-100% (75). External ear abnormalities include absent or small lobes with an overall distinct “cup” shape (75). Temporal bone CT scans reveal multiple internal ear malformations in patients including semicircular canal abnormalities, cochlear hypoplasia, and Mondini malformations (80). In addition, dysfunction of the cranial nerve VIII is commonly reported in CHARGE patients

(80). Taken together, these structural and/or sensory changes can result in severe to total hearing loss in patients (79).

Other common phenotypes

In addition to the cardinal features of the CHARGE acronym, the spectrum of clinical presentations can include several additional developmental abnormalities. One such abnormality can be found in cranial nerve development.

Although generally underappreciated, cranial nerve anomalies occur in 86-99% of CHARGE syndrome patients (75). The dysfunction of several cranial nerves contributes to several defects including hyposmia/amosmia, facial palsy, internal strabismus, and also sensorineural deafness (80). Craniofacial developmental defects are also found in a large proportion of patients with 33-48% of patients presenting with either cleft lip or cleft palate (75). Furthermore, many CHARGE syndrome patients present with limb abnormalities. Limb abnormalities can

! #'! include features such as hypoplastic nails, polydactyly, tibial anomalies, and brachydactyly (79). At one point, it was predicted that one-third of all patients had a limb or bone defect (83), however, these defects are rarely considered in more current patient cohorts making the true frequency of these defects undetermined.

On rare occasions, CHARGE syndrome patients can also present with immunological problems including T-cell deficiency and thymic aplasia (84, 85).

Mutation spectrum

In 2004, Vissers et al were able to identify the CHD7 gene as causative for

CHARGE syndrome. Through a series of array comparative genomic hybridization experiments on two key patients, they identified a commonly disrupted region of chromosome 8 containing the CHD7 gene (3). Since the identification and subsequent confirmation of CHD7, approximately 60-70% of all tested CHARGE syndrome patients have found to be positive for a mutation in this gene (80). The majority of identified mutations are nonsense or frame-shift mutations (~78%) with missense and splice-site mutations to a lesser extent

(~19%). Complete or partial deletions or duplications are very rare, occurring in approximately 3% of all cases (73). This last mutational class is comprised of only 20 cases with 11 cases of whole gene deletions, 2 whole gene duplications, and 7 exonic deletion/duplication (73, 86-88). Translocations of CHD7 are extremely rare, as only 3 cases have been reported in the literature (3, 73, 89).

Mutations in CHD7 can be found throughout the entire length of the gene with at least one reported mutation in every exon (73, 90). There are no known mutation hotspots, but there are two known recurrent mutations that have been

! #(! reported in over 10 patients each. These mutations include c.1480 C>T located in exon 2 and c.7879 C>T located in exon 36 (73). Interestingly, there is also no enrichment of CHD7 patient mutations found in the functional protein domains.

Approximately 30% of mutations lay within these domains, which is only slightly higher than expected given that the functional domains comprise only 25% of the

CHD7 amino acid sequence (73). Surprisingly, there are no strong correlations between patient genotype and clinical phenotype (80). This can be further illustrated by monozygotic twin studies in which both siblings have the same

CHD7 mutation, but highly variable severity of phenotypes (91, 92). However, it has been reported that patients with missense mutations tend to have milder phenotypes (92).

The vast majority of CHD7 mutations arise de novo, but there have been

18 total cases of familial transmission (73). For many of these cases, parents have very mild phenotypes and are not aware of their mutation status until after giving birth to children with more severe phenotypes (75). However, approximately one third of these cases are the result of either somatic and germline mosaicism (73, 92-94).

Clinical overlap with other syndromes

CHARGE syndrome has features that overlap clinically with several other congenital disorders including 22q11 deletion syndrome, Kallman syndrome,

VACTERL syndrome, Goldenhar syndrome, and SOX2 anophthalmia (75, 95).

22q11 deletion syndrome is comprised of two associated genetic syndromes including velocardiofacial syndrome and DiGeorge syndrome and is believed to

! #)! heavily involve haploinsufficiency of the gene TBX1 (75). It shares a high degree of overlap in clinical presentation such as congenital heart defects, developmental delays, renal abnormalities, growth retardation, and ear anomalies resulting in hearing loss (75). To illustrate this high degree of clinical overall, a study by Corsten-Janssen et al found that many patients diagnosed with 22q11 deletion syndrome have CHD7 mutations and vice versa (95).

Interestingly, a mutant mouse model of Tbx1 and Chd7 double haploinsufficiency was shown to have a synergistic effect with enhanced abnormalities of thymus and semicircular canal development suggesting an overlap in developmental pathway regulation (96).

Animal models of CHARGE syndrome

Through targeting the expression of CHD7 homologs, multiple animal models of CHARGE syndrome have been developed. Mutant mouse models have been the most extensively developed, using multiple methods such as N- ethyl-N-nitroso (ENU) mutagenesis and gene-trapping technology (67, 68).

Characterization of Chd7 mutant mice reveals they have developmental abnormalities in almost the all the same organ systems as human patients (67,

68, 97). It has been reported in these studies that Chd7 heterozygous mice are have keratoconjunctivitis sicca eye abnormalities, craniofacial abnormalities including cleft palate, cardiac edema with ventricular septal defects, genital abnormalities, and inner ear abnormalities including malformed or absent semicircular canals. They also commonly display head-shaking and head- bobbing behaviors that are consistent with vestibular dysfunction. Supporting that

! #*! embryonic development is extremely sensitive to CHD7 expression levels, homozygous null mutant mice die during mid-gestation at approximately E10.5

(68).

Non-mammalian vertebrate models of reduced CHD7 expression similarly result in developmental malformations in many of the same organ systems as mammals indicating that its function is highly conserved. Both zebrafish and

Xenopus models of CHARGE syndrome have been developed through the use of morpholino targeting (54, 69, 98). These models display abnormalities in eye organization, missing or malformed otoliths, and heart defects including truncus arteriosis and cardiac outflow tract defects. These also have evidence of cranial neural crest defects with craniofacial cartilage malformations and abnormal positioning of the cranial nerves. Additionally, it was reported in the zebrafish models that Chd7 loss led to abnormalities of the vertebrae including reduced mineralization and also abnormal segmental vasculature (69).

The role of the Drosophila CHD7 ortholog, kismet, is also under study.

The Drosophila Kismet protein is evolutionarily highly related to the subfamily III

CHD members CHD6-9; however, the functions of the Kismet protein have been proposed to be a combination of both CHD7 and CHD8 (44, 45, 99, 100). Kismet has been shown to be a modifier of several signal transduction pathways such as

Ras, Notch, and Hedgehog (101-103), required for immediate recall memory, and the regulation of RNA polymerase II elongation activity through H3K27me modulation (99, 100). Homozygous null mutants are embryonic lethal, indicating a critical need for Kismet during early development (99). However, conditional

! $+! knockdown of kismet expression using RNAi leads to postural defects and an inability to fly, revealing a requirement for Kismet in muscle cells. Interestingly, reduction of Kismet expression specifically in the nervous system results in structural neural abnormalities similar to those seen in other models. These animals display defects in the developing peripheral nervous system structures and wing vein differentiation defects (99).

Regulation of rDNA expression

CHD7 was previously reported to localize to the nucleolus of the cell and its expression promotes the transcription of rRNA (61, 64, 65). Targeting of

CHD7 was accompanied by several changes in cell-cycle regulation including increased p21 expression and reductions of both protein synthesis and cellular proliferation (64). However, it is unclear whether these changes in cell-cycle regulation are due to direct changes of nucleoplasmic gene expression with loss of CHD7 or if these are downstream effects of decreased rRNA levels in the nucleolus.

The nucleolus itself is a subnuclear structure whose primary role is ribosome biogenesis. This specialized compartment has other functions involving cell cycle control, stress response, and coordination of ribonucleoproteins (104).

However, its role in producing ribosomes for protein synthesis makes the nucleolus crucial for cell growth and proliferation (104-106). One step in this crucial role of cell growth and proliferation is the proper production of rRNA. The importance of this role is evident as misregulation of rRNA production results in

! $"! several diseases such as Treacher-Collins syndrome, dyskeratosis congenital, and perhaps even CHARGE syndrome (107).

The nucleolus and building a ribosome

The nucleolus is the largest subnuclear structure and is formed predominately around sites of active transcription from rDNA repeats, or more specifically, nucleolar organizing regions (NORs). In humans, there are NORs located on the five acrocentric chromosomes 13, 14, 15, 21, and 22 (108). The nucleolus is not a membrane-bound organelle, but rather a clustering of the

NORs along with their recruited nucleolar protein components (109).

The mammalian nucleolus is comprised of three sub-structures and this morphology correlates with the multiple steps in ribosome biogenesis (110).

These sub-structures include the fibrillar center, the dense fibrillar center, and the granular component. Transcription of rRNA occurs in the fibrillar center and it is then further processed in the outer dense fibrillar center. The granular component is the assembly substructure combining the processed rRNA transcripts into the nascent preribosomal subunits (110).

Structure and transcriptional regulation of rDNA

As mentioned previously, the human rDNA genes are located on the acrocentric chromosomes and form NORs (104, 111). The number of rDNA repeats can be variable across organisms ranging from approximately 400 repeats in humans to almost 10,000 in the lungfish (112). The rDNA genes are arranged in stretches predominantly in tandem from head-to-tail but also non- canonical palindromic repeats. Each rDNA repeat is approximately 43 kb in

! $#! length, consisting of 13-14 kb coding for the 45S pre-rRNA with the remainder composed of non-coding intergenic spacers (IGS) (111). The IGS contains several regulatory elements including the promoter, repetitive enhancer elements, and transcriptional terminators that function in conjunction with the transcription factor TTF-1 to halt elongating RNA polymerase I (RNA Pol I)

(Figure 1-2). In mouse rDNA repeats, a secondary spacer promoter is also located in the IGS and codes for a small intergenic transcript known as the promoter-associated RNA (pRNA) that contributes to rDNA repeat inactivation

(111).

The transcriptional activity from the rDNA to form rRNA is a highly regulated process. The rRNA is transcribed by the RNAPol I and requires a pre- initiation complex containing the upstream binding factor (UBF) and a promoter selectivity factor (SL1) (113). RNA Pol I produces a single 45S pre-rRNA molecule that is subsequently processed by snoRNAs to remove both external and internal transcribed spacers (111, 114). This produces one molecule each of the 18S, 5.8S, and 28S rRNA to be incorporated in the ribosomal subunits (111).

The fourth rRNA molecule required by the ribosome, 5S, is encoded by separate genes outside the nucleolus and is transcribed by RNA Polymerase III (115).

While the essential structure of the rDNA repeat remains the same in all cells, several mouse studies indicate that rDNA variants due exist with variable

IGS lengths and sequence polymorphisms. Interestingly, these studies also demonstrate that the rDNA variants are regulated independently and also in a tissue-dependent manner (116, 117).

! $$!

Figure 1-2. Simple schematic of a single mouse rDNA gene. Transcriptional machinery for rRNA synthesis binds at the rRNA promoter. A single 45S molecule is transcribed containing the three rRNA species separated by two internal transcribed spacers (ITS) and capped by two external transcribed spacers (ETS) highlighted in blue. These spacer regions are subsequently removed during rRNA processing. The upstream space promoter is required for the promoter-associated RNA (pRNA). The coding region of the rDNA is separated by a long non-coding intergenic spacer (IGS).

! $%! Epigenetic regulation of rDNA

In addition to regulation of rDNA variants by RNA Pol I activity, rDNA repeats are regulated epigenetically and can exist in either an active or inactive state based on chromatin features (108). The number of active and inactive rDNA repeats varies between different cell types, reflecting different metabolic needs of the cell during development and differentiation (118-120). This regulatory mechanism is especially important as unrestricted rDNA expression can result in genomic instability with uncontrolled cellular expression and growth

(121).

Active and silent rDNA repeats are epigenetically demarcated and regulated by DNA methylation and histone modifications. While both promoters of active and inactive rDNA repeats have degrees of DNA methylation, DNA hypermethylation at the promoter and enhancer correlates with transcriptional repression of rDNA (111). In addition to DNA methylation, histone tail modifications can also indicate the activity level of an rDNA repeat.

Transcriptionally silent rDNA genes are nucleosome dense and associated with repressive marks including H3K9me3, H3K27me3, and H3K20me. Expressed rDNA repeats lack regular nucleosomes are generally associated with the activating histone mark H3K4me3 and H3/H4 acetylation (111).

Epigenetic silencing of rDNA repeats and ultimately rRNA expression is catalyzed by the nucleolar remodeling complex (NoRC). This complex is comprised of two protein subunits – the SNF2H ATPase chromatin remodeler and the TTF-1-interacting protein-5 (TIP5) (118). To target and silence rDNA

! $&! repeats, the pRNA transcribed from the spacer promoter is utilized by NoRC to recruit DNA methyltransferases and histone deacetylases to the pre-rRNA promoter (111, 122). Additionally, mouse SNF2H activity shifts pre-rRNA promoter nucleosomes to reduce access to a critical UBF binding site and lower

RNA Pol I activity (111).

FBXL10 is an epigenetic modifier of rDNA expression

In addition to NoRC, other proteins can epigenetically modify rDNA repeats and alter rRNA transcriptional activity. The gene FBXL10 (NDY1,

JHDM1B, KDM2B) located on chromosome 12 codes for a 1336 amino acid protein. The structure of this protein contains several protein domains including

F-box, Jumonji C (JmjC), CXXC-zinc finger, and a PHD domain (Figure 1-3).

Taken together, it has been proposed that FBXL10 and its family members are a class of histone demethylases, and they can be found in almost all organisms

(123).

There are two isoforms of Fbxl10. The short isoform, which is lacking the catalytic JmjC domain, is expressed ubiquitously throughout embryonic development. The long isoform, which has been the most studied, is expressed in early embryonic development, but concentrated mainly in the testes, thymus, spleen, and brain of the adult mouse. Interestingly, Fbxl10 expression is high in the developing neural tube and specifically in neural progenitor cells and the neural crest. While heterozygous Fbxl10 mutant mice are completely wild-type, homozygous null mutants displayed neural tube defects, retinal colobomas, and tail curling (124).

! $'!

Figure 1-3. Schematic of FBXL10 protein domain composition. Fbxl10 contains four protein domains including a F-box domain (yellow), JmjC domain

(blue), a CXXC-ZF domain (pink), and PHD domain (green). The F-box and PHD domains are sites of protein-protein interaction, although, it was recently found that PHD domains of FBXL10 can bind methylated lysine residues of histone H3 and exhibits E3 ligase activity (125, 126). The CXXC-ZF domain recognizes non-methylated CpG DNA and can act as a DNA-binding domain (123). The

JmjC domain is the catalytic domain and acts to remove methyl groups from lysine residues on histone tails (123).

! $(! In a study by Frescas et al, it was discovered that FBXL10 is a regulator of chromatin state at rDNA repeats (127). FBXL10 was shown to contain a nucleolar localization signal and especially bound at the transcribed rDNA repeats. A series of siRNA and over-expression experiments demonstrated that by binding to rDNA FBXL10 acts as a negative regulator of rRNA transcription by modulating H3K4me3 levels. Consistent with levels of rRNA expression correlating with cellular growth, over-expression of FBXL10 also resulted in smaller cells and a significant decrease in BrdU incorporation suggested that cellular proliferation was stunted. The Drosophila homolog dKDM2 was also shown to have a similar function in regulating nucleolar structure through the modulation of H3K4me3 levels (128).

While studies in multiple organisms provide evidence that FBXL10 is a negative regulator of rRNA transcription, the functions of this protein outside the nucleolus are still somewhat controversial. Multiple lines of evidence support a nuclear of function of FBXL10 as a regulator cellular senescence through modulation of several potent cell cycle genes such as c-Jun and the

Ink4/Arf/Ink4b locus (124, 126, 129, 130). Fbxl10 regulates the silencing of the

Ink4/Arf/Ink4b genes by promoting the expression of Ezh2 and, ultimately,

H3K27me3 levels at this locus (131). This regulation of cell cycle senescence does appear to be context dependent as Fukuda et al report that Fbxl10 can function as positive or negative regulator of cellular proliferation depending on the developmental stage and cell type (124). Another area of contention is the histone methylation specificity of FBXL10. While multiple groups concur that

! $)! FBXL10 is a histone demethylase, conflicting evidence makes it unclear as to whether FBXL10 exclusively targets H3K4me3 or if it can target another histone modification, such as H3K36me2, for demethylation (131-134).

Dysregulation of ribosome biogenesis

The process of building a ribosome for protein synthesis requires extreme precision likened to assembling a machine. It requires many components fashioned together in a step-wise fashion in order to form a fully functional unit.

The completed ribosome is composed of two subunits containing approximately

80 proteins and four separate rRNA transcripts. However, the process requires several hundred additional accessory proteins responsible for processing the rRNA transcripts, adding nucleotide modifications to the individual rRNA species, and assembling the protein units themselves (135). Dysregulation of any of these individual processes can have an impact on the overall ribosome biogenesis pathway and ultimately growth and development as seen in the class of diseases collectively known as ribosomopathies (107).

The nucleolar stress response

Dysregulation of the ribosome biogenesis pathway can be caused by any number of abnormalities and cellular changes. These changes can include ribosomal protein depletion or mutation of genes coding for these ribosomal protein components (136), abnormal transcription or processing of rRNA (137,

138), or even environmental exposure to certain drugs that can block RNA

Polymerase I activity such as Actinomycin D (139) (Figure 1-4).

! $*!

Figure 1-4. Overview of the nucleolar stress response. Multiple genetic and environmental factors have the ability to induce the nucleolar stress response.

Regardless of the stress source, the activation of the stress response can result in the increased stabilization and activation of p53 and, ultimately, result in drastic changes in cellular fate.

! %+! In the past few years, these individual nucleolar stressors and how the cell reacts to them have become an area of intense investigation. One mechanism utilized by the cell to combat these various disruptions of the ribosomal biogenesis pathway is the nucleolar stress response. The nucleolar stress response is a signaling cascade originating from the nucleolus of the cell to ultimately halt cell proliferation and/or induce cellular apoptosis. The most commonly studied aspect of the nucleolar stress response pathway heavily relies on the stabilization and activation of the tumor suppressor protein p53.

Interestingly, studies have shown that the stabilization of p53 for this stress response can occur completely independently of DNA damage. In fact, several lines of study have shown that p53 activation is dependent on nucleolar disruption for activation and a series of highly complex regulatory interactions

(139). In a normal cellular environment, p53 protein levels are kept low by the E3 ubiquitin ligase MDM2 (HDM2 in humans), which targets p53 for proteosomal degradation. Upon stress, the structure of the nucleolus collapses, allowing for the dispersal of proteins into the nucleoplasm. These proteins, such as ARF and ribosomal protein components, bind to the MDM2 protein and inhibit its ability to target p53 for destruction, consequently resulting in the increase expression of p53 target genes (Figure 1-5) (140). Several in vivo animal models with mutations in the ribosomal biogenesis pathway have been key to understanding how important this mechanism is and how inappropriate activation of the stress response results in developmental abnormalities from increases in cell death and proliferation loss (136-138, 141, 142). Interestingly, all cells may not have the

! %"!

Figure 1-5. Disruption of ribosomal biogenesis alters nucleolar structure resulting in the stabilization of p53. Under normal cellular conditions, ribosomal proteins are localized to the nucleolus to partake in ribosome biogenesis. Disruption of ribosome biogenesis results in the release of these proteins, including ARF, into the nucleus and alters the MDM2/p53 feedback loop. The proteins bind and cause steric hindrance of MDM2 from tagging p53 for proteosomal degradation. The result of this stabilization typically induces the expression of p53 target genes such as p21 to halt cellular proliferation.

! %#! same nucleolar stress response when faced with ribosomal disruptions. In has been recently described in two different lines of study that the nucleolar stress response can be activated independently of p53 stabilization. In a study by

Donati et al, it was described that the release of the ribosomal protein RPL11 was sufficient to disrupt gene expression and induce cell-cycle arrest without p53 expression or stabilization (143). This was preceded by another study describing the proto-oncogene kinase PIM1 and its ability to instigate cell-cycle arrest by increasing p27 expression levels (144). While both studies reported decreases in cellular proliferation with ribosomal biogenesis disruption, changes in cell death were not commented on. It would be interesting to further understand these p53- independent pathways of the nucleolar stress response to see if they help steer cellular fate towards either cell death or arrest.

Ribosomopathies

The process of building a ribosome is absolutely vital for cellular survival, and so, it may be be surprising that any disruption in this process is compatible with life. And yet, for the past 15 years, it is slowly coming to light how alterations in ribosome biogenesis can play a large role in a spectrum of these clinically unique disorders. Ribosomopathy research began primarily with those diseases affecting the bone marrow and blood development including Diamond-Blackfan anemia (DMA), Schwachman-Diamond syndrome (SDS), and 5q-syndrome (107,

145). Since these early studies the list of ribosomopathies has expanded to include diseases such as Treacher Collins syndrome (TCS), North American

! %$! Indian childhood cirrhosis (NAIC), and Bowen-Conradi syndrome (BCS) (146,

147).

While these diseases commonly affects one component in the process of building a ribosome, what is surprising is that these diseases result in distinct clinical presentations. Model system studies into the molecular etiology of NAIC and TCS reveal potential common defects into rRNA production and, yet, target different organ systems such as the liver and craniofacial development respectively (146-148). This has become the conundrum of understanding the pathogenesis for ribosomopathies – how do mutations affecting components of the same cellular pathway, which are present in all cell types, result in very tissue specific problems? More confounding, some abnormalities, such as the anemia complications seen in DBA, don’t present in patients until almost one year after birth when ribosomes have been long into production (149).

Several articles have been published highlighting potential explanations for this tissue proclivity for ribosomopathies (150, 151). One potential explanation is that the affected tissues are actively dividing and thus highly sensitive to mutations affecting ribosome assembly. However, some dismiss this idea as all organs in early embryonic development are rapidly dividing (151). Another idea includes the notion of a “specialized” ribosome, or rather, the idea that the ribosome and its regulatory constituents change between tissue types. With this thought, mutations in specific ribosomal components resulting in haploinsufficiency may disrupt biogenesis in only a small set of cell types and result in tissue specific defects.

! %%! An alternative explanation is that not all cell types are responding to changes in ribosome biogenesis in the same way i.e. the nucleolar stress response. Several animal model studies of ribosomopathies show some degree of the p53-mediated nucleolar stress response involvement (142, 152, 153).

These models, using both mice and zebrafish, show significant increases in apoptosis with the targeting of ribosomal biogenesis proteins. To further illustrate the role of p53 in the pathogenesis, several of these models of both TCS and

DBA can be phenotypically rescued upon knockdown of p53 expression (142,

154). However, it is important to note, that knockdown of p53 does not rescue the developmental phenotypes of all mutated ribosomal proteins such as mutations of the mouse Rpl38 and Rps6 (150). As for more less studied ribosomopathies such as BCS and NAIC, it is still unclear at this time the degree of p53 involvement during pathogenesis.

In addition to a wide clinical spectrum of developmental abnormalities, several ribosomopathy diseases have an increased risk for cancer. SDS patients have an increased risk of approximately 20-30% in developing myelodysplastic syndromes (MDS), or similarly to 5q-syndrome, the development of acute myeloid leukemia (AML) at later stages (107). At present, it is less clear whether there is an increased cancer risk in DBA patients. A wide range of cancer types has been reported in these patients ranging from AML/MDS to colon cancer

(107) making patterns difficult to distinguish. Overall, it is not hard to imagine that changes in ribosomal regulation could be related to neoplasia given the close connection between ribosomes and proliferation. However, it is still not evident

! %&! whether this relationship extends to all ribsomopathies or to those with that suffer from bone marrow failure.

Summary and Research Aims

Disruptions in CHD7 expression are closely linked with the congenital disorder CHARGE syndrome. Mutations in this gene give rise to a constellation of developmental abnormalities targeting multiple organ systems. Evidence shows that CHD7 is a chromatin remodeler and functions to modulate gene expression levels; however, we are still struggling to understand the full context- dependency of CHD7 function and how it interacts with other transcriptional regulators during development (51, 53).

For example, it is still not clearly understood how many different cell types or tissue types are affected by alterations in CHD7 expression and how reductions in CHD7 give rise to the developmental abnormalities presented by

CHARGE syndrome patients. It was hypothesized by several groups that

CHARGE syndrome is due to abnormalities of the migratory neural crest (155,

156). While abnormalities in the neural crest may explain part of the clinical spectrum, it does not account for all the phenotypes (81). Randall et al illustrated this by showing specific restoration of Chd7 expression to the neural crest does not rescue heart structural abnormalities in the mouse (58). Strong evidence shows that derivatives of the ectodermal lineage are also affected, but again, this may not account for all abnormalities (60, 61, 63).

! %'! Furthermore, our understanding of CHARGE syndrome pathogenesis is further complicated by multiple genomic functions of the CHD7 protein. Several independent lines of evidence demonstrate that CHD7 can interact with a variety of transcription factors to regulate the expression of key genes of cell identity and differentiation (54, 56, 57). However, a secondary function of CHD7 is reported to modulate the levels of rRNA transcription from the nucleolus (64). It is unclear which of these functions, transcriptional regulation in the nucleoplasm or nucleolus, plays a more prominent role in development or, more likely, if it is the combination of both functions.

To answer these questions, the goal of these studies was to develop a tractable model system in which we could modulate gene expression of CHD7 and its potential interaction partners to understand how changes transcriptional regulation affects growth and development on an embryonic scale. To this end, we modeled CHD7 expression changes using the zebrafish. The zebrafish offers several advantages to study early embryonic development such as embryos are transparent and develop externally, they are laid in large clutch sizes, and gene expression can be quickly targeted through morpholino technology (157). The zebrafish chd7 homolog is located on chromosome 2 and is 55 kb long.

As described in the following chapter, targeting of the zebrafish chd7 homolog results in developmental abnormalities in several organ systems commonly affected in CHARGE syndrome patients. We demonstrate further that these changes in gross morphology are also accompanied by large decreases in cellular proliferation, although it was unclear whether this was due to alterations

! %(! in cell cycle regulatory genes or rRNA expression levels. Regardless of the upstream molecular mechanism, we wanted to test whether these changes in cellular proliferation were causative for the CHARGE-like developmental abnormalities in the chd7 morphants. After reasoning that increases in global proliferation can be modulated through rRNA levels, we targeting a known negative regulator of rRNA expression, Fbxl10. Through the modulation of

Fbxl10, we significantly restored the abnormal phenotypes and improved cellular proliferation. While it is still not fully elucidated whether changes in nucleolar expression or nucleoplasmic gene expression are causative for the CHARGE-like phenotypes, we propose that ultimately dysregulation in cellular proliferation plays a role in CHARGE syndrome pathogenesis.

! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! %)! Chapter 2

Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a

zebrafish model of CHARGE syndrome

Stephanie A. Balow1, Lain X. Pierce1, Gabriel E. Zentner1,4, Patricia A. Conrad1,

Stephani Davis5, Hatem E. Sabaawy5, Brian M. McDermott Jr.1,2, and Peter C.

Scacheri1,3

Departments of 1Genetics and Genome Sciences, 2Otolaryngology-Head and

Neck Surgery, 3Case Comprehensive Cancer Center, Case Western Reserve

University, Cleveland, Ohio, USA, 4Basic Sciences Division, Fred Hutchinson

Cancer Research Center, Seattle, Washington, USA, 5Rutgers Cancer Institute of

New Jersey, Robert Wood Johnson Medical School, New Brunswick, New

Jersey, USA

A modified version of this chapter was previously published as:

Balow SA, Pierce LX, Zentner GE, Conrad PA, Davis S, Sabaawy HE, McDermott, Jr BM, Scacheri PC (2013). Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a zebrafish model of CHARGE syndrome. Developmental Biology 382: 57-69.

! %*! Abstract

CHARGE syndrome is a sporadic autosomal-dominant genetic disorder characterized by a complex array of birth defects so named for its cardinal features of ocular coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. Approximately two-thirds of individuals clinically diagnosed with CHARGE syndrome have heterozygous loss- of-function mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7), an ATP-dependent chromatin remodeler. To examine the role of Chd7 in development, a zebrafish model was generated through morpholino

(MO)-mediated targeting of the zebrafish chd7 transcript. High doses of chd7 MO induce lethality early in embryonic development. However, low dose-injected embryos are viable, and by 4 days post-fertilization, morphant fish display multiple defects in organ systems analogous to those affected in humans with

CHARGE syndrome. The chd7 morphants show elevated expression of several potent cell-cycle inhibitors including ink4ab (p16/p15), p21 and p27, accompanied by reduced cell proliferation. We also show that Chd7 is required for proper organization of neural crest-derived craniofacial cartilage structures.

Strikingly, MO-mediated knockdown of the jumonji domain-containing histone demethylase fbxl10/kdm2bb, a repressor of ribosomal RNA (rRNA) genes, rescues cell proliferation and cartilage defects in chd7 morphant embryos and can lead to complete rescue of the CHARGE syndrome phenotype. These results indicate that CHARGE-like phenotypes in zebrafish can be mitigated

! &+! through modulation of fbxl10 levels and implicate FBXL10 as a possible therapeutic target in CHARGE syndrome.

! &"! Introduction

CHARGE syndrome is an autosomal dominant genetic disorder that affects 1 in 10,000-18,000 newborns worldwide (73, 92). CHARGE is an acronym for ocular coloboma, heart defects, atresia of the choanae, retardation of growth and development, genital anomalies, and ear malformations/deafness.

The clinical presentation of CHARGE syndrome is highly variable and may include additional features such as cleft lip/palate, cranial nerve dysfunction, kidney anomalies, and rare limb anomalies (80). Two-thirds of cases of CHARGE syndrome are caused by spontaneous mutation of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7), an ATP-dependent chromatin remodeler (3, 91). Most CHD7 mutations are nonsense, frameshift, or splice-site, predicted to lead to loss of protein function, and thus CHARGE syndrome is likely due to reduced dosage of CHD7 (73, 94). Consistent with haploinsufficiency as the genetic mechanism underlying CHARGE syndrome, mice that are homozygous for Chd7 null mutations die around embryonic day

10.5, but heterozygous Chd7 mutants are viable and recapitulate many features of CHARGE syndrome, including heart defects, choanal atresia, postnatal growth retardation, genital abnormalities, abnormal semicircular canals, and cleft palate

(67, 68).

CHD7 is a member of the CHD family of proteins. Nine proteins comprise this family in vertebrates, and all nine contain tandem N-terminal chromodomains and a central conserved SNF2-like ATPase domain presumed to mediate chromatin remodeling. In addition to the chromodomains and ATPase domain,

! &#! CHD7 contains two BRK domains of unknown function and a SANT-like domain that may mediate DNA and/or histone binding (9). CHD7 is a nuclear protein and binds to gene enhancer elements and promoters, functioning as a transcriptional co-regulator (52, 53, 57). CHD7 cooperates with PBAF (polybromo- and BRG1- associated factor-containing complex) to regulate genes important for formation and migration of neural crest, including TWIST and SOX9 (54). In mouse neural stem cells, CHD7 collaborates with SOX2 to regulate a common set of target genes including Jag1, Gli3, and Mycn. Mutations in these and other genes co- regulated by CHD7 and SOX2 cause clinical malformation syndromes that show some clinical overlap with CHARGE syndrome (57). Thus, it is hypothesized that dysregulated expression of genes normally regulated by CHD7 during development gives rise to the developmental defects observed in CHARGE syndrome.

In addition to its role as a transcriptional regulator in the nucleoplasm,

CHD7 localizes to the nucleolus (64, 65), where it associates with rDNA and functions as a positive regulator of rRNA transcription. Cell proliferation is tightly coupled to protein synthesis, ribosome biogenesis, and rRNA production (158-

161). Accordingly, siRNA-mediated knockdown of CHD7 in cultured cells suppresses protein synthesis and cell proliferation (64). Affected tissues from

Chd7 mutant mouse embryos also show deficiencies in rRNA levels as well as cell proliferation (64, 162, 163). These findings raise the possibility that the pathogenesis of CHARGE syndrome is related to that of other human disorders caused by deficiencies in ribosomal biogenesis. Collectively known as the

! &$! “ribosomopathies”, these disorders include Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, Treacher Collins syndrome, and myelodysplastic syndrome (107). Despite these discoveries, it remains unclear if the multiple anomalies in CHARGE syndrome are due to dysregulated expression of nucleoplasmic gene targets, rRNA, or the combination of both deficits.

Here, we developed a zebrafish model of CHARGE syndrome through morpholino-mediated targeting of the zebrafish chd7 homolog. At 4 days post fertilization (dpf) chd7 morphant fish show multiple defects in organ systems analogous to those affected in humans with CHARGE syndrome. The defects in the chd7 morphant fish are accompanied by a general deficiency in cell proliferation at the early stages of development, associated with elevated expression of potent cell cycle inhibitors including ink4ab, p21, and p27.

Remarkably, reduction of the Fbxl10/Kdm2bb histone demethylase, a negative regulator of rRNA transcription, restores cell proliferation in the chd7 morphants, with concomitant rescue of CHARGE-like phenotypes. Our findings implicate cell proliferation deficiencies in the pathogenesis of CHARGE syndrome, and suggest that elevation of rRNA levels maybe a viable strategy for therapeutic intervention in CHARGE syndrome.

! &%! Results

Organization of the zebrafish chd7 gene

The sole zebrafish chd7 gene is located on chromosome 2 (Zv9 Ensembl).

Of the five annotated transcripts, one is non-protein coding, two contain only the first two or three exons, and the remaining two are full-length. Both of the full- length transcripts have identical predicted protein coding sequences and have an exon-intron structure similar to that of human CHD7. These full-length transcripts code for a zebrafish Chd7 protein of 3140 amino acids, which is slightly longer than the human homolog of 2997 amino acids. Additionally, the zebrafish Chd7 protein contains a similar complement of protein domains including tandem N- terminal chromodomains, a central SNF2-like ATPase/helicase domain, and a C- terminal BRK domain (Figure 2-1,A). The N-termini of zChd7 and hCHD7 are less conserved; however, the remainder of Chd7, including all of the functional domains, is highly similar. Overall, the aligning portion of the zChd7 sequence demonstrates 69% identity to hCHD7 at the amino acid level.

chd7-morpolino gene targeting recapitulations major features of CHARGE syndrome

To model CHD7 haploinsufficiency in zebrafish, an antisense morpholino

(MO) was designed to produce a mis-spliced chd7 transcript containing a premature stop codon. Specifically, the MO was targeted to the junction of chd7 intron 15 and exon 16 to induce production of a transcript either missing exon 16 or one including intron 15 (Figure 2-1,B, upper). We performed RT-PCR analysis

! &&!

Figure 2-1. Morpholino targeting of the zebrafish chd7 RNA induces an aberrant transcript. (A) Schematic of the human CHD7 and zebrafish Chd7

! &'! proteins with the location of the predicted protein domains. (B) Schematic of the zebrafish un-spliced chd7 transcript (upper) and the exon/intron splice site targeted by the chd7 morpholino (black bar). Schematic of the chd7 morphant transcript (lower) with the location of the predicted induced nonsense mutation

(black arrow). Chromatogram of the sequenced morphant transcript PCR product reveals a nonsense mutation (red highlight). (C) Agarose gel with the amplified

PCR products of both wild-type and morphant chd7 transcripts from Std control morphants and chd7 morphants. (D) Graph of qRT-PCR data quantifying the expression of the chd7 morphant transcript. Error bars represent standard error of the mean (SEM) (n = 4). Significance was determined by a Student’s two- tailed t-test and significant p-values are noted p < 0.01 (**).

! &(! using multiple combinations of primers designed to amplify transcripts containing exon 15 mis-spliced to exon 17. However, such transcripts were not detected in chd7 MO-injected embryo, raising the possibility that the chd7 MO blocks splicing of exons 15 and 16, leading to intron inclusion. We therefore performed RT-PCR analysis using multiple primer sets designed to amplify transcript containing an intron 15 inclusion. The expected wild-type transcript was amplified with cDNA from control embryos. The wild-type transcript was also present in chd7 morphant embryos, even upon treatment with high doses of chd7 MO. However, chd7 morphant embryos also expressed a larger transcript, consistent with intron inclusion (Figure 2-1,C). The levels of the chd7 morphant transcript increased in an MO dose-dependent manner (Figure 2-1,D). Direct sequence analysis of the morphant transcript cDNA revealed inclusion of intron 15. In silico translation of the morphant transcript predicts mis-incorporation of a premature stop codon at

1383 amino acids past the first codon (Figure 2-1,B, lower), presumably leading to degradation of the morphant chd7 message via nonsense-mediated decay.

The chd7 morphants exhibited CHARGE-like defects in an MO dose- dependent manner. Specifically, high doses of chd7-MO (5-10 ng) induced lethality within 24 hours post-fertilization (hpf), reminiscent of embryonic lethality observed in homozygous null Chd7 mice (67, 68). Low-doses of chd7-MO (2.5 ng) yielded viable fish that were indistinguishable from wild-type and control- injected embryos at 3 days post-fertilization (dpf). However, multiple defects were apparent at 4 dpf (Figure 2-2,A-D). Approximately 95% of low-dose chd7-

MO injected embryos showed an abnormal phenotype (Figure 2-2,E-L). chd7

! &)! morphant fish developed defects in organs analogous to those affected in humans and mice with heterozygous CHD7 mutations (Table 2-1). Defects included pectoral fin hypoplasia (~60%), eye abnormalities (70%), and abnormalities in both otolith morphology and number (approximately 25% and

10% respectively) (Figure 2-2,E-L). Moreover, ~60% of chd7 morphants presented with heart defects including pericardial edema, weak heartbeat with reduced circulatory flow, and occasionally a lack of proper heart tube folding.

Importantly, 64% of chd7 morphants also exhibited craniofacial defects involving the nasal region and jaw (Figure 2-3,A-B). None of these defects were apparent upon injection of a chd7 MO containing a 5-bp mismatch to the endogenous chd7 gene, indicating that the observed defects are specific to chd7 targeting. To further verify specificity, we tested a second splice-blocker chd7 MO, and a third chd7-MO designed to block translation. Both the second splice-blocker and chd7-translation blocker yielded similar phenotypes to that seen with the original splice blocker MO, although the translation blocker yielded more severe heart defects, and ear defects were observed at a higher frequency. No aspects of chd7 morphant phenotype were rescued upon co-injection of chd7 and p53 morpholinos, indicating that the observed defects in the chd7 morphants are unlikely to be related to non-specific MO-mediated cell death. Lastly, we note that the observed phenotype resembles that reported in a separate study in which the chd7 gene was targeted using a different splice-blocker MO (69, 98).

Overall, the chd7 morphants displayed defects in many of the same organs affected in humans and mice with CHD7 mutations, including the ear, eye, heart,

! &*!

Figure 2-2. Zebrafish chd7 targeting results in CHARGE-like phenotypes.

Whole-embryo lateral (A,C) and dorsal (B,D) views of representative Std control and chd7 morphant embryos at 4 dpf. The chd7 morphants display pectoral fin defects (E-F), eye abnormalities including under-developed or missing anterior eye structures (G-H), changes in otolith morphology (I-J), and pericardial edema

(K-L). Missing or abnormal structures are highlighted with an asterisk (*) and/or dashed line. Structures are also highlighted in Std morphants for comparison. HT

! '+! = heart, L = lens, OT = otolith, PE = pericardial edema, PEC = pectoral fin, R = retina.

! '"!

Table 2-1. CHD7 mutant phenotypes comparison across multiple species.

! '#! craniofacial region, and limbs. Thus, the requirement for CHD7 in the development of these organs appears to be conserved between mice, humans, and zebrafish, and suggests that MO-mediated targeting of chd7 creates a suitable system in which to examine Chd7 function in development.

chd7 morphants develop defects in neural crest-derived craniofacial cartilage

Early zebrafish craniofacial structure is dependent on proper migration of the multipotent cranial neural cells from the neural tube into the pharyngeal arches. These cells differentiate to form several tissues of the developing cranium such as the cranial nerves, bones, and cartilage (164). Based on these findings and published studies implicating a deficiency in neural crest cell migration in CHARGE syndrome (54, 69), we performed a detailed morphological analysis of craniofacial cartilage in low-dose chd7 morphant embryos.

Compared to Standard MO-injected controls, chd7 morphants showed wide range of craniofacial cartilage abnormalities (Figure 2-3,F-G). In approximately

50% of chd7 morphants, the first pharyngeal arch comprising Meckel’s cartilage and the palatoquadrate were morphologically normal. However, Meckel’s cartilage was located posteriorly to that observed in control morphants. In addition, the ceratobranchial cartilages were absent, and the ceratohyal was malformed (less V-shaped) compared to controls. In the majority of chd7 morphants, the ceratobranchial arches were undetectable at this resolution.

However, microscopic analysis of sagittal sections revealed that the first

! '$!

Figure 2-3. chd7 morphants display variable defects in craniofacial cartilage development. (A-B) Bright-field lateral views of representative Std control morphants and chd7 morphants at 4 dpf. (C-D) Lateral and ventral views

! '%! of Std morphants with wild-type craniofacial cartilage structures at 4 dpf. (F-G)

Representative lateral and ventral views of the average chd7 morphant phenotype and is categorized as underdeveloped. The ceratohyal cartilages of the chd7 morphant are malformed and form a more linear shape. The five ceratobranchial cartilages were also undetectable with Alcian blue staining. (I-J)

Lateral and ventral views of a severe chd7 morphant phenotype detected a highly underdeveloped neurocranium with the anterior and branchial arches absent. (E,H,K) Schematic views of the zebrafish craniofacial cartilage excluding the neurocranium. A solid red line indicates that the structure is present but malformed; while, a dashed red line indicates that the structure is absent. AC = auditory capsule, CB = ceratobranchial, CH = ceratohyal, EP = ethmoid plate,

ME = Meckel’s cartilage, N = notochord, PC = parachordal, PEC = pectoral fin,

PQ, palatoquadrate, T = trabecula cranii.

! '&!

Figure 2-4. Sectioning of chd7 morphants reveals moderate to severe craniofacial cartilage abnormalities. (A-C) Alcian blue cartilage staining of

10"m sectioned zebrafish embryos at 4 dpf. Staining revealed wild-type structures of Std morphants (A), while chd7 morphants (B-C) had many of the cartilaginous structures absent. Sectioning confirmed the common chd7

! ''! morphant phenotype does not develop all of the ceratobranchial cartilages (B).

Staining of severe chd7 morphants only detected some development of the neurocranium (C). AC = auditory capsule, CB = ceratobranchial, CH = ceratohyal, EP = ethmoid plate, ME = Meckel’s cartilage, PC = parachordal, PQ

= palatoquadrate, T = trabecula cranii.

! '(! ceratobranchial arch was occasionally present (Figure 2-4). In more severely affected chd7 morphants, the only craniofacial cartilage structure observed was a severely underdeveloped neurocranium with malformed parachordal and trabecula cranii cartilages (Figure 2-3, I-J). Interestingly, parachordal and trabecula cranii cartilages are partially derived from mesoderm, while the more anterior cartilage structures that were typically absent in the severe chd7 morphants are mostly derived from neural crest (165). These results suggest that chd7 plays a critical role in the formation of cranial neural crest-derived cartilage tissues.

Chd7 is required for normal cellular proliferation during zebrafish development

In E9.5-E10.5 mouse embryos, haploinsufficiency of CHD7 is associated with reduced proliferation of olfactory neural stem cells and cells of the otic epithelium and ganglion (162, 163). Moreover, we previously showed that siRNA-mediated knockdown of CHD7 in cultured cells attenuates their proliferation (64). These proliferative deficiencies occur without concomitant increases in cell death, indicating that CHD7 regulates cell proliferation and not apoptosis. We tested for proliferative defects in the chd7 morphants through immunofluorescence analysis of phosphorylated histone H3 at serine 10 (P-H3), a marker of mitosis (Figure 2-

5,A-B). Compared to control embryos, the total area positive for cellular P-H3 expression in chd7 morphants (25 hpf) was reduced by 37% in the anterior

! ')!

Figure 2-5. chd7 targeting impairs cellular proliferation. (A-B) Lateral views of representative P-H3 stained zebrafish morphants at 25 hpf. (C) Quantification of the area occupied by P-H3 positive cells in chd7 morphants relative to P-H3 positive cells in Std morphants (n = 5-6). (D-E) Graphs of P-H3 positive cell counts taken from confocal images of both the head and tail regions of Std and chd7 morphants (n = 4-6). (F-H) Graphs of qRT-PCR data measuring gene expression of several cell cycle regulator genes at 25 hpf in chd7 morphants

! '*! relative to Std morphants (n = 5-6). (I) Expression of pre-rRNA in chd7 morphants at two separate morpholino dosages relative to Std morphants at 8 hpf (n = 3). All error bars represent SEM. Significance for all graphs was determined with a Student’s two-tailed t-test and significant values are noted p <

0.01 (**) and p < 0.001 (***).

! (+! region of the embryo (Figure 2-5,C). In addition, counts of P-H3 immuno-positive cells showed a 47% reduction in the total number of proliferating cells in chd7 morphants compared to controls (Figure 2-5,D-E). A reduction in the number of proliferating cells was detected not only in the anterior region of the embryo

(Figure 2-5,D), where chd7 levels are high at this stage of development, but also the tail region (Figure 2-5,E), where chd7 levels are low. Zebrafish chd7 expression is ubiquitous in early development and becomes more anteriorized around 3 dpf (166). Thus, the observed proliferative deficiencies are probably related to Chd7’s role during early development, prior to the time at which chd7 expression is restricted to the anterior region of the zebrafish embryo.

Similar to the findings in mice and cultured cells, the proliferative defects in the chd7 morphants occurred in the absence of apoptosis, as determined through TUNEL-assays (data not shown). The proliferative deficiencies were accompanied by elevated expression of potent cell-cycle inhibitors, including p21, p27, and ink4ab (the zebrafish homolog of both p15 and p16 (167)) (Figure

2-5,F-H). Because we have previously shown that decreases in rRNA correlate with reduced proliferation, we also quantified the levels of 45S pre-rRNA, the product of Pol I transcription of rDNA that is ultimately processed into the mature

18S, 5S, and 28S ribosomal subunits (64, 111). No significant differences in pre- rRNA levels were detected between chd7 morphants and controls, even in embryos injected with high doses of chd7 MO (Figure 2-5,I). Thus, either chd7 does not regulate rRNA in the zebrafish, or the effect is restricted to specific cell types or other stages of development. Overall, these findings indicate that Chd7

! ("! is required for normal cellular proliferation in the developing zebrafish embryo, although it is currently not clear from these data that the effect is mediated through rDNA regulation.

Fbxl10 regulates rRNA levels during zebrafish embryogenesis

Cell proliferation rates are tightly coupled to ribosomal RNA levels.

Though our results were inconclusive as to whether zebrafish Chd7 regulates rRNA, we set out to modulate rRNA levels in chd7 morphants, reasoning that this might restore the proliferative deficiencies. To achieve this, we chose to knockdown the homolog of a known repressor of rRNA genes in mammals:

FBXL10 (also known as NDY1, JHDM1B, and KDM2B). FBXL10 is a jumonji domain-containing histone demethylase that represses rRNA genes in the mammalian nucleolus, suppressing cell proliferation (127). Other studies contradict these findings, suggesting that FBXL10 increases proliferation by directly suppressing cell-cycle inhibitors in the nucleoplasm, including p15Ink4b, p16Ink4a, and p19Arf (129, 131, 132). Similarly to chd7, fbxl10 (kdm2bb) is expressed ubiquitously throughout early zebrafish embryogenesis. By 4 dpf, fbxl10 expression is restricted to the anterior embryo including the retina and central nervous system (168). Using a splice-blocker morpholino, we knocked down fbxl10 transcript levels by 35 to 50% (Figure 2-6,A-B). When 5 ng of MO were injected, the fbxl10 knockdown yielded viable fish without an obvious gross morphological abnormalities or defects in craniofacial cartilage structures

! (#!

Figure 2-6. Targeting of zebrafish fbxl10 transcript modulates pre-rRNA expression. (A) Schematic of the un-spliced fbxl10 transcript and the location of the fbxl10 morpholino (black bar). Injection of the morpholino results in an exclusion of exon 2 in the mature fbxl10 transcript and is predicted to induce a nonsense mutation (black arrow). Exclusion of exon 2 in the fbxl10 morphant transcript was confirmed by PCR. (B) Graph of qRT-PCR data measuring the

! ($! expression of wild-type fbxl10 relative to the expression in Std control morphants at 8 hpf (n =4). (C-D) Representative lateral (C) and dorsal (D) bright-field images of fbxl10 morphants. (E-F) Lateral views of representative P-H3 stained zebrafish morphants at 25 hpf. (G) Quantification of the area occupied by P-H3 positive cells in fbxl10 morphants relative to P-H3 positive cells in Std morphants (n = 12-

13). (H) Graph of qRT-PCR expression data for 45S pre-rRNA relative in fbxl10 morphants relative to Std morphants (n = 4). (I) Graph of qRT-PCR data measuring ink4ab expression relative to Std morphants (n = 6). Error bars in all graphs represent SEM. Significance determined by a Student’s two-tailed t-test and significant values are noted p < 0.05 (*) and p < 0.01 (**).

! (%!

Figure 2-7. Modulation of fbxl10 expression results in normal development of gross anatomical and craniofacial structures. (A-B) Lateral and dorsal bright-field images of 4 dpf fbxl10 morphants. (C-D) Whole-mount lateral and ventral representative images of fbxl10 morphant craniofacial structure. (E)

Sagittal sectioning of Alcian blue stained craniofacial cartilage in a representative fbxl10 morphant. AC = auditory capsule, CB = ceratobranchial, CH = ceratohyal,

ME = Meckel’s cartilage, PC = parachordal, PQ = palatoquadrate, T = trabecula cranii.

! (&! (Figure 2-6,C-D and Figure 2-7). Additionally, fbxl10 knockdown did not impact cell proliferation (Figure 2-6,E-G). However, higher doses of fbxl10 morpholino induced early embryonic lethality by 24 hpf. Also, chd7 expression was also found to be normal in fbxl10 morphants when measured by qRT-PCR (not shown). We next tested if Fbxl10 regulates rRNA in the nucleolus or cell cycle genes in the nucleoplasm. 45S pre-rRNA levels were 30% higher in embryos injected with fbxl10-MO than control embryos (Figure 2-6,H). ink4ab levels were not significantly different between controls and fbxl10 morphants (Figure 2-6,I).

These results support the reported mammalian function of Fbxl10 as a repressor of rRNA genes in the nucleolus.

Rescue of chd7 morphant phenotype upon knockdown of fbxl10

Having established that Fbxl10 represses rRNA levels in the fish, we tested the effects of knocking down both fbxl10 and chd7, through co-injection of chd7 and fbxl10 morpholinos. Co-injection of fbxl10-MO and the chd7-translation blocker MO induced embryonic lethality within 24 hpf. However, co-injection of fbxl10-MO with the chd7 splice blocker MO suppressed the morphological defects induced by chd7 morpholino alone. Specifically, co-injection of chd7 and fbxl10 morpholinos reduced the penetrance of pericardial edema (13% from

58%), eye abnormalities (11% from 73%), pectoral fin defects (8% from 55%), craniofacial defects (27% from 64%), and otolith abnormalities (0% from 25%)

(Figure 2-8,A). In addition to the improvement of gross morphology discussed

! ('!

Figure 2-8. Modulation of fbxl10 expression rescues CHARGE-like phenotypes and improves cellular proliferation defects. (A) Graph of the percentage of observed CHARGE-like phenotypes across the different zebrafish morphants. Significance was determined by chi-square tests. (B-E) Lateral and

! ((! ventral views of representative cartilage staining in the chd7/fbxl10 double morphants compared to controls (B) at 4 dpf including morphants with normal phenotypes (C), mild malformations of the ceratohyal (D), and chd7/fbxl10 double morphants with severe ceratohyal malformations and ceratobranchial cartilage were undetectable (E). (F) Graph of the frequency of the observed craniofacial cartilage phenotypes in the developing zebrafish. Zebrafish were categorized on the severity of the craniofacial cartilage defect. “Malformed” morphants had all cartilages present but displayed morphological changes in the ceratohyal. “Underdeveloped” morphants had no detectable ceratobranchial arches in addition to a malformed ceratohyal. A morphant was categorized

“Absent” with no detectable anterior and branchial arches. A highly underdeveloped neurocranium was present in these morphants. (G-H) Lateral views of representative P-H3 stained zebrafish Std and chd7/fbxl10 morphants at

25 hpf. (I) Quantification of the area occupied by P-H3 positive cells in chd7 morphants (re-plotted from Figure 4C) and chd7/fbxl10 morphants relative to P-

H3 positive cells in Std morphants (n = 7). (J) Representative image of the amplified PCR products of both wild-type and morphant chd7 transcripts from 8 hpf across the panel of morphants. Significant p-values in all graphs are noted p

< 0.05 (*), p < 0.01 (**), p < 0.001 (***). AC = auditory capsule, CB = ceratobranchial, CH = ceratohyal, EP = ethmoid plate, ME = Meckel’s cartilage,

PQ = palatoquadrate.

! ()!

Figure 2-9. Changes in cellular proliferation correlate with embryonic head size. (A) A diagram indicating the locations of head measurements taken across the morphant zebrafish head region. Width measurements were taken across the forebrain (i) and midbrain (ii). The length of the zebrafish head was also measured from the anterior tip to the pectoral fins (iii). (i-iii) Graphs of the

! (*! individual measurements for each morphant (n=7) correspond with the location on the diagram. All error bars represent SEM. Significance for all graphs were determined with a Student’s two-tailed t-test and significant values are noted p <

0.05 (*), p < 0.01 (**), p < 0.001 (***).

! )+! above, we also observed a significant restoration in overall head size in the chd7/fbxl10 double morphants compared to the chd7 morphant (Figure 2-9).

Forty-six percent of co-injected morphants showed restoration of the craniofacial cartilage to wild-type or near wild-type morphology with only minor malformations in the second pharyngeal arch (Figure 2-8,B-C and Figure 2-10,A). Compared to the single chd7 morphants (Figure 2-3,F-G), the chd7/fbxl10 morphants had more anteriorly developed Meckel’s cartilage, a wild-type or near wild-type ceratohyal morphology, and all ceratobranchial arches were detectable upon

Alcian blue staining. The remaining 54% showed craniofacial cartilage abnormalities similar to those observed in chd7 single morphants, but in general, these abnormalities were less severe than those seen in the single chd7 morphants (Figure 2-8,D-F). For example, the chd7/fbxl10 double morphants had a similar proportion of morphants categorized to be malformed compared to the chd7 single morphants due to more linear ceratohyal morphologies (Figure 2-

8,D and Figure 2-10,B). However, only a small proportion of the chd7/fbxl10 double morphants were scored as underdeveloped with both an inverted ceratohyal cartilage and undetectable ceratobranchial arches (Figure 2-8,E and

Figure 2-10,C). This indicates a significant restoration of the ceratobranchial arches in the double morphants. The craniofacial cartilage structures in the majority of single fbxl10 morphants injected in parallel were again indistinguishable from the standard-MO controls (Fig. 2-8,F).

We also analyzed cell proliferation though quantitative analysis of P-H3 immuno-positive cells in morphant embryos. Relative to controls, the percentage

! )"!

Figure 2-10. Sagittal sectioning of 4 dpf chd7/fbxl10 double morphants indicates variable restoration of ceratobranchial cartilage development.

Bright-field images of 10"m sagittal sections of representative chd7/fbxl10 double morphants. The chd7/fbxl10 double morphants were classified into three categories based on the degree of restoration in craniofacial development. These images are sections from representative double morphants from each category including normal (A), malformed (B), and underdeveloped (C). AC = auditory

! )#! capsule, CB = ceratobranchial, CH = ceratohyal, EP = ethmoid plate, ME =

Meckel’s cartilage, PC = parachordal, PQ = palatoquadrate, T = trabecula cranii.

! )$! of mitotic cells within measured areas increased from 63% in chd7 single morphants to 78% in co-injected embryos (Figure 2-8,G-I). Importantly, these percentages reflect average counts from multiple co-injected embryos, from which the degree of rescue was variable. In fact, analysis of individual P-H3 immunostained embryos showed complete to near-complete restoration of cell proliferation in approximately two-thirds of all embryos analyzed (n = 29). Overall, these data indicate that knockdown of fbxl10 can mitigate chd7-morphant phenotypes in zebrafish. Importantly, co-injected embryos retained the chd7 morphant transcript, indicating that the effects of Fbxl10 depletion genuinely trump those of Chd7 depletion, and that rescue is not simply due to loss of the chd7 morphant transcript (Figure 2-8,J).

Analysis of rRNA and cell cycle genes in fbxl10/chd7 double morphants

To gain insights into the mechanism underlying the fbxl10-mediated rescue, we compared the levels of rRNA, p21, p27, and ink4ab in fbxl10/chd7 co- injected morphant embryos to those in chd7 single morphants and standard- injected embryos. These experiments are inherently difficult to analyze for several reasons. First, 46% of co-injected embryos show rescue by 4 dpf, and, at the 25 hpf time point at which embryos are collected to perform the assay, one cannot distinguish rescued embryos from those that will fail to show rescue. In addition, embryos must be pooled to obtain sufficient quantities of RNA for the analysis. Nonetheless, we found that pre-rRNA levels were similar between co- injected and control-injected embryos, indicating that the fbxl10-mediated

! )%!

Figure 2-11. Gene expression changes in cell-cycle regulators in chd7/fbxl10 double morphants. (A) Expression of pre-rRNA in chd7/fbxl10 double morphants and fbxl10 single morphants (re-plotted for comparison from

Figure 5F) relative to Std morphants at 8 hpf (n = 3). (B-D) Graphs of qRT-PCR data measuring gene expression of several cell cycle regulator genes at 25 hpf across the panel of morphant embryos relative to Std morphants (n = 5-6).

Expression data for chd7 morphants re-plotted here from Figure 4D-F for comparison. Error bars represent SEM. Significance for all graphs was determined with a Student’s two-tailed t-test and significant values are noted p <

0.05 (*), p < 0.01 (**), p < 0.001 (***).

! )&! increases in rRNA are attenuated through targeted knockdown of chd7 (Figure 2-

11,A). Thus, even though reduced rRNA levels were not detected in the chd7 single morphants, elevated rRNA levels in the fbxl10 morphants are clearly attenuated upon knockdown of chd7, suggesting that Chd7’s role as a positive regulator of rRNA may be context specific. p27 levels, elevated in the chd7 single morphants, were also attenuated upon co-injection of fbxl10 and chd7 morpholinos (Figure 2-11,B). p21 and ink4ab levels in chd7 single morphants were not significantly different from co-injected embryos (Figure 2-11,C-D).

However, these results reflect transcript levels measured from pooled co-injected embryos, of which 46% were rescued and 54% remained morphant. Thus, the levels of p21 and ink4ab shown in the plots, which are clearly trending downward, may be much lower in the 46% of embryos that were rescued. Based on these findings, we propose that elevated rRNA levels, induced upon knockdown of fbxl10, leads to suppression of cell cycle inhibitors activated upon chd7 targeting, thereby alleviating the chd7-associated cell proliferation defect.

! )'! Discussion

To study the role of Chd7 during development, we generated a zebrafish model of CHARGE syndrome through MO-mediated gene targeting. The chd7 morphant phenotype is highly MO dose-dependent, with high doses leading to early embryonic lethality and lower doses yielding viable fish with craniofacial defects as well as malformations of the eye, heart, otoliths, and pectoral fins.

The chd7 morphants also show cranial cartilage abnormalities, consistent with a defect in developing neural crest, the proposed cell type of origin for many of the anomalies observed in CHARGE syndrome patients (54, 58, 66, 69, 155). The chd7 morphant embryos also display widespread deficiencies in cellular proliferation, accompanied by elevated levels of potent cell-cycle inhibitors.

Remarkably, the gross morphological alterations, cartilage abnormalities, and cell proliferation deficiencies that define the chd7 morphant phenotype are all restorable with knockdown of Fbxl10, a repressor of rRNA. We propose that the mechanism of rescue is due to suppression of cell cycle inhibitors that are activated upon chd7 targeting, possibly through global modulation of rRNA levels.

To date, zebrafish, frogs, and mice have been used to study the role of

CHD7 in development (54, 60, 67-69, 97, 98). The overall phenotype and the organ systems affected in the chd7 morphants reported here are consistent with those previously observed in the other models (Table 2-1). For example, defects in neural crest development and malformed craniofacial cartilage structures were previously reported in both zebrafish and frogs. Similarly to our zebrafish model,

! )(! the frog model also shows facial width compression (54). As in the previously described zebrafish, frog, and mouse models, chd7 morphants described here show eye, heart, and otolith abnormalities. However the specific anomalies described vary somewhat between the models. With respect to the eyes for example, our zebrafish models show an underdeveloped lens, the previously described zebrafish models show retinal disorganization, the frog models develop colobomas, and the mouse models present with keratoconjunctivitis sicca (54, 67, 69, 97). The underlying basis for these differences is not known, but interestingly, this phenotypic variability is highly reminiscent of that seen among human patients with CHARGE syndrome and warrants further study.

To better understand the contribution of the proliferative deficiencies to the overall phenotype of the chd7 morphants, we knocked down the levels of fbxl10/kdm2bb, a histone demethylase that normally suppresses cell proliferation and has a similar spatiotemporal expression pattern to chd7 (127, 131, 132, 166,

168). To our knowledge, this is the first report of a zebrafish fbxl10 morphant model, and the first report that fbxl10 modulates rRNA levels in the zebrafish.

Fifty percent reduction of fbxl10 did not yield any gross morphological abnormalities. However, more substantial decreases of fbxl10 induced early embryonic lethality by the end of the segmentation period (24 hpf). This phenotype is somewhat reminiscent of that observed in Fbxl10 mutant mice.

Specifically, fbxl10 heterozygotes develop normally, while fbxl10-null homozygotes die during late embryogenesis or shortly after birth (124).

! ))! It has been demonstrated in several model systems that decreases in

CHD7 levels impair cellular proliferation in multiple cell types (64, 162). Our studies not only corroborate these findings, but also implicate proliferative deficiencies as the basis for the developmental anomalies observed in the zebrafish model of CHARGE syndrome. The rescue of a multiple congenital anomaly syndrome caused by a deficiency in rRNA biogenesis is not unprecedented. Treacher-Collins Syndrome (TCS) is congenital disorder of craniofacial development caused by mutations in TCOF1 and POLR1D (169-

171). These mutations lead to reduced rRNA biogenesis, and similarly to

CHARGE syndrome, neural crest is implicated as the cell type of origin for most of the associated anomalies in TCS. In mouse models of TCS, impaired ribosome biogenesis triggers the nucleolar stress response, activating p53 and leading to proliferative deficiencies and apoptosis of neural crest cells.

Moreover, inhibition of p53 blocks neural crest apoptosis and rescues the craniofacial defects (142). In our study, several genes associated with the nucleolar stress response, including p21, p27 and rRNA itself, responded upon

MO-targeting of chd7 and fbxl10. These findings lead us to hypothesize that the cell proliferation defects observed in the chd7 morphants are due to activation of the nucleolar stress response, like in TCS. However, MO-mediated knockdown of p53 failed to rescue the chd7 morphant phenotype. Additionally, we previously showed that proliferative deficiencies induced upon knockdown of CHD7 in cultured cells were accompanied by changes in the levels of rRNA and p21, but not p53 (64). Thus, if the nucleolar stress response is the basis of the

! )*! proliferative defect seen in the chd7 morphants, and the mechanism of rescue is due to suppression of the nucleolar stress response, it is likely to be p53- independent.

Together with the proposed role for CHD7 as a regulator of rRNA transcription in the nucleolus, the multiple anomalies in CHARGE syndrome are thought to due to insufficiencies in cell specification and proliferation during development. Our findings in the zebrafish suggest that restoring the cell proliferative deficiencies at the early stages of embryogenesis, even in the context of the other cellular deficits, could be sufficient to attenuate or altogether bypass the developmental defects associated with CHD7 mutation. Our studies lay the foundation to test this hypothesis in higher vertebrates, either through targeted modulation of FBXL10 or other genes that regulate rRNA levels, or genes that directly regulate the cell cycle. It is also noteworthy that histone demethylases are particularly amenable to targeting with small-molecule inhibitors (172, 173). If the findings here on Fbxl10 are validated in higher vertebrates, we might be able to tap into this growing area of therapeutic research for application to CHARGE syndrome.

! *+! Materials and Methods

Zebrafish maintenance

Wild-type Tuebingen (TÜ) zebrafish (Danio rerio) embryos were raised at

28°C. The zebrafish were raised on 14-hour light cycle and 10-hour dark cycle.

Protein sequence comparison

Human CHD7 (NP_060250.2) and zebrafish Chd7

(ENSDART00000016208) protein sequences were aligned using the NCBI

BLAST blastp suite to determine the degree of amino acid overlap between the two sequences (174). To retrieve the predicted domain composition of each protein sequence, we used the NCBI Conserved Domain Database (175).

Morpholino design and injection

A splice-blocking morpholino, 5’- ACCTACAATGAAGGAAATAGGCCGT-

3’, a 5-bp mismatch control morpholino, 5’-

ACGTAGAATCAAGCAAATACGCCGT-3’, a second confirmation splice-blocking morpholino, 5’-TGTGCCTGGAGGCAACAGCACAAAC-3’, and a translation- blocking morpholino, 5’-GGCTCATCATGCCTGGGTCAGCCAT-3’ were designed against the zebrafish chd7 transcript (ENSDART00000016208). To reduce fbxl10 expression, we used a splice-blocking morpholino designed against the zebrafish fbxl10 transcript (ENSDART00000102530), 5’-

ACAACACCTGAGAACAGAAGCAGGA-3. To target p53 expression, we used a previously characterized morpholino (176). To control for phenotypes resulting

! *"! from the injection procedure alone, a standard control morpholino that has no target or biological activity within the zebrafish was used in all experiments (Gene

Tools). Phenotypes between standard morphants and those injected with the 5- bp mismatch chd7 morpholino were comparable. Zebrafish embryos received 2.5 ng of chd7 morpholino, 5 ng of standard control morpholino, 4 ng p53 morpholino, or 5 ng fbxl10 morpholino unless concentration is indicated otherwise. The chd7/fbxl10 double morphants received a simultaneous injection of 2.5 ng chd7 morpholino and 5 ng fbxl10 morpholino. The chd7/p53 double morphants received a simultaneous injection of 2.5 ng chd7 morpholino and 4 ng p53 morpholino. This p53 morpholino dosage was slightly higher than that recommended for testing for non-specific morpholino related cell death (177). For all co-injection experiments, chd7 and fbxl10 or chd7 and p53 morpholino single injections were performed in parallel as an additional control. All morpholinos were designed and manufactured by Gene Tools (Philomath, Oregon).

Morpholinos were dissolved in sterile water to stock solution of 65 mg/ml. 5 mg/ml or 10 mg/ml working solutions were made by diluting the morpholino in water and 2% phenyl red. Zebrafish embryos were then injected at the 1 or 2 cell stage with using a microinjector.

Scoring of zebrafish morphant phenotypes

Zebrafish embryos were incubated at 28°C in fish water until the desired developmental stage was reached. The chd7 morphant phenotypes were monitored and scored using a Leica S6E stereomicroscope between 4-6 dpf. The

! *#! bright-field images of chd7 morphant phenotypes were taken at 6 dpf and were images on a Leica DM6000. Significance was assessed by chi-square contingency test.

RNA extraction, cDNA synthesis, and qRT-PCR

Total RNA was extracted by homogenizing 20-30 pooled embryos at 8 hpf or 25 hpf in Trizol reagent (Invitrogen). Embryos were homogenized by drawing them through a 21G 1 # needle and 1 ml syringe (BD) approximately 20 times.

RNA was further purified via the Trizol Reagent protocol (Invitrogen) and was re- dissolved in RNase-free water. From the total purified RNA, cDNA was synthesized using a High-capacity cDNA Archive Kit (Applied Biosystems). Using quantitative polymerase-chain reaction (PCR), gene expression was measured in triplicates across a set of three biological replicates. PCR reactions were performed using SybrGreen or Taqman chemistry on an ABI 7300 real-time thermocycler. Designed primer sequences for PCR reactions are listed in Table

2. Expression of the 45S rRNA transcript measured using a previously published primer set (138). Expression of several genes was amplified using several

Taqman assays (Invitrogen) including p27 (ID: Dr03101119_ml) and b-actin1 (ID:

Dr0332610_m1). Permission for the ink4ab Taqman probe (ID: AJ1RUB6) was provided graciously by Dr. Hatem Sabaawy.

Cartilage staining of whole mounts

! *$! At 4 dpf, zebrafish were anesthetized with tricaine and fixed with cold 4% paraformaldehyde solution (PFA). Zebrafish cartilage was stained with Alcian blue and Alzarian Red as previously described (178). Zebrafish were imaged in glycerol and bright-field images were taken using a Leica MZ10F fluorescent microscope.

Cartilage sectioning

Zebrafish morphants were anesthetized with tricaine and fixed with cold

4% PFA at 4 dpf. The sample preparation, sectioning, and Alcian blue staining of all zebrafish embryos was performed by Histoserv, Inc. (Germantown, MD). All embryos were sectioned at 10"m.

Whole-mount antibody labeling

At 25 hpf, zebrafish embryos were anesthetized with tricaine and fixed with cold 4% PFA overnight at 4°C. Fixed embryos were rinsed several times with PBS and permeablilized overnight at room temperature with 3% Triton in

PBS. Embryos were rinsed in PBS again and blocked in 5% normal goat serum in PBS (blocking solution) for 3 hours at room temperature. Embryos were then incubated with rabbit anti-phosphorylated Histone H3 (phospho-ser10) antibody

(1:200, Cell Signal #9701) diluted in blocking solution overnight at 4°C. Embryos were rinsed several times over 6 hours in blocking solution and incubated overnight at 4°C with goat anti-rabbit AlexaFluor 488 (1:200, Invitrogen A-11008).

Embryos were rinsed in blocking solution for 2 hours. Zebrafish were stored in

! *%! VECTASHIELD mounting medium (VECTOR Laboratories). Fluorescent images were taken on a Leica MZ10F microscope after mounting zebrafish in 0.1% agarose.

Quantification of proliferation

Quantification of the cellular area expressing fluorescently labeled P-H3 was performed using Adobe Photoshop as previously described (179). Mitotic cells were quantified by calculating the average fluorescent area (pixel2). Five cells within each fluorescent image were selected using the Photoshop Magic

Wand Tool. On selection of the five mitotic cells, cells of similar fluorescent intensity were selected using the “Select Similar” command with a stringency factor of 20. Cells were not selected for based on intensity; however, selected cells in the plane of focus tended to be slightly higher in fluorescent intensity. The average pixel area was calculated for each image and the process was repeated across three separate z-planes for each zebrafish embryo. The three averaged pixel areas from the z-planes were again averaged to give a total average pixel area for each individual embryo. The total average pixel areas from multiple zebrafish embryos were plotted and statistical significance was determined using a Student’s t-test.

To count the number of P-H3-positive cells, zebrafish embryos were imaged on a Leica SP2 confocal microscope. Z-plane images were taken throughout the embryo at 40"m steps to avoid capturing cells in multiple z- planes. The cells were then manually counted by an individual whom was blinded

! *&! to the identity of the morpholino used in the experiment, to avoid bias. The data were then stratified by the morpholino used (Standard versus chd7), and the results were plotted and tested for statistical significance using a Student’s two- tailed t-test.

! *'!

Table 2-2. PCR primers designed to measure gene expression.

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! *(! Chapter 3

Dissecting CHD7 functions in a human iPS cell model of CHARGE

syndrome

! *)! Introduction

A recent technological advance in the field of human genetics and disease modeling is the discovery of induced pluripotent stem (iPS) cells. For the past several decades, researchers have been searching for methods to restore developmental potential to terminally differentiated cells (180). In 2006,

Takahashi and Yamanaka reported that four key transcriptional factors (Klf4,

Sox2, Oct3/4, and c-Myc) have the ability to induce differentiated mouse embryonic fibroblasts to a reprogrammed pluripotent stem cell state (181). It was demonstrated that these cells could be differentiated into cells of all three germ layers and that they have similar expression profiles to embryonic stem cells.

With advances in this reprogramming strategy, these cells provide an alternative to the controversial use of embryonic stem cells and can be used for multiple applications including drug screening, development of cell replacement therapies, and disease modeling (182).

From our work in the zebrafish and others, there are two outstanding questions that are key to a better understanding of CHARGE syndrome pathogenesis. First, what developmental cell types are the most affected by changes in CHD7 expression? Although CHD7 is expressed globally in the early stages of embryonic development, only a subset of organ systems is affected by changes in protein expression (53, 66, 69), which is consistent with our zebrafish results. Second, are there changes in cellular proliferation, as we observed in the zebrafish, in these specific developmental cell types? More importantly, are they relevant to the pathogenesis of CHARGE syndrome? In the zebrafish studies

! **! presented here, restoration of cellular proliferation improved the chd7 morphant phenotypes in all organ systems suggesting that a role for CHD7 in maintaining cellular proliferation is essential for embryonic development. Previous mouse studies have measured a similar occurrence, but we currently do not know whether these proliferation changes are a global phenomenon or restricted to particular tissue types (96, 162). Through the use of this technology, we can begin to address the roles of CHD7 during human embryonic development and begin to explore which cell types are the most CHD7-dosage sensitive.

The iPS cell system offers several advantages making it an ideal system in which we can address the necessary questions to understand human development. Patient-specific cell lines are obtained through relatively simple skin biopsies and reprogrammed to an induced pluripotent state. These cells open up countless paths of investigation and have already been employed to study a multitude of developmental genetic disorders such as Rett Syndrome, dysmyelinating disorders, Long QT syndrome, as well as many others (183). For some of these iPS cell lines, it has even been shown that in vitro differentiation to pure populations of disease relevant cell types results in specific disease features (182). Overall, iPS cell technology allows us the advantage of studying the genetic contribution of the donor patient at the individual level to understand their unique genetic mutations in any cell type (184).

Furthermore, a population of iPS cells is by definition pluripotent. In other words, the population of cells has the potential to be differentiated into all three embryonic germ layers (ectoderm, mesoderm, endoderm) and their further

! "++! derivative cell types (181). Through this differentiation process, iPS cells allow for the advantage of not only acquiring a large number of cells for experimentation, but also these cells can be studied in a pure population. Multiple cell types could theoretically be isolated using the zebrafish model system through cell dissociation and sorting or adapting the INTACT (isolation of nuclei tagged in specific cell types) method for zebrafish. However, to obtain a pure population of cells would require a very large number of morphant embryos, making the zebrafish model systems difficult and inefficient (185).

Overall, the utilization of iPS cells would allow for further investigation of the cellular proliferation defects in chd7 morphants and also additional studies into the nuclear and nucleolar functions of CHD7 in a developmental context. To this end, we present here our initial characterization of iPS cell lines derived from two separate CHARGE syndrome patients. These cells will provide the basis for these potential investigations and may reveal a deeper understanding into the complexity of CHD7 function and its regulation during development.

! "+"! Results

Early characterization and preliminary data in CHARGE syndrome patient- derived iPS cells

To begin our characterization of CHD7 functions during development, we first needed a population of cells with a CHD7 mutation to compare with controls.

Instead of inducing a random mutation in the CHD7, we chose to use fibroblast cells from skin biopsies of two separate CHARGE syndrome patients. Both of the

CHARGE syndrome patients were positive for CHD7 mutations with each mutation targeting a different region within the gene. CHARGE syndrome Patient

A carries a nonsense mutation while Patient B carries a missense mutation

(Figure 3-2). While the nonsense mutation in Patient A presumably results in nonsense-mediated decay of the CHD7 transcript, the missense mutation in

Patient B requires a more nuanced interpretation. There are several possibilities in which a missense mutation could result in CHD7 dysfunction: disruption of protein-protein interactions, decreased stability, or changes in protein structure and/or folding or localization. The mutation is not located within any predicted protein domains nor within a nuclear localization signal (http://nls- mapper.iab.keio.ac.jp). However, the amino acid is predicted to be buried within the CHD7 protein structure (PredictProteinOpen, http://ppopen.informatik.tu- muenchen.de). Thus, a change from an uncharged to a positively charged amino acid residue may possibly impact overall structure and folding. The affected residue in Patient B is highly conserved across vertebrates suggesting that it is important for CHD7 function. Despite the difference in mutation type and location,

! "+#!

Figure 3-1. The locations of CHD7 mutations in CHARGE syndrome patient- derived iPS cell lines. The CHD7 gene from each patient was Sanger sequenced to confirm the mutation locations. The A->T mutation in Patient A results in a premature stop codon at amino acid 752. The G->A mutation in

Patient B results in a missense mutation. The location of each amino acid residue change is indicated above the CHD7 protein schematic.

! "+$! both patients were clinically diagnosed with many of the same features including ocular colobomas, developmental delays, genital abnormities, ear abnormalities, and craniofacial defects.

To use the patient fibroblast cells lines to study the importance of CHD7 expression in multiple cell lineages, the cell lines were first reprogrammed to pluripotency (186, 187). This was achieved by infecting cell lines with a lentivirus containing an integrating expression construct coding for all four of the

Yamanaka transcription factors – OCT4, c-MYC, KLF4, and SOX2. After approximately one month, reprogrammed colonies of patient cells were isolated and expanded. To ensure that the cell lines were reprogrammed to a pluripotent state, cells were harvested and RNA collected to measure expression for two markers of pluripotency including the genes OCT4 and NANOG (Figure 3-3).

In addition to measuring marker genes for cell pluripotency, early characterization of the CHARGE syndrome patient lines included expression measurements of the CHD7 gene. Compared to a human embryonic stem cell line and a control iPS cell line, CHD7 gene expression in the two patient lines was reduced by approximately 50% (Figure 3-4). However, the levels of protein expression in these cells lines were reduced by almost 90% compared to the two controls. This surprising decrease in protein expression levels compared to the levels of RNA expression suggests that there are multiple levels of yet uncovered regulation in the expression of the CHD7 protein and warrants further investigation.

One potential explanation for this striking difference is that CHD7 may

! "+%!

Figure 3-2. Reprogrammed CHARGE syndrome patient cells express multiple markers of pluripotency. Two cell colonies were selected from each patient’s cell line to confirm cellular reprogramming to an induced pluripotent state. Relative expression of OCT4 and NANOG was measured using qRT-PCR and normalized to a CHD7 mutation-negative iPS control line.

! "+&!

Figure 3-3. CHD7 expression is significantly reduced in CHARGE syndrome patient-derived iPS cells. (A) Relative gene expression of CHD7 was measured by qRT-PCR and normalized to a CHD7 mutation-negative iPS control cell line.

(B) Western blot analysis of CHD7 protein expression. (C) Densitometry quantification of CHD7 protein expression relative to a control H9 human embryonic stem cell line.

! "+'! function in a positive-feedback loop to promote its own expression. In this scenario, a mutation in a single CHD7 allele may result in insufficient protein to function in chromatin remodeling while promoting its own expression. In support of this idea, previous ChIP-seq studies in mouse embryonic stem cells from our laboratory indicated that CHD7 localizes within 1 kb of its promoter (53).

Interestingly, this potential regulatory mechanism may exist in a subset of undifferentiated cells types in both mouse and humans. CHD7 binding peaks can be found within the promoter region and/or gene body of human embryonic stem cells and mouse neural stem cells (57, 188). However, this was not true in the human K562 myelogenous leukemia cell line (188). If the altered protein in

Patient B affected this potential feedback interaction, it may also help explain why the missense mutation could result in both reduced gene expression and protein expression. Another possibility is that additional proteins, whose expression is dependent on CHD7 levels, could also function in this potential feedback interaction. Alternatively, the discrepancy between RNA and protein expression could be a combination of qRT-PCR primer positioning and a lag in transcription degradation by nonsense-mediated decay.

Additionally, our preliminary studies on CHD7 also indicate that protein localization within the cell is cell-type dependent. Immunofluorescence studies revealed that in iPS cells CHD7 is localized mainly in the cytoplasm of the cell with diffuse staining in the nucleus (Figure 3-5). Contrary to these findings, CHD7 localization is mainly nuclear upon cellular differentiation and neither of these cell types show dense staining in the nucleolus of the cell contrary to previously

! "+(! reported studies (64, 65). Interestingly, these CHD7 protein localization patterns correlate with rRNA expression in iPS cells (Figure 3-6). This further supports that CHD7 is localizing within the cell to the rDNA promoting rRNA transcription, but in a context-dependent fashion.

! "+)!

Figure 3-4. CHD7 protein localizes to different cell sub-compartments in different cell types. Immunofluorescence staining for CHD7 and the nucleolar marker Nucleolin. Combined panels also include the overlay of DAPI staining of the nucleus.

! "+*!

Figure 3-5. Decreases in CHD7 expression do not affect 45S pre-rRNA expression in iPS cells. Relative expression of the 45S precursor rRNA was measured using qRT-PCR and normalized to a control human embryonic stem cell line.

! ""+! Discussion

From our previous work studying the function of Chd7 in development, we reported that targeting of zebrafish chd7 expression results in global cellular proliferation decreases. We further demonstrated that a partial restoration of cellular proliferation could rescue the complete chd7 morphant phenotype, which included developmental defects in CHARGE syndrome relevant organ systems.

While this finding could have promise for downstream therapeutics, it is still unclear how relevant these findings are to human embryonic development or, more namely, CHARGE syndrome pathogenesis. To this end, we developed a human model of early embryonic development using both wild-type iPS cell lines and CHD7 mutant iPS cell lines derived from the fibroblasts of CHARGE syndrome patients. Prior to investigating the relevance of cellular proliferation defects in the patient cell lines, we first set to characterize CHD7 expression and its localization patterns within these cells.

After successful reprogramming of the two CHARGE syndrome patient cell lines, we measured both gene and protein expression of CHD7. Despite the difference in mutation type, both patient lines had reduced CHD7 expression of approximately 50%. Furthermore, the CHD7 protein expression in both lines was reduced by almost 90% compared to control cell lines. The lack of concordance between gene expression and protein levels was highly unexpected. Upon further investigation, several CHD7 binding sites were found proximal to the

CHD7 gene promoter in previously characterized cell lines raising the possibility for CHD7 regulation through a positive feedback loop (57, 188). However,

! """! another potential explanation for this dramatic reduction of CHD7 expression is that it is an artifact of the reprogramming process. To test this possibility, we measured CHD7 expression in the original patient fibroblast lines. However, neither protein expression nor gene expression were detected in the CHARGE syndrome patient or control lines (data not shown). This suggests that CHD7 is not expressed in fibroblasts and thus we could not conclude whether the reduced

CHD7 expression is an artifact. However, as we acquire a wider panel of patient lines to characterize, we can begin to test whether or not this phenomenon holds true in multiple iPS cells lines or just for those presented here.

During our initial investigations, we also observed a change in the CHD7 subcellular localization pattern between cell types. Previous reports have found

CHD7 to be mainly localized to the nucleus and nucleolus of the cell consistent with CHD7 as a chromatin remodeler (64, 65). Despite the numerous nuclear localization signals predicted by the CHD7 amino acid sequence, we observed

CHD7 to be localized highly to the cytoplasm in iPS cells and was altered upon differentiation into the nucleus. Interestingly, CHD7 did not localize to the nucleolus in either of these cell types. Upon measuring pre-rRNA levels in the iPS cells, there was no difference in expression suggesting that pre-rRNA expression levels may only be regulated by CHD7 in specific cell types with nucleolar localization. To test this hypothesis, further experimentation is required and needs to be expanded to a panel of differentiated cell types including cell lines with known CHD7 mutations such as the CHARGE syndrome patient lines.

! ""#! Although our ultimate goal is understand the relevance of proliferation changes in CHARGE syndrome pathogenesis using patient iPS lines, we present here the preliminary characterization studies of these cells. These initial experiments resulted in novel findings into the potential regulatory mechanisms of both CHD7 expression and regulation within this early embryonic development. Future experimentation for this iPS cell resource to uncover the relevance of proliferation changes during development is discussed further in the following chapter.

! ""$! Materials and methods

Reprogramming of fibroblasts to induced pluripotent stem cells

CHARGE syndrome patient lines were reprogrammed to iPS cells by the

Case Western Reserve University Pluripotent Stem Cell Facility. Briefly, fibroblast cells were infected with a lentivirus containing an integrating expression vector to induce the exogenous expression of four transcription factors – OCT4,

KLF4, SOX2, and c-MYC. After approximately one month, several iPS colonies from each line were selected for further characterization. This methodology was previously described (186, 187).

Cell maintenance

The iPS cell lines were grown and maintained on a feeder layer of irradiated mouse embryonic fibroblasts (iMEFs). iMEFs were plated one day prior to passaging on gelatin-coated plates and fed with MEF media (DMEM, 10%

FBS, 1% Glutamax, 1% Non-essential amino acids, supplemented with 10ng/ml

FGF). iMEF feeder layer was conditioned at least one hour prior to iPS passaging with hES media (DMEM F12, 20% KSR, 1% Non-essential amino acids, 1% Glutamax, $-mercaptoethanol, supplemented with 10ng/ml FGF). Cells were fed daily with fresh hES media and passaged every 4 days using TrypLE

(Invitrogen).

Sanger sequencing of CHD7 mutations

! ""%! Genomic DNA (gDNA) was isolated from iPS cell lines using the Puregene

DNA Isolation Kit (Gentra Systems). gDNA was amplified by PCR across the previously identified mutations in each line. Amplified PCR products were submitted to the departmental DNA Sequencing Core for Sanger sequencing.

RNA extraction, cDNA synthesis, and qRT-PCR

Total RNA was extracted from iPS cell lines using the Trizol reagent and protocol (Invitrogen). From the total purified RNA, cDNA was synthesized using a

High-capacity cDNA archive kit (Applied Biosystems). Gene expression was measured by qRT-PCR as previously described in Chapter 2. Gene expression was amplified using several Taqman chemistry assays (Invitrogen) including

CHD7 (ID: Hs00215010_ml), OCT4 (Hs00742816_s1), NANOG

(Hs02387400_g1), and $-ACTIN (Hs00242273_m1). Human pre-rRNA was measured using SybrGreen chemistry with the following primers: Forward –

GAACGGTGGTGTGTCGTTC, Reverse – GCGTCTCGTCTCGTCTCACT.

Western blot analysis

For the iPS cell protein western blot analysis, 40"g of whole cell extracted protein was separated using a 3-8% Tris-acetate gel and transferred to a 0.45"m nitrocellulose membrane. The protein was detected using an %-CHD7 antibody

(Cell Signaling, #6505, 1:1000) or an %-$-actin antibody (Novus, NB600-501,

1:5000) in 5% milk/PBS-T. The membrane was incubated with HRP-conjugated secondary antibodies and the signal detected using a Supersignal West Dura

! ""&! Extended Duration Substrate kit (Thermo Scientific). Densitometry quantitation of the protein bands was performed using ImageJ software (NIH).

Immunofluorescence

To measure the localization of CHD7 and Nucleolin, cell lines were fixed using 4% PFA at approximately 70-80% confluency. After permeabilization with

0.2% Triton X-100 in 1X PBS, cells were blocked with 10% normal goat serum in

1X PBS, and then incubated overnight with %-CHD7 (Cell Signaling, #6505,

1:200) and %-Nucleolin (Abcam, AB13541, 1:500) antibodies. To detect the protein, cells were then incubated with Alexa Fluor secondary antibodies

(Invitrogen, A-11008, A-11005, 1:200) and DAPI stained (Sigma-Aldrich, D9542,

1"g/ml).

! ""'! Chapter 4

Discussion and Future Directions

! ""(! Summary

The chromatin remodeler CHD7 is an evolutionary conserved protein required for embryonic growth and development in a wide range of organisms.

Accordingly, abrogation of CHD7 function results in a constellation of developmental abnormalities affecting multiple organ systems. In humans, CHD7 mutations are responsible for approximately two-thirds of cases of CHARGE syndrome, a complex developmental disorder. In light of this, previous studies have focused on CHD7 and understanding its molecular roles in several specific cell types and tissues. However, it is currently unknown how and if these individual molecular functions of CHD7 interplay on the scale of whole embryonic development. With this question in mind, we set to test the relevance of these functions using the zebrafish as a model for early embryonic growth and development.

The targeting of the zebrafish Chd7 homolog resulted in a spectrum of morphological abnormalities and cellular defects. Although the targeting of chd7 did not result in the precise phenotypes seen in human CHARGE syndrome, there was a remarkable degree of overlap in the affected organ systems (Table

2-1). The chd7 morphants displayed abnormalities in the developing heart, otoliths of the ears, anterior eye, and pectoral fins. It was especially interesting that chd7 morphants also developed a spectrum of craniofacial defects in cranial neural crest-derived tissues.

Similarly to previous studies (64, 162), chd7 targeting in the zebrafish also results in significant decreases in cellular proliferation. However, these changes

! "")! in proliferation were not restricted to a subset of tissues, and were rather seen throughout the embryo in regions of both high and low chd7 expression and correlated with increased expression of the cell-cycle regulators ink4ab, p21, and p27. We next sought to test the importance of this conserved CHD7 function in regulating cellular proliferation by modulating the global expression levels of rRNA transcription, as CHD7 has been shown to regulate rRNA levels in human and mouse (61, 64). By knocking down expression of fbxl10, a histone demethylase that negatively regulates rRNA transcription (127), we successfully increased the levels of rRNA expression and ultimately increased cellular proliferation in chd7 morphant zebrafish embryos. The restoration of cellular proliferation in chd7 morphants resulted in a significant rescue of both morphological and molecular abnormalities including a significant reduction in p27 gene expression. This illustrates the importance of Chd7 regulating cellular proliferation during zebrafish embryogenesis and also implicates a potential etiology for CHARGE syndrome.

To address the relevance of these findings in human development, we established multiple iPS cell models from the fibroblasts of CHARGE syndrome patients. However, before investigating this question, we first characterized these reprogrammed cell lines. Preliminary experiments revealed that CHD7 protein expression was reduced by approximately 90% in both lines regardless of CHD7 mutation type. Furthermore, our data suggests that subcellular localization patterns of the CHD7 protein can vary between cell types. Also, these localization patterns may correlate with the levels of rRNA expression.

! ""*! Collectively, these cells will not only provide an ideal model to test the relevance of CHD7 in regulating cellular proliferation, but more importantly, allows us to broaden our investigations to characterize the functions and regulatory mechanisms of CHD7 throughout early human embryonic differentiation.

Discussion and future directions

Implications of zebrafish chd7 morphant model studies

The results presented here establish that Chd7 is required for the direct or indirect regulation of cellular proliferation during zebrafish embryogenesis.

However, these studies raise several questions regarding the specific spatiotemporal expression of Chd7 during development and how these findings may be applicable to higher vertebrates and, more specifically, human development.

Does chd7 regulate cellular proliferation in specific tissues?

From these studies, we can make two observations regarding changes in cellular proliferation resulting from chd7 targeting. First, targeting of chd7 expression results in a global decrease of approximately 40% in cellular proliferation in the developing embryo. Furthermore, upon complete rescue of the chd7 morphant morphological phenotype via fbxl10 targeting, there is a significant but incomplete restoration in the number of proliferating cells. One possibility for this selective rescue is that the expression patterns of chd7 and fbxl10 are not completely overlapping. In situ hybridization studies of fbxl10 and

! "#+! chd7 in zebrafish embryos suggest some discordance in between these genes in expression timing and tissue during later stages in development (98, 168).

However, both genes do share a similar pattern of global expression during embryonic development with gradual restriction to specific tissues such as the developing neuroepithelium and brain (midbrain/hindbrain). Additionally, both fbxl10 and chd7 are both highly expressed in tissues of developmental importance, especially in the context of CHARGE syndrome, such as neural progenitor cells and the neural crest (54, 124, 162). Taken together, these pieces of evidence suggest there is a subset of cells or tissue types, such as the neural crest and neural progenitor cells, that express both chd7 and fbxl10 that are potentially more greatly affected by changes in proliferation yet can be rescued by targeting fbxl10. Overall, this indicates that a smaller population of cell types requires crucial regulation of proliferation and plays a role in the pathogenesis of

CHARGE-like phenotypes in the zebrafish.

What is the molecular mechanism of chd7 morphant rescue by fbxl10 knockdown?

Through the modulation of fbxl10 expression, the zebrafish chd7 morphant phenotype can be fully restored to wild-type during early development. We targeted fbxl10 expression by reasoning that the reduction of a negative regulator of rRNA transcription, a key factor in cell proliferation, could be used to restore levels of global cellular proliferation in chd7 morphant embryos. However, it is still unclear to us the exact molecular mechanism by which this occurs, as changes

! "#"! in nucleoplasmic gene expression from fbxl10 knockdown could also be a contributor.

We propose two potential mechanisms of rescue based on these data

(Figure 4-1). The first potential mechanism is through the direct modulation of rRNA transcription levels in the developing embryo. Previous studies in Chd7 mouse mutants show significant reductions in rRNA expression, but in a subset of affected tissues. These changes were only detectable after tissue isolation instead of whole embryonic expression studies (64). Although we were unable to detect global embryonic decreases in rRNA transcription with the targeting of chd7 expression alone, we observed clear attenuation of global rRNA levels with the targeting both the chd7 and fbxl10 transcripts. Together, this suggests that zebrafish Chd7 has a conserved role in regulation rRNA expression and ultimately cellular proliferation.

Taking this into consideration, we propose that knocking down fbxl10 expression in chd7 morphants does not restore rRNA expression levels in all cell types, but only in a specific set of developmentally crucial cell types. This idea is not unprecedented. Several well-characterized ribosomopathies have well characterized defects in the ribosome biogenesis pathways, but each genetic disorder has a unique clinical presentation with only a small subset of cell types heavily affected by both ribosome and proliferation decreases (147). Given that rRNA transcription and subsequent ribosome biogenesis are considered universal “housekeeping” processes, these tissue-specific defects are puzzling.

To further investigate this potential model, it would be helpful to identify

! "##!

Figure 4-1. Model of interactions potentially involved in the molecular mechanism of rescue in chd7/fbxl10 double morphants. Solid lines indicate previously reported direct interactions between proteins while dashed lines indicate indirect interactions.

! "#$! developmentally relevant cell types in which both chd7 and fbxl10 are expressed, but also function as regulators of rRNA transcription. Recent work from human cell lines and mouse models has already begun investigation into these regulators (60, 61, 124, 162). Interestingly, it appears that both genes are expressed in cells of the neural crest lineage, but it has yet to be determined whether they specifically regulate rRNA expression in this cell type.

A second potential mechanism of rescue in chd7/fbxl10 double morphants could be through the interactions and feedback mechanisms between cell-cycle regulators. With changes in chd7 expression, we observed a significant increase in several cell-cycle inhibitor genes including p27, p21, and ink4ab. Although we measured the expression of several potent cell cycle regulators, our studies were not all-inclusive. There is still the possibility that chd7 may play a larger role in cell cycle regulation depending on the developmental context undetectable by our method of measuring gene expression. Through the targeting of fbxl10 expression, these increased cell-cycle regulators could be countered by increases in rRNA expression or, alternatively, through the modulation of its nucleoplasmic gene targets such as Ink4a/Arf/Ink4b and c-jun (124, 129, 130,

132). Although it is unclear whether rRNA levels can supersede increases in cell- cycle regulator expression (189), there is overwhelming evidence that the inverse is true through the nucleolar stress response pathway (137, 138, 141, 190). To further investigate the potential of this mechanism of rescue, further studies should include RNA-seq global expression profiling to examine changes cell- cycle gene expression in affected organ systems and cell types for both chd7

! "#%! and fbxl10 morphants. This could be accomplished by dissecting out individual tissues or through the isolation of individual cell types utilizing cell-sorting methods.

Are these findings applicable to mammalian development?

Like other model organisms, the zebrafish was chosen for these studies to model a congenital disorder that cannot be directly investigated in humans. We chose the zebrafish due to the many advantages it offers to study early embryonic development and model morphological changes that occur in

CHARGE syndrome. But with the future goal of uncovering the molecular etiology of CHARGE syndrome, we must ask whether this pathogenic change in cellular proliferation is relevant to mammalian development and, more specifically, human development. There is evidence of proliferation changes in

Chd7+/- mutant mouse models, but these are studies focused on isolated, but developmentally relevant, tissues of interest (60). Expanded studies would be required with the mouse model to determine whether changes in cellular proliferation are a global phenomenon similar to the zebrafish or truly restricted to only a subset of tissue types.

A second approach is to focus on whether the reduction in fbxl10- mediated rescue of CHARGE-like developmental abnormalities can be recapitulated in other model systems. As discussed, CHD7 loss of expression models and its homolog has been previously described in both mouse and

Drosophila (58, 67, 68, 99, 162). If these model organisms were crossed with

! "#&! FBXL10 mutant strains, it may prove to be insightful as to whether changes the changes in cellular proliferation are pathogenic. There are however multiple caveats to this approach. First, loss of CHD7 expression in both mouse and drosophila does not fully recapitulate the spectrum of developmental abnormalities as seen in humans. Furthermore, the functions of FBXL10 and its homologs are not fully understood in a developmental context making interpretation of these potential experiments difficult. For example, FBXL10 is shown to be both a positive and negative regulator of cellular proliferation in neural crest and neural progenitor cells, respectively (124). While it is generally observed that FBXL10 regulates the negative regulators INK4A/ARF/INK4B, these expression changes are reported only in mouse embryonic fibroblasts

(124, 129, 132). This leaves the context dependency for FBXL10 to function as a positive or negative regulator of cellular proliferation unknown.

Future experiments investigating the nucleolar and nuclear functions of

CHD7 using iPS cells as a developmental model of CHARGE syndrome

Multiple lines of evidence from previous studies have shown that changes in CHD7 expression results in changes in both nucleoplasmic gene expression and rRNA transcription in the nucleolus (53, 54, 56, 57, 61-65). While the studies presented here suggest a large role for CHD7 in regulating cellular proliferation, it is still unclear to what extent the nucleoplasmic and nucleolar functions of CHD7 play a role in this regulation and ultimately organism development. With the advantages of iPS cell technology, we can potentially begin to uncover changes

! "#'! in nucleoplasmic gene and nucleolar rDNA regulation with the added benefit of performing experiments in several developmental lineages.

(1) Uncovering the biological relevance of CHD7 in the nucleolus

A first step in understanding the developmental relevance of CHD7 at the rDNA and to further our understanding the potential molecular mechanism of rescue in the zebrafish chd7/fbxl10 double morphants is to characterize how relevant the function of CHD7 is to proper differentiation and ultimately CHARGE phenotypes. There are several questions we can ask. Are the molecular changes in zebrafish, such cellular proliferation deficits and alterations in cell-cycle regulator gene expression, also found in the human iPS cell models of CHARGE syndrome and in differentiated cell lineages? Does the function of CHD7 in regulating rRNA expression hold true in all cell types or only a subset? And lastly, if there are cellular proliferation defects present in these cell lineages compared to controls, can these defects be restored through the modulation of rRNA expression?

To answer these questions, initial experiments would include both controls and CHARGE patient iPS lines that are differentiated into endoderm, ectoderm, and mesoderm by a series of established protocols (191). These differentiated germ layers would be characterized using a series of experiments including immuno-fluorescence for CHD7 protein localization, ChIP-seq or ChIP-PCR at the rDNA, BrdU assays for changes in cellular proliferation rates, and RNA expression studies to measure expression of both rRNA levels and cell-cycle regulator genes. If changes in protein nucleolar localization and rRNA expression

! "#(! were indeed found in a subset of tissue types this would support our previous hypothesis that rRNA expression may be crucial in specific cell lineages. A caveat to these experiments is that we may not see changes in these early developmental lineages. To address this, further studies would include looking at a more specified panel of differentiated cell lineages. As most of the affected tissues in CHARGE syndrome patients are ectodermal derivatives, we hypothesize that this lineage would be the most highly affected with changes in cellular proliferation and rRNA expression with CHD7 mutations. Another consideration is that with the reduction of CHD7 protein levels, we are assuming that ChIP signal will also decrease evenly across the genome. There is a possibility that with decreases in CHD7 protein levels, the protein may be shuttled to critical loci (i.e. potentially the rDNA). While this potential bias would be biologically interesting, we would need to be aware for proper interpretation of global analyses.

(2) Enhancer and expression profiling of multiple cell lineages with CHD7 mutations

Through the use of iPS cell technology, we can also begin an in-depth systemic investigation into the roles of CHD7 on nucleoplasmic gene regulation during development. CHD7 localizes to thousands of enhancer loci across the genome to modulate nucleoplasmic gene expression in a context-dependent fashion (53). A few individual studies have focused on gene expression changes with CHD7 loss (52, 53, 57). Unfortunately, these studies have focused on expression changes within a single cell type and it is unclear how relevant these

! "#)! cell types are in relation to CHARGE syndrome. In other words, studies have not yet systematically measured gene expression changes or changes in regulatory pathways that may occur during the gradual process of cell differentiation and specification in organismal development.

Furthermore, it is unclear how changes in CHD7 levels affect the chromatin state at target enhancer loci resulting, potentially altering gene expression. Enhancer elements can exist in multiple epigenetic regulatory states that generally correlate with the transcriptional activity of their gene targets (192,

193). These states (active, poised, inactive) are defined by the presence or absence of specific histone tail modifications (Figure 4-2). These chromatin states at enhancers can fluctuate during development, reflecting changes in cell type-specific gene expression (194). CHD7 is reported to localize to all three of these enhancer classes in mouse embryonic stem cells (mESCs), but it is not clear whether this trend holds true throughout development. Additionally, it is not fully understood whether CHD7 is required for maintaining these individual enhancer states. Given that CHD7 is a chromatin remodeler (51), it might help maintain nucleosome depletion at these enhancer loci. A study by Feng et al showed that loss of CHD7 results in decreased H3K4me3 at promoters of the

Sox4 and Sox11 genes in mouse neural stem cells leading to reduced expression (62). While this change in the chromatin state was not specifically at enhancers, it supports the hypothesis that CHD7 can be required for the maintenance of specific chromatin marks at regulatory elements.

! "#*!

Figure 4-2. Multiple histone tail modifications demarcate enhancer activity and expression of target genes. Regulatory enhancer regions are reported to exist in three epigenetic states correlating with expression of their target genes

(192, 193). Active enhancers are demarcated by the histone tail modifications

H3K27ac and H3K4me1. A second intermediary state or poised state is demarcated by H3K4me1 alone and is associated with low levels of gene expression. Inactive enhancers are characterized by H3K27me3 and H3K4me1 and generally associated with transcriptionally repressed genes.

! "$+! Interestingly, exploratory studies from our lab have shown that gene targets of CHD7-bound poised enhancers in mouse ESCs are enriched for expression changes in the CHD7 mutant mouse inner ear. Given the evidence that CHD7 may have a role in maintaining the epigenetic state enhancers and promoters, it would interesting to further investigate whether these gene expression changes within the mouse inner ear are correlated with differences in the enhancer epigenetic state. If so, investigation into these epigenetic changes may provide a molecular mechanism for gene dysregulation with loss of CHD7 and may help predict expression changes in other downstream affected tissues.

With the CHARGE patient iPS cells and controls, we could pursue these questions from multiple angles. Through ChIP-seq and RNA-seq, we could begin our investigation by assessing the distribution of CHD7 across the genome and gene expression profiling in both wild-type and mutant lines. This could be done in all 3 germ layers and multiple derivative tissues with a focus in cell types of the neuroectodermal lineage. Alternatively, these studies could be pursued using mouse embryonic stem cells and compare to isolated tissues affected in Chd7 mutant mice.

To further explore this possibility of CHD7 regulating the chromatin state at enhancers, multiple data sets could be integrated including chIP-seq data from histone tail modifications to demarcate enhancer activity (H3K4me1, H3K27ac, and H3K27me3). The integration of RNA expression studies would be vital to determining whether any change in the enhancer state truly correlates with changes in gene expression.

! "$"! Summary remarks

The goal of this work was to understand how loss of function of the chromatin remodeler CHD7 alters organismal development. Using the zebrafish as a model of early embryonic development, the studies presented in Chapter 2 demonstrate that loss of chd7 function results in phenotypes in organ systems affected in CHARGE syndromes, due at least in part to a global decrease in cellular proliferation.

However, from this body of work and others, we are still left with many questions about the molecular mechanisms of CHD7 function, the understanding of which is vital for greater understanding of CHARGE syndrome pathogenesis.

First, it is largely unclear what genes are regulated by CHD7 in developmentally relevant tissues and second, how interactions with other chromatin remodeling and modifying complexes and transcription factors may play a role in targeting

CHD7 to specific genes. Investigations into individual CHARGE-relevant cell lineages in various model systems have revealed multiple protein interactions that regulate the expression of cell type-specific genes (54, 56, 57). Furthermore, while it is clear that CHD7 associates with enhancer elements, it is unclear what role CHD7 plays in enhancer-mediated regulation of gene expression. CHD7 utilizes energy derived from ATP hydrolysis to slide nucleosomes in vitro and so it is presumed that it remodels chromatin in vivo. Given that nucleosome remodeling plays a role in enhancer activation (195), CHD7 could promote open chromatin by sliding nucleosomes to expose transcription factor binding sites at

! "$#! enhancers. Alternatively, CHD7 could maintain an open chromatin state established by other remodelers, such as BRG1 (54).

The experiments proposed here can help begin to answer these questions and forward our biological understanding of CHD7 function and, ultimately,

CHARGE syndrome. By using iPS cell lines as a model of human development, we can study the role of CHD7 in enhancer biology, uncover gene expression targets, and further expand upon cell-specific protein interactions using proteomics approaches.

However, these proposed studies are not limited to our understanding of

CHARGE syndrome pathogenesis. The majority of these questions are key to understanding the role of CHD7 in human development, but can be applied to the biology of other chromatin remodelers and their roles in human disease (6). As such, further investigations in to CHD7 function may prove useful in a much larger context. For example, it was recently described that several chromatin remodelers interact and can collaborate in a step-wise to regulate chromatin structure and ultimately gene expression (196). Strong interactions have already been identified between CHD7 and a member of the SWI/SNF2 family of chromatin remodelers, BRG1. Therefore, a deeper understanding of CHD7 function may prove to useful to understanding BRG1 functions and its associated disease, Williams syndrome (7). Taken together, these studies and proposed future experiments provide a novel means for assessing the functions of CHD7 in regulating chromatin structure and may be applied to other disease relevant chromatin regulators to gain insight into disease pathogenesis.

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