Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations

Home , ANK3, CBARA1, DYNC1I1, KIF13A, KIF1B, KIF2A

by Jelena Perovanovic

M.S. in Molecular Biology and Physiology, September 2009, University of Belgrade M.Phil. in Molecular Medicine, August 2013, The George Washington University

A Dissertation submitted to

The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

August 31, 2015

Dissertation directed by

Eric P. Hoffman Professor of Integrative Systems Biology

The Columbian College of Arts and Sciences of The George Washington University certifies that Jelena Perovanovic has passed the Final Examination for the degree of

Doctor of Philosophy as of May 5, 2015. This is the final and approved form of the dissertation.

Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations

Jelena Perovanovic

Dissertation Research Committee:

Eric P. Hoffman, Professor of Integrative Systems Biology, Dissertation Director

Anamaris Colberg-Poley, Professor of Integrative Systems Biology, Committee Member

Robert J. Freishtat, Associate Professor of Pediatrics, Committee Member

Vittorio Sartorelli, Senior Investigator, National Institutes of Health, Committee Member

iii Acknowledgments

I am deeply indebted to countless individuals for their support and encouragement during the past five years of graduate studies.

First and foremost, I would like to express my gratitude to my mentor, Dr. Eric P.

Hoffman, for his unwavering support and guidance, and keen attention to my professional development. This Dissertation would not have been possible without the critical input he provided and the engaging environment he created. I would also like to thank my Dissertation Committee of Dr. Anamaris Colberg-Poley, Dr. Robert J. Freishtat,

Dr. Vittorio Sartorelli and Dr. Ashley Hill for challenging and guiding me throughout the development of this Dissertation.

I am grateful to my Master thesis mentor, Dr. Nikola Tucic at University of

Belgrade, Serbia, who taught me to observe biological phenomena through the lens of evolution and encouraged me to pursue my doctoral degree in the US.

Furthermore, I would like to thank Hoffman lab members for their friendship, help and support throughout the years, Center for Genetic Medicine at Children’s National

Medical Center and Institute for Biological Sciences at the George Washington

University, especially Dr. Linda Werling, Dr. Anne Chiaramello and Marc Wittlif.

I owe much gratitude to my parents, Milan and Sladjanka Perovanovic, and brother, Rade, for their continuous love and support throughout my life.

Finally, I would like to thank my beloved Nikola for his unconditional love, patience and support.

iv Abstract of Dissertation

Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations

The nuclear envelope is important to myogenesis, where the transition of lamin B to lamin A/C during terminal myogenic differentiation is needed for correct muscle development. Gene mutations of inner nuclear envelope components (LMNA, EDM) cause muscular dystrophy phenotypes (Emery-Dreifuss muscular dystrophy [EDMD]; limb-girdle muscular dystrophy). There is considerable allelic and clinical heterogeneity of the laminopathies, where distinct LMNA mutations selectively affect striated muscle, adipose, peripheral nerve, or multiple systems. These nuclear envelope components provide structural support to eukaryotic nuclei, but emerging data suggest key roles in

DNA replication, transcription and chromatin organization. Previous studies in our lab have shown that patients with EDMD show specific alterations of MyoD/Rb pathways compared to other dystrophies (Bakay et al., 2006), and further that emerin null mice show a specific block in myogenesis during terminal differentiation of myoblasts (Melcon et al., 2006). In this dissertation it is shown that mutations in LMNA and loss of emerin causing EDMD lead to disruption of developmentally appropriate chromatin-nuclear envelope associations during myogenesis. We scanned for genomic loci interacting with lamin A protein during myogenic differentiation using DamID-seq, and determined the effect of disease-associated amino acid changes on these protein/chromatin interactions

(p.R453W Emery-Dreifuss Muscular Dystrophy; p.R482W Familial Partial

Lipodystrophy). Lamin A missense mutations caused both promiscuous formation of lamin A associated heterochromatin domains (LAD), and decreased the length of WT

v LADs. These epigenetic alterations corresponded with pervasive presence of lamin A p.R452W (EDMD) at myogenic loci that was not seen with WT and p.R482W (FPLD).

This finding was validated by DNA methylation studies in EDMD patient myogenic cells

(WT vs. p.H222P). Also, appropriate exit from the cell cycle was perturbed by both mutations, with CDK1 and RB1 loci showing failure to attain LAD-mediated heterochromatin formation by DamID-seq and Chromatin Immuprecipitation sequencing

ChIP-seq (H3K9me3 heterochromatin mark) analyses. Epigenetic findings in EDMD-AD

(lamin A missense) were shared with the clinical phenocopy EDMD-XR (lamin A- associated emerin loss of function), with ChIP-seq of emerin null myogenic cells showing loss of heterochromatin marks at Sox2 and pluripotency pathway members. The failure of epigenetic remodeling at Sox2 was validated by mRNA (in vitro, and in EDMD patient muscle biopsies), and by lamin A p.H222P ChIP-qPCR. The alterations of SOX2 epigenetics may be upstream of the observed myogenic and cell cycle changes, as overexpression of SOX2 inhibited myogenic differentiation in vitro. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming caused by altered chromatin tethering to the nuclear periphery.

vi Table of Contents

Acknowledgments ...... iv

Abstract of Dissertation ...... v

List of Figures ...... ix

List of Tables ...... x

List of Abbreviations ...... xi

1 Chapter: Introduction ...... 1

1.1 Overview ...... 1 1.2 Diseases linked to mutations in LMNA ...... 6 1.3 Molecular role of LMNA in normal and pathogenic conditions ...... 16 1.4 Current molecular laminopathy models ...... 28 1.5 Significance ...... 29 2 Chapter: Material and Methods ...... 30

2.1 Cell culture ...... 30 2.2 qRT-PCR ...... 32 2.3 Chromatin immunoprecipitation assay and ChIP-seq ...... 34 2.4 ChIP-seq data analysis ...... 35 2.5 DamID sequencing ...... 35 2.6 Bioinformatics analysis ...... 41 2.7 Methylation analysis ...... 41 2.8 Expression data ...... 42 2.9 Overexpression and myogenic differentiation experiments ...... 42 3 Chapter: Results ...... 43

3.1 Summary ...... 43

vii 3.2 Introduction ...... 44 3.3 Results ...... 47 3.3.1 Lamin mutations alter genomic LADs of human muscle cells ...... 47 3.3.2 EDMD mutations show allele-specific alteration of epigenomic programming of myogenic differentiation ...... 56 3.3.3 Delayed E2F cell cycle suppression is predicted by lamin A mutation (EDMD-AD) ...... 76 3.3.4 Genome-wide prioritization of ChIP-seq alterations identifies aberrant persistence of pluripotency programs in emerin null cells...... 84 3.3.5 Targeted validation shows inappropriate transcriptional upregulation of Sox2 pluripotency pathways in emerin null myogenic cells in vitro and in vivo...... 103 3.3.6 Gain-of-function of Sox2 cell fate perturbations are specific to the disease-type of lamin A/C mutation...... 112 3.3.7 Wild-type lamin A protein directly interacts with SOX2 locus and this interaction is altered by LMNA missense mutation ...... 115 3.3.8 Overexpression of SOX2 perturbs myogenesis ...... 115 4 Chapter: Data interpretation and integration into models of pathogenesis ...... 119

5 Chapter: Discussion ...... 123

5.1 Choice of techniques: DamID vs. ChIP ...... 123 5.2 Lamin A mutations show tissue specificity in chromatin tethering to nuclear lamina ...... 125 5.3 Failed epigenetic remodeling in emerin null myogenic cells ...... 130 5.4 Building an integrated model of nuclear envelope molecular pathogenesis ..... 131 5.5 Conclusion ...... 135 5.6 Future directions ...... 137 6 Chapter: References ...... 139

viii List of Figures

Figure 1…………………………………………………………...... 3

Figure 2…………………………………………………………...... 17

Figure 3…………………………………………………………...... 26

Figure 4…………………………………………………………...... 39

Figure 5…………………………………………………………...... 48

Figure 6…………………………………………………………...... 50

Figure 7…………………………………………………………...... 52

Figure 8…………………………………………………………...... 57

Figure 9…………………………………………………………...... 66

Figure 10…………………………………………………………...... 74

Figure 11…………………………………………………………...... 77

Figure 12…………………………………………………………...... 80

Figure 13…………………………………………………………...... 82

Figure 14…………………………………………………………...... 98

Figure 15…………………………………………………………...... 101

Figure 16…………………………………………………………...... 104

Figure 17…………………………………………………………...... 107

Figure 18…………………………………………………………...... 113

Figure 19…………………………………………………………...... 117

Figure 20…………………………………………………………...... 133

ix List of Tables

Table 1…………………………………………………………...... 7

Table 2…………………………………………………………...... 15

Table 3…………………………………………………………...... 33

Table 4…………………………………………………………...... 55

Table 5…………………………………………………………...... 60

Table 6…………………………………………………………...... 69

Table 7…………………………………………………………...... 85

Table 8…………………………………………………………...... 109

Table 9…………………………………………………………...... 110

x List of Abbreviations

BAF Barrier to autointegration factor

CaaX motif C' is Cysteine, 'a' is an aliphatic amino acid, and 'X' is variable

ChIP-seq Chromatin Immunoprecipitation Sequencing

CMT Charcot-Marie-Tooth disease d day

DAM DNA adenine methyltransferase

DamID-seq DNA adenine methyltransferase identification by sequencing

DCM Dilated cardiomyopathy

DMEM Dulbecco's Modified Eagle Medium

EDL Extensor digitorum longus muscle

EDMD Emery-Dreifuss muscular dystrophy

EDMD-AD Emery-Dreifuss muscular dystrophy autosomal dominant

EDMD-XR Emery-Dreifuss muscular dystrophy X linked

ESCs Embryonic stem cells

FACE2 Farnesylated proteins-converting enzyme 2

FBS Fetal bovine serum

FC Fold change

FPLD Familial partial lipodystrophy gDNA Genomic DNA

GEO Gene Expression Omnibus

GFP Green fluorescent protein

HDAC Histone deacetylase

xi HGPS Hutchinson-Gilford progeria syndrome

HHSS Hand-heart syndrome Slovenian type

HI-FBS Heat inactivated fetal bovine serum hrs hours hTERT Human telomerase

ICMT Isoprenylcysteine carboxymethyltransferase

IFNγ Interferon gama

INM Inner nuclear membrane

IPA Ingenuity pathway analysis

LADs Lamina associated domains

LBR Lamin B receptor

LEM domain Lap Emerin Man shared domain

LGMD1B Limb-girdle muscular dystrophy type 1B

LMNA Lamin A/C

MADA Mandibuloacral dysplasia with type A lipodystrophy

MEFs Mouse embryonic fibroblasts

MHC Major histocompatibility complex

NE Nuclear envelope

ORF Open reading frame qPCR Quantitative polymerase chain reaction qRT-PCR Quantitative reverse transcription polymerase chain reaction

TE Tris EDTA (Ethylenediaminetetraacetic acid)

TSS Transcription start site

xii

1 Chapter: Introduction

1.1 Overview

The nuclear envelope defines the cell nucleus, and is the major cellular feature that distinguishes prokaryotes from eukaryotes. This evolutionary discriminator emerges with greater specialization of cells, and thus the nucleus plays a role in cellular diversity and differentiation. The nuclear envelope (Figure 1) is a double membrane system contiguous with the endoplasmic reticulum. A dense meshwork of intermediate filaments, called the nuclear lamina, provides structural and mechanical support to the double membrane structures, and together they make up the nuclear envelope.

Traditionally, the nuclear envelope and nucleus has been attributed with the main role of separating the genetic material (DNA and associated proteins, termed chromatin) from the rest of the cytoplasm. Recent studies have implicated the nuclear envelope in processes governing chromatin organization and remodeling, including gene silencing, transcriptional and differentiation patterns, RNA export and protein import, as well as epigenetic remodeling and memory.

Gene mutation is a powerful tool to study the effect of loss-of-function or gain-of- function of the encoded proteins on cellular and organismal biology. The first gene mutation of nuclear envelope components was discovered in the mid 1990’s, when

Bione et al. identified that loss of function mutations in a gene encoding for the nuclear envelope protein emerin caused Emery-Dreifuss muscular dystrophy (Bione et al.,

1 1994). Soon after Bonne at al. described gain-of-function mutations in the gene coding for lamin A/C, a key intermediate filament component of the nuclear lamina, causing the same EDMD phenotype (Bonne et al., 1999). Patients with EDMD show a muscular dystrophy with progressive muscle wasting and weakness, as well as cardiac conduction block, requiring a pacemaker.

Currently, there are at least 11 clinically distinct disorders that show disease specific mutations in LMNA (Worman and Bonne, 2007) (Table 1). There are also 9 additional protein components of the nuclear envelope (Table 2) that show mutations and corresponding phenotypes (Schreiber and Kennedy, 2013). The entire group of nuclear envelope disorders is often called ‘laminopathies’, although this term is most appropriate for lamin A/C protein mutations.

3 Figure 1. Schematics of the eukaryotic cell and nuclear envelope components.

Eukaryotic cells are defined by nuclear envelope that separates genetic material from the rest of the cytoplasm and represents a continuous structure with the endoplasmic reticulum (scheme on the left). Nuclear envelope is composed of nuclear membrane

(double layer shown in pink) and nuclear lamina (dashed red line). The nuclear membrane is a double lipid bilayer and contains various transmembrane proteins

(emerin shown in blue among others). Nuclear lamina is an intermediate filament meshwork composed of two lamins networks- lamin B and lamin A/C (dashed red line).

Nuclear lamina interacts with heterochromatin (yellow beads marked by red methyl marks) and shows very little interaction with euchromatin (yellow beads marked by green methyl marks).

4 While it is clear that different LMNA mutations cause a strikingly wide range of human disorders, the molecular and biochemical pathogenesis of these disorders is not well understood, with no therapeutic approaches currently used in patients. The relationship between specific mutations and resulting clinical phenotypes is further complicated by ‘phenocopies’ – distinct genes that appear to cause the same disease. A relatively common laminopathy that affects skeletal and cardiac muscle is Emery-

Dreifuss Muscular Dystrophy (EDMD). EDMD has two underlying genetic causes

(phenocopies), both involving components of the nuclear envelope. EDMD can be caused by dominant mutations in LMNA that results in the production of an altered version of its protein products (gain of function). EDMD is also caused by loss of function of the emerin protein encoded by the X-linked EMD gene. Emerin is an inner nuclear membrane protein that interacts with nuclear lamina and acts as transcriptional repressor. Current laminopathies models focus either on impairment of the structural integrity of intermediate filaments and nuclear envelope (Lammerding et al., 2004; Zhang et al., 2007) or regulation of cell type-specific gene expression through failed interaction with different regulatory proteins (pRb, Oct-1, SREBP-1) (Bakay et al., 2006; Demmerle et al., 2012; Melcon et al., 2006; Prokocimer et al., 2009). Nevertheless, most existing pathogenesis models do not seem to explain range of phenotypes and apparent tissue- specific developmental features.

Our previous model of EDMD suggested that a key aspect of the molecular pathogenesis might involve poorly timed or coordinated transition from mitotically active to inactive (terminally differentiated state) during myogenesis (Bakay et al., 2006; Melcon et al., 2006). We showed that appropriate binding of hypophosphorylated and/or

5 acetylated Rb to nuclear envelope via lamin A/C was delayed in EDMD muscle. This interaction (or lack of it) was commensurate with critical mitotic/post-mitotic shift (Bakay et al. 2006; Melcon et al. 2006). The model suggested that Rb-nuclear envelope interaction was necessary for the HDAC1 release from MyoD and initiation of myogenic differentiation via p300 and CREB1 (Bakay et al. 2006; Melcon et al. 2006). This model has obtained further support from others (Meaburn et al. 2007).

1.2 Diseases linked to mutations in LMNA

LMNA has been associated with the largest and most diverse number of disease- linked mutations in the human genome (Burke and Stewart, 2013). Here, we review different lamin A/C mutations that have been linked to phenotypes that involve inappropriate muscle development, inadequate distribution of adipose tissue, peripheral nervous system defects and accelerated aging (Table 1).

6 Table 1. LMNA allelic heterogeneity

Myodystrophy Cardiomyopathy, dilated, 1A Emery-Dreifuss muscular dystrophy 2, AD Emery-Dreifuss muscular dystrophy 3, AR Muscular dystrophy, congenital Muscular dystrophy, limb-girdle, type 1B Heart-hand syndrome, Slovenian type Malouf syndrome Peripheral nerve disease Charcot-Marie-Tooth disease, type 2B1 Lipodystrophy Lipodystrophy, familial partial, 2 Mandibuloacral dysplasia Accelerated aging Hutchinson-Gilford progeria Restrictive dermopathy, lethal

7 Phenotypes affecting muscle

Emery-Dreifuss muscular dystrophy (EDMD).

EDMD is a progressive muscle wasting disorder featured by relatively benign myopathic changes in certain skeletal muscles and early contractures at the neck, elbows, and Achilles tendons (Emery, 2000). A life-threatening feature of the disease is cardiac conduction defects (Emery, 2000). The most common histological features of

EDMD are variation in fiber size, internally positioned nuclei and smaller type 1 fibers

(Brown et al., 2008; Sewry et al., 2001). Nuclear staining of EDMD patient muscle biopsies shows abnormal aggregation of chromatin and chromatin detachment from the nuclear membrane (Sewry et al., 2001).

Emery-Dreifuss muscular dystrophy was first defined as an X-linked recessive disorder caused by loss of function mutations in the gene encoding for emerin (Bione et al., 1994). Bonne and collegues described a phenocopy disorder caused by gain-of- function mutations in the emerin binding partner, lamin A/C (Bonne et al., 1999). More recently, dominantly inherited mutations in both SYNE1 and SYNE2 genes encoding for nuclear envelope proteins nesprin 1 and 2 have been found in patients with an EDMD phenotype (Zhang et al., 2007). The EDMD patients show variable inheritance patterns depending on the underling genetic cause: autosomal dominant (LMNA, SYNE1,

SYNE2), and X-linked recessive (EMD). In rare cases, certain homozygote mutations in

LMNA can cause an autosomal recessive atypical form of EDMD (Di Barletta et al.,

2000).

Benedetti et al. carried out genotype/phenotype correlations in 27 individuals with

LMNA mutations (Benedetti et al., 2007). One group of patients showed late-onset phenotypes that were attributed to the loss of of function of the LMNA protein in the

8 heterozygous state (haploinsufficiency). These mutations are usually associated with truncations of the protein (Benedetti et al., 2007). Missense LMNA mutations in the second group of patients followed dominant-negative or toxic gain-of-function mechanisms that were underlying the more severe early phenotypes. The molecular pathogenesis (e.g. how mutations of nuclear envelope components disrupt muscle structure and function) remains poorly understood. Proposed mechanisms include structural impairments in nuclear membrane integrity (Lammerding et al., 2004; Zhang et al., 2007) and defects in cell signaling and transcriptional regulation (Bakay et al., 2006;

Demmerle et al., 2012; Melcon et al., 2006; Prokocimer et al., 2009).

Dilated cardiomyopathy (DCM)

DCM is the most common form of cardiomyopathy and is associated with cardiac dilatation and reduced systolic function. DCM is one of the major causes of sudden cardiac death (Fatkin et al., 1999; Levitas et al., 2010). Dilated cardiomyopathy can be caused by coronary artery disease and myocarditis, or genetic causes (often called idiopathic DCM) (Fatkin et al., 1999). Mutations in more than 60 genes have been associated with idiopathic dilated cardiomyopathy (Tesson et al., 2014). Most commonly

DCM is associated with mutations in following genes: TTN, LMNA, MYH7, MYH6,

SCN5A, MYBPC3, TNNT2, BAG3, ANKRD1, and TMPO. Inheritance patterns are typically autosomal recessive with age-related penetrance (Hershberger and Morales,

2013; Levitas et al., 2010). Mutations in LMNA account for ~7% of all idiopathic DCM cases (Tesson et al., 2014). Mutations are mostly located in the part of the gene encoding for head and rod domains of lamin A and C proteins with no obvious hot spots,

9 similar to other diseases affecting the striated muscle (Tesson et al., 2014). Furthermore, mutations in another nuclear envelope component - lamina associated protein 2α

(encoded by TMPO) are associated with 1.2% of DCM cases.

The underlying molecular pathogenesis of LMNA gene mutations leading to cardiomyopathy is poorly understood. Studies in Lmna H222P/H222P mice harboring this

EDMD mutation develop DCM with atrio-ventricular conduction defect. Expression profiling in hearts of these mice showed abnormal activation of ERK1/2 and JNK signaling pathways implicated in various aspects of cardiac function (Muchir et al.,

2007a). These findings led to the hypothesis that LMNA mutations inhibit signaling that has a protective role in cardiac functioning (Lu et al., 2011). However, the mice studied were homozygous for the mutation, whereas human EDMD patients are heterozygous, thus there may be differences in pathogenesis of loss of function (mouse) vs. gain of function (human) of the same mutation.

Hand-heart syndrome Slovenian type (HHSS)

HHSS is a progressive cardiac conduction defect disease, and it is associated with sudden death due to ventricular tachycardia, dilated cardiomyopathy, and a type of brachydactyly with mild hand involvement and more severe foot involvement (Sinkovec et al., 2005). It is caused by a heterozygous splice site mutation resulting in truncated lamin A/C protein. This mutation introduces a frame shift in the LMNA mRNA leading to a premature termination codon in exon 10 and production of a truncated protein of 550 amino acids (p. E536fsX14) with 14 new amino acids at its C-terminal end (Renou et al.,

2008).

10 Rare muscle phenotypes

In some cases LMNA mutations produce phenotypes that include muscle phenotypes, as well as syndromic presentations affecting multiple organ systems.

Congenital muscular dystrophy shows intermediate phenotype including features of

EDMD2, LGMD1B and progeria (in some cases) with an early disease onset (infancy and childhood) with an autosomal dominant inheritance pattern (Kirschner et al., 2005;

Quijano-Roy et al., 2008). Limb-girdle muscular dystrophy 1B is a disorder that exhibit overlapping phenotype with EDMD2, dilated cardiomyopathy and lipodystrophy (Brodsky et al., 2000). LGMD1B is consistent with the first group Benedetti et al. described with late disease onset and heterozygous LMNA truncation as an underlying cause

(homozygous LMNA p.Y259* is lethal) (Benedetti et al., 2007). Malouf syndrome is dilated cardiomyopathy and hypergonadotropic hypogonadism with phenotypes overlapping with lipodystrophy and premature aging (McPherson et al., 2009). Malouf syndrome also includes premature ovarian failure, and progressive facial and skeletal changes. It is caused by heterozygous missense mutations in the LMNA (p.A57P and p.L59R) (Chen et al., 2003; McPherson et al., 2009).

Phenotypes affecting adipose tissue

Familial partial lipodystrophy (FPLD)

FPLD shows atypical distribution of subcutaneous adipose tissue. Patients gradually lose adipose tissue from the upper and lower extremities as well as from the gluteal and truncal regions, which results in a muscular appearance of these individuals

(Dunnigan et al., 1974; Spuler et al., 2007). In some FPLD patients, accumulation of fat is limited to the face and neck, causing a cushingoid appearance. FPLD also manifests as a metabolic disorder. Metabolic abnormalities include insulin-resistant diabetes

11 mellitus with velvety hyperpigmentation of the skin and hypertriglyceridemia, with females being more severally affected than males (Garg, 2000). The onset of the disease is in late childhood or early adult life (Shackleton et al., 2000). The histological features of FPLD patients’ skeletal muscle include Type 1 and 2 muscle fiber hypertrophy and nonspecific myopathic changes (Spuler et al., 2007). In most cases

(80%), FPLD is caused by a dominant heterozygous p.R482W LMNA mutation but there are other mutation throughout the LMNA coding region that are associated with FPLD

(Tesson et al., 2014).

Mandibuloacral dysplasia with type A lipodystrophy (MADA)

MADA is an autosomal recessive disorder associated with growth deficit, and craniofacial and skeletal anomalies (Kosho et al., 2007). Some cases show progeroid features. Metabolic features of the disease can include insulin resistance and diabetes

(Garavelli et al., 2009). It is caused by homozygous p.R527H LMNA mutation (recessive inheritance pattern). It should be noted that MADA can show a high degree of clinical variability where some homozygous LMNA MADA mutations result in phenotypes that overlap with HGPS (p.S573L) and EDMD with progeroid features (p.R471C). 85% of

MADA cases caused by LMNA mutations are homozygous for a change at the p.527 residue (Tesson et al., 2014). MADA type B is caused by mutation in the ZMPSTE24 gene that encodes for enzyme involved in lamin A/C processing and is mutated in some progeria cases.

12 Peripheral nerves

Charcot-Marie-Tooth disease (CMT)

CMT is a clinically and genetically heterogeneous group of motor and sensory neuropathies. These rare disorders are characterized by progressive distal sensory loss that mainly affect lower limbs (Auer-Grumbach et al., 2003). CMT patients show weakness and atrophy in distal muscle that is associated with sensory loss, and high- arched feet (Bird, 2015). Based on the inheritance pattern and molecular genetics, CMT hereditary neuropathy is classified into 5 groups: Type 1, 2, and 4, X type 1, and rare intermediate form. There are at least 37 genes that have been implicated in CMT pathology; some of the genes are listed (CMT 1- PMP22, MPZ; CMT2- KIF1B, MFN2,

LMNA, GDAP1; Intermediate Form- DNM2, GNB4; CMT4- MTMR2, SFF2, EGR2;

CMTX- GJB1). Mutations in LMNA cause a type 2B1 of Charcot-Marie-Tooth disease.

This is the axonal form, with a normal or slightly reduced nerve conduction function

(Bouhouche et al., 1999; De Sandre-Giovannoli et al., 2002). It is caused by homozygous mutation in the LMNA gene (p.R298C), showing an autosomal recessive inheritance pattern.

Accelerated aging (progeria)

Hutchinson-Gilford progeria syndrome (HGPS)

HGPS is a rare disorder showing accelerated aging. Patients with HGPS show abnormally short stature and facial features of aged person, early loss of body weight and hair, with features of osteolysis, scleroderma and lipodystrophy. Cardiovascular defects lead to early death of progeria patients. These patients have normal cognitive

13 development and the onset of the disease is usually within the first year of life

(Hennekam, 2006). The majority of patients with HGPS show de novo heterozygous dominant mutations in the LMNA gene. These patients harbor the identical de novo substitution (C to T transition), that results in a silent mutation at codon 608 within exon

11 (p.G608G) (De Sandre-Giovannoli et al., 2003). This substitution creates an exonic consensus splice donor sequence and results in activation of a cryptic splice site and deletion of 50 amino acids of prelamin A protein.

14 Table 2. Mutations in lamin A/C-associated proteins

Disease type Disease Genes Reference Lipodystrophy Partial lipodystrophy LMNB2 (Hegele et al., 2006) Adult onset leukodystrophy LMNB1 (Padiath et al., 2006) Peripheral (Gros-Louis et al., nerve disease Spinocerebral ataxia type 8 SYNE1 2007) SYNE1, (Bione et al., 1994; Emery-Dreifuss Muscular SYNE2, EMD, Liang et al., 2011; Dystrophy TMEM43, Zhang et al., 2007) (Bione et al., 1994; Myodystrophy SYNE1, Hodgkinson et al., SYNE2, EMD, Dilated Cardiomyopathy 2013; Taylor et al., TMEM43, 2005; Zhang et al., TMPO 2007) Buschke-Ollendorff (Hellemans et al., syndrome MAN1 2004; Zhang et al., Melorheostosis (LEMD3) 2009) Bone disease Osteopoikilosis Greenberg skeletal (Hoffmann et al., LBR dysplasia 2002; Waterham et Pelger-Huet anomaly al., 2003)

15 1.3 Molecular role of LMNA in normal and pathogenic conditions

Molecular structure

Nuclear lamins belong to type V intermediate filaments family and are characterized by tripartite domain organization typical for intermediate filaments

(Prokocimer et al., 2009). These domains include a central α-helical rod domain made of four coiled-coil segments (1A, 1B, 2A, 2B). The rod domain is flanked by non-helical short head and longer tail domains (Figure 2) (Burke and Stewart, 2013; Prokocimer et al., 2009). Nuclear lamins are divided into two main types: type A (lamin A and lamin C) encoded by LMNA gene (Fisher et al., 1986; McKeon et al., 1986) and type B (B1 and

B2) encoded by LMNB1 and LMNB2 respectively (Lin and Worman, 1993; Peter et al.,

1989; Vorburger et al., 1989). Additionally, humans have testis-specific lamin C2 and B3 encoded by LMNA and LMNB2 and minor lamin AΔ10 expressed in some somatic cells

(Burke and Stewart, 2013). Finally, mature A- and B- type lamins likely polymerize into separate intermediate filament networks that are formed parallel to the inner nuclear membrane (Dechat et al., 2008).

17 Figure 2. Lamin A/C domains, mutations and protein binding regions.

Nuclear lamins are type V intermediate filaments and are characterized by tripartite domain organization. Structural domains include central α-helical rod domain (green) made of four coiled-coil segments (1A, 1B, 2A, 2B). Non-helical short head (yellow) on the N terminus and longer tail domain (blue) on the C terminus surround the rod domain

(green). Nuclear localization signal (red circle) is located within the tail domain. Residue numbers corresponding to each structural domain are shown below the scheme of structural domains. Locations of structural domains that interact with nuclear envelope protein, transcription factors and chromatin are shown in the lower panel. Locations of missense mutations causing EDMD (red) and FPLD (blue) that are relevant for this dissertation are shown in the top panel.

18 Post-translational processing of nuclear lamins

Nuclear lamins go through key post-translational maturation steps that involve covalent binding of a lipid moiety via a CaaX motif at the C terminus of the protein ('C' is

Cysteine, 'a' is an aliphatic amino acid, and 'X' is variable). The initial step involves the farnesylation of the cysteine residue in the CaaX box by farnesyl transferase.

Farnesylation of CaaX Cys residue is followed by proteolytic cleavage of the aaX residue by either farnesylated proteins-converting enzyme 2 (FACE2) (lamin B1 and B2) or zinc metallo-endoprotease (ZMPSTE24) (lamin A). Processing of lamin B is finalized by carboxy methylation by isoprenylcysteine carboxymethyltransferase (ICMT) (Kitten and

Nigg, 1991), while processing of lamin A into mature form requires additional ZMPSE24 cleavage step, which removes farnesylated Cys together with additional 15 amino acids

(Corrigan et al., 2005; Pendás et al., 2002; Rusiñol and Sinensky, 2006). Lamin C is an alternatively spliced isoform of lamin A that lacks the CaaX motif, and is therefore not subjected to farnesylation.

Lamins also undergo phosphorylation that plays role in modulating interactions between lamins and histone H2A/H2B dimer (Mattout et al., 2007). Studies in Drosophila melanogaster have shown that lamin is phosphorylated at three residues: S25, S595 and

T432 or T435 (Schneider et al., 1999). Substitution of T432 and T435 (TRAT sequence) with alanine, dramatically reduces the binding of lamin to the histones (H2A/H2B dimer), suggesting that phosphorylation plays a role in lamin-histone binding (Mattout et al.,

2007). Mutations in human lamin A/C at the position of S22 and S392 prevent phosphorylation at these sites and block the disassembly of the nuclear lamina during mitosis (Heald and McKeon, 1990). Moreover, phosphorylation of residues T19, S22 and

S392 in lamin A and C and S636 in lamin A, significantly increases during mitosis

19 providing further evidence for the role of phosphorylation in lamin disassembly during mitosis (Chen et al., 2013; Prokocimer et al., 2009).

Lamina associated proteins (LAPs)

The nuclear lamina is in contact with the inner nuclear membrane (INM) through various INM proteins (Figure 1). Mammalian INM has over 50 different proteins that are mostly uncharacterized (Wilson and Foisner, 2010). Initially, Senior et al. defined three

INM proteins that co-fractionated with lamins during high salt and nonionic detergent extraction (Senior and Gerace, 1988). These first discovered lamina-associated polypeptides were LAP1 (Martin et al., 1995), LAP2 (Furukawa et al., 1995) and lamin B receptor (LBR) (Worman et al., 1990). Later, more LAP proteins were discovered and the term was extended to lamin binding proteins found in nuclear lumen (e.g., LAP2α).

The INM proteins are defined by LEM domain. The LEM domain is the 40-residue helix- loop-helix motif found in prokaryotic and eukaryotic DNA/RNA binding proteins (Cai et al., 2001). The LEM domain directly binds Barrier to Autointegration Factor (BAF) that is known chromatin and lamin binding partner that interacts with DNA and histones

(Margalit et al., 2007). It is essential for heterochromatin tethering to the nuclear envelope (Berk et al. 2013). The LEM proteins are highly conserved between mammals

(7 genes encodes for LEM proteins), nematodes (3 LEM proteins) and fruit flies (4 genes), suggesting their importance in nucleus functioning (Wilson and Foisner, 2010).

Nuclear lamins provide structural support

The nuclear envelope and lamina are highly organized and regulated structures that separate genetic material from the rest of the cytoplasm (Hetzer, 2010). Mutations in nuclear envelope components lead to deformities in nuclear shape and have multiple downstream effects on chromatin organization and signaling (Shumaker et al., 2006).

20 When subjected to mechanical strain, lamin A/C–deficient nuclear lamina shows impairment in nuclear mechanical properties and strain-induced signaling (Lammerding et al., 2004). More specifically, this study found that the Lmna null mouse embryonic fibroblasts (MEFs) exhibit increased levels of nuclear deformation and show defective

NF-kB signaling in response to mechanical stress (Lammerding et al., 2004). Similar findings were seen in Emd null cells (Lammerding et al., 2005). Other structural proteins

(e.g., SYNE1 and SYNE2) also cause an Emery-Dreifuss muscular dystrophy (EDMD type 3). Zhang et al. proposed that disruptions of structural NE components, specifically nesprin/lamin/emerin interactions, underlie the pathogenesis of EDMD and demonstrate the structural importance of nuclear lamins. Finally, mutations in nuclear lamins cause catastrophic nuclear envelope collapse in cancer cell micronuclei, reduce nuclear functioning and induces major DNA damage (Hatch et al., 2013). A key issue in interpreting these knock-out (loss-of-function) studies with regards to relevance to human disease (missense gain-of-function mutations; LMNA, SYNE1, SYNE2) is the distinct mechanisms of biochemical pathogenesis. In most biochemical disease states, there are marked differences in loss-of-function vs. gain/change-of-function (toxic protein) in terms of effects on cell biology. Thus, the gain-of-function missense mutations seen in most laminopathies may involve other biochemical defects than physical effects on the nuclear envelope. Indeed, this is a goal of the current dissertation to dissect these potential differences.

Nuclear lamins during development

Nuclear lamins show differential expression during both cellular differentiation and organismal development. The onset of lamin A/C expression is highly variable, where some cell types, including cells of the central nervous system, begin with lamin

21 A/C expression only after birth (Stewart and Burke, 1987). On the other hand, particular stem cells and certain cells of the haematopoietic system, never express these lamin

A/C (Röber et al., 1989). Constantinescu et al. showed that undifferentiated mouse and human ESCs express lamins B1 and B2 but not lamin A/C and that lamin A/C expression is coordinated with induction of differentiation, after downregulation of pluripotency markers (Tra-1-60, Tra-1-81, and SSEA-4)(Constantinescu et al., 2006).

Earlier, Röber et al. showed that in developing mouse embryos, lamin A/C expression first appears at embryonic day 12 in muscle cells of the trunk, head and the limbs while its expression in certain tissues was obeserved only after birth (epithelia of lung, liver, kidney and intestine, heart and brain) (Röber et al., 1989). Additionally, Houliston et al. showed that unfertilized mouse eggs had lamin A/C expressed at much higher levels in comparison to 8-cell embryos and blastocysts (Houliston et al., 1988).

Absence of lamin A/C from embryonic stem cells (ESCs) could provide explanation for dispersed nuclear shape observed in these cells (Meshorer and Misteli,

2006). Additionally, embryonic stem cells show abundance of less condensed euchromatin and general absence of heterochromatin (Arney and Fisher, 2004;

Bernstein et al., 2006). Thus, tissue differentiation is concomitant with increased lamin

A/C expression and restricted transcriptional expression predicted by increasing levels of heterochromatinization. Mutations in LMNA (and certain lamin associated proteins) specifically affect terminal differentiation of postmitotic tissue, suggesting that lamin A/C is necessary for these processes. Together these data suggest that lamin A/C expression coincides with lineage commitment and that it may limit cell plasticity to promote differentiation.

22 Nuclear lamins in DNA chromatin assembly

The nuclear lamina is increasingly recognized for its importance in heterochromatin organization (Brickner, 2011). Chromatin (DNA) interacts with the nuclear envelope through lamina associated domains (LADs) that cover very large genomic domains and are generally associated with heterochromatin (Guelen et al.,

2008; Peric-Hupkes et al., 2010; Reddy et al., 2008; Wu and Yao, 2013). Additionally,

LADs are characterized by relative absence of histone modifications associated with active gene transcription (euchromatic marks) (Wu and Yao, 2013) and include mostly, but are not limited to transcriptionally inactive genes (Lund et al., 2013). Peric-Hupkes et al. showed that genes interact with the nuclear lamina in a cell-type specific fashion

(Peric-Hupkes et al., 2010). Upon differentiation, transcript units showing de novo expression lose their nuclear lamina association, while transcript units associated with pluripotency (stem cell-associated) get shifted to nuclear periphery and become LAD- associated (Peric-Hupkes et al., 2010). Knock-out of lamin A/C and LBR from the nuclear envelope leads to an inverted chromatin architecture with movement of heterochromatin away from the periphery towards the nuclear lumen (Solovei et al.,

2013).

Both transcriptionally inactive heterochromatin and transcriptionally active euchromatin shows specific histone marks (post-translational modifications of the histone tails). The nuclear lamina appears to provide an environment for heterochromatic formation, and associated histone marks. Towbin et al. have shown that step-wise formation of H3K9me3 heterochromatic foci using H3K9me1/2 as substrate occurs at the nuclear periphery, suggesting that lamina plays active role in epigenetic remodeling (Towbin et al., 2012).

23 Taken together, these findings provide evidence for nuclear envelope involvement in chromatin remodeling and downstream processes such as gene expression and tissue differentiation. Further, it might be predicted that mutations of the nuclear envelope proteins (as observed in Emery-Dreifuss muscular dystrophy) may perturb chromatin remodeling. Consistent with this, mutations in lamin A/C (AD-EDMD and HGPS) cause heterochromatic detachment from the nuclear lamina detected by electron microscopy (Fidziańska and Hausmanowa-Petrusewicz, 2003; Goldman et al.,

2004). Also, disease-linked mutations in lamin impair tissue specific reorganization of heterochromatin by inadequately retaining the genomic regions that normally exhibit tissue-specific activation (Mattout et al., 2011).

Nuclear lamins in transcriptional regulation

It has been shown that EDMD mutations in lamin A/C disrupt transcriptional fingerprints during terminal differentiation of myogenic cells. We showed that appropriate binding of hypophosphorylated and/or acetylated Rb to nuclear envelope via lamin A/C was delayed in EDMD cells both in vivo and in vitro. This interaction (or lack of it) was commensurate with critical mitotic/post-mitotic shift (Bakay et al., 2006; Melcon et al.,

2006). The model suggested that Rb-nuclear envelope interaction was necessary for the

HDAC1 release from MyoD and initiation of myogenic differentiation via p300 and

CREB1 (Bakay et al. 2006; Melcon et al. 2006). This model has obtained further support from others (Meaburn et al. 2007). Yao et al. have reported that spatial segregation of core transcription components away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts provides evidence for nuclear lamina involvement in promoter selectivity during differentiation (Yao et al., 2011). Mouse model expressing EDMD-causing LMNA mutation, showed aberrant activation of MAPK

24 pathways in heart tissue, isolated cardiomyocytes and cultured myoblasts (Muchir et al.,

2007a). These cardiac findings were validated in emerin null mouse model (Muchir et al.,

2007b).

Progeroid phenotypes also show perturbation of transcriptional programs with aberrant expression of Notch (Scaffidi and Misteli, 2006) and Wnt signaling (Espada et al., 2008), two major stem cell signaling pathways, directly linking mutations in lamin A/C to the stem cell function. Together, mutations in LMNA cause tissue-specific signaling and transcriptional abnormalities of progenitor cells and could explain inefficient terminal differentiation seen in laminopathies.

26 Figure 3. Tissue specific LMNA associated phenotypes. Mutations in LMNA affect terminal differentiation of adipose (blue), skeletal and cardiac muscle (red), neuronal

(yellow) and bone (purple) tissue. Disease phenotypes associated with different cell lineages are outlined in Table 2.

27 1.4 Current molecular laminopathy models

Summarizing the literature to date, as presented above, two models have been proposed to connect molecular and biochemical defects (laminopathies) with cellular dysfunction and clinical phenotypes (Figure 3). The first model proposes that mutations in nuclear envelope components impair the structural integrity of intermediate filaments and nuclear envelope, with downstream defects in mechano-transduction (Lammerding et al., 2004). This model is supported by evidence that mutations in nesprin-1 and -2, spectrin-repeat proteins that link inner nuclear membrane to the outer nuclear membrane and cytoskeleton, can also cause EDMD-like phenotypes (Zhang et al., 2007). The second model focuses on cell- and tissue-specific regulation of chromatin remodeling and downstream transcriptional patterns (Bakay et al., 2006; Demmerle et al., 2012;

Melcon et al., 2006; Prokocimer et al., 2009). This model suggests that defects of the nuclear envelope disturb the timing and specificity of chromatin-envelope interactions, and is supported by evidence suggesting perturbation of stem cell differentiation and tissue-specific gene expression (Van Berlo et al., 2005; Muchir et al., 2007b). Consistent with the second model, lamins A and C have been implicated in regulation of cell type- specific gene expression during adult stem cell differentiation (Gotzmann and Foisner,

2006) affecting Rb/MyoD and TGF-β/Smad signaling (Bakay et al., 2006; Van Berlo et al., 2005). It should be noted that these models are not mutually exclusive and both mechanisms contribute to the disease pathophysiology.

A key unresolved question is how different LMNA missense mutations cause such clinically distinct, tissue-restricted clinical phenotypes. These marked phenotypic differences, often for missense mutations neighboring each other, seem poorly explained

28 by the structural model. On the other hand, a gain-of-function model where different missense mutations in LMNA alter cell- and tissue-specific euchromatin-heterochromatin transitions is more satisfying. The missense mutations in LMNA may also cause a dominant-negative mechanism of action of nuclear lamina structures. Again, while theoretically possible, this structural model would not seem to justify the remarkably variable tissue-restricted phenotypes (a dominant-negative loss of function of lamin A/C might be expected to affect all cell and tissue types similarly). A second unresolved question is regarding the molecular mechanisms driving the close phenotypes seen with emerin loss-of-function and specific lamin A/C gain-of-function mutations (EDMD).

1.5 Significance

The observed tissue specificity of LMNA mutations remains an unsolved puzzle.

It is still not clear how different LMNA mutations that do not seem to follow any obvious protein domain-specific pattern specifically affect particular tissue types. Dissection of the pathophysiology of nuclear envelope disorders is complicated by the diverse roles lamin A/C plays in various cellular processes, ranging from structural and developmental to chromatin remodeling, signaling and cell fate maintenance. The goal of this dissertation is to provide evidence for the interplay between the specific cell fates of the lineage-specific cell types and the biochemical role of lamin A in euchromatin- heterochromatin transitions. Based upon this research, laminopathies are established as an epigenetic group of disorders and opens a new avenue towards the development of novel therapeutic approaches.

29 2 Chapter: Material and Methods

2.1 Cell culture

Conditionally immortalized H2K myoblasts

For the following experiments, conditionally immortal satellite cell-derived cell- line- H2K was used. The H2K cell line was generated from the H2Kb-tsA58 immortomouse (Morgan et al., 1994). Both emerin and wild-type mice are on the Black 6

(BL6) background. The transgenic mice harbor a temperature sensitive immortalizing simian virus 40 T-antigen gene (tsA58) under the control of a gamma interferon (IFNγ)- inducible major histocompatibility complex (MHC) Class I promoter. Depending on culture conditions, the cells either exhibit continuous growth or alternatively terminally differentiate into myotubes. Emerin-null H2K mice were generated in our research center by Dr. Cohen and Dr. Partridge by breeding emerin-null (Melcon et al., 2006) and H-

2KbtsA58 mice on the BL6 background. Cells were harvested at following time points: contact-inhibited myoblasts - 80-90% confluent myogenic cells grown under permissive conditions; differentiating myotubes for 24, 48, 72, and 96 hrs (d1 to d4) - 80-90% confluent cells grown under permissive conditions for 24hrs, and induced to differentiate by serum withdrawal, non-permissive temperature and absence of IFNγ.

Single fiber-derived satelite cell isolation and primary myotubes differentiation

Emerin null and wild-type mice aged 9 weeks were sacificed and the extensor digitorum longus (EDL) muscle was dissected. Muscles were digested in 0.2% collagenase type 1/DMEM (LifeTechologies) and individual myofibers were dissociated and washed, as described perviously (Gnocchi et al., 2009).To induce satellite cell

30 activation, 6-12 myofibers were cultured on matrigel coated 6 well plate in DMEM

(LifeTechnologies) containing 10% (v/v) horse serum (LifeTechnologies), 10% (v/v) fetal bovine serum (LifeTechologies) 1% (v/v) chick embryo extract (US Biological), 2% L-

Glutamine (Sigma) and 1% (v/v) penicillin/streptomycin solution (Sigma) at 37°C in 5%

CO2. After myofibers removal activated myoblasts were proliferated for five days. To induce differentiation, myoblasts were plated on matrigel-coated dishes and kept in

DMEM containing 5% horse serum (LifeTechnologies), 2% L-Glutamine (Sigma) and 1%

(v/v) penicillin/streptomycin solution (Sigma) at 37°C in 5% CO2. Cell were differentiated for 24 hrs and harvested for downstream applications.

Immortalized MyoD-converted human myoblasts

EDMD and control patient fibroblast were obtained from skin biopsies. Cells were immortalized using a lentiviral vector containing the sequence encoding the catalytic subunit of human telomerase (hTERT) as previously described (Auré et al., 2007).

Inducible myogenic conversion was obtained using a lentiviral vector contining a murine

MyoD under the control of the Tet-On inducible construct as decribed previously

(Chaouch et al., 2009). Immortalized EDMD, FPLD and control fibroblasts were proliferated to 90% confluence followed by MyoD induction by doxycycline (2 μg/ml).

Four days after MyoD induction, myotubes were harvested for quantitative reverse transcription PCR (qRT-PCR) and ChIP-qPCR experiments.

Immortalized human skeletal myoblasts

Human skeletal myoblasts were proliferated in Skeletal Muscle Cell Growth

Medium (PromoCell) according to manufacturer’s instructions except for fetal bovine serum (FBS, Invitrogen) that was adjusted to 20%. Differentiation was induced at high

31 cell density by serum starvation (DMEM (Life Technologies) in the presence of

Gentamycin 50 μg/ml and 10 μg/ml Insulin).

HEK293T cells

HEK293T cells were grown according to standard protocols in DMEM medium supplemented with 10% heat inactivated FBS (HI FBS, Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 2% L-glutamine (Invitrogen).

2.2 qRT-PCR

Total RNA was isolate using Trizol according to manufacturer’s instructions. Two hundred nanograms of total RNA were reverse transcribed into single-stranded cDNA and processed for quantitative PCR (SYBR Green). Primers used can be found in Table

32 Table 3. Primer sequences used for RT-qPCR and ChIP-qPCR experiments

Mouse primers

ChiP-qPCR primers Name Forward primer Reverse primer Cdk1 5’-TCAAGAGTCAGTTGGCGCCC-3’ 5’-CACACCGCAGTTCCGGACTG-3’

Human primers

RT-qPCR primers Name Forward primer Reverse primer SOX2 5’-TAGTGGTACGTTAGGCGCTT-3’ 5’-TCTTGCCAGTACTTGCTCTC-3’ HOXB1 5’-TCAGAAGGAGACGGAGGCTA-3’ 5’-CAGGGTGTTTCCTTGTCCTC-3’ GATA6 5’-TTGTGGACTCTACATGAAACTCCA-3’ 5’-TTATGTTCTTAGGTTTTCGTTTCCTG-3’

ChIP-qPCR primers Name Forward primer Reverse primer HOXB1 5’-AAGGCAGCTGGTGCTATTGT-3’ 5’-TCCTCCTTTCCCTTCCAACT-3’ GATA6 5’-GGGACAGGGATTCTTTTGTTGG-3’ 5’-TGATTCACCAGAGGTCTCAAGCC-3’

DamID-qPCR primers Name Forward primer Reverse primer SOX2 5’-TGGTACGGTAGGAGCTTTGC-3’ 5’-GCAAGAAGCCTCTCCTTGAA-3’

33 2.3 Chromatin immunoprecipitation assay and ChIP-seq

ChIP-qPCR and ChIPSeq were performed with modification following Mayers lab protocol (Mayers lab ChIP-seq protocol). Briefly, cells were lysed in Farnham buffer (5 mM PIPES pH 8.0; 85 mM KCl; 0.5% NP-40) and subsequently in RIPA buffer (1x PBS;

1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS). Chromatin was sheared by 12x15s sonication with 15s break in between intervals in 1.5 ml microcentrifuge tubes using

Sonifier Cell disruptor 350. Fragmentation of chromatin was inspected on 1% agarose gel. 50 μg of chromatin/DNA were immunoprecipitated overnight at 4⁰C with 3 µg of antibody against H3K9me3 (Abcam 8898) and H2K27me3 (Abcam 6002). As a control we used 5 μg of sheared, non-precipitated chromatin – input. Samples were incubated with magnetic beads (Dynabeads, Invitrogen) for 6 hrs and washed successively in buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-

100); buffer II (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1%

Triton X-100); buffer III (10 mM TrisHCl pH 8.0, 250 mM LiCl, 1% NP-40; 1% sodium deoxycholate, 1 mM EDTA) and Tris-EDTA (pH 8.0). All washes were performed at 4⁰C for 10 mins. qPCR primers used could be found in Table 3. The results were reported as qPCR values normalized to input chromatin (genomic DNA) and presented as percentage of input. To ensure specific PCR amplification, every real time PCR run was followed by a dissociation phase analysis (denaturation curve), to confirm the presence of a single dissociation peak. For ChIP-seq, chromatin was precipitated as described above, libraries were prepared using Mondrian SP system and run on Illumina HiScan platform with multiplexed four samples per lane.

34 2.4 ChIP-seq data analysis

De-multiplexed .fastq files were aligned to reference genome (mm9) using Bowtie

(Langmead et al., 2009) and uniquely mapped reads were kept for further analysis.

Peaks were called using SICER algorithm (Zang et al., 2009) with parameters set as follows: window size 1000 bp,gap size 3000 bp and FDR1e-2. Genomic distribution of

ChIP peaks and tag densities around TSS were determined using HOMER platform using following commands: perl annotatePeaks.pl wt.bed mm9 > output.txt and annotatePeaks.pl tss.txt mm9 -size 10000 -hist 100 -d wt.bed emd.bed > outputfile.txt.

Peaks specific to cell type were determined using Bedtools Suite: bedtools intersect -a

A.bed -b B.bed –v. Differentially enriched peaks were intersected to generate union file using bedtools intersect and annotated with read counts from individual .bam files using bedtools annotate counts –i union.bed -files F1.bam F2.bam FN.bam. GO of biological processes of peaks showing reduction/absence in H3K9me3 enrichment at all time points were identified using GREAT -cis regulatory region prediction tool (McLean et al.,

2010) while top canonical pathways were generated by Ingenuity Pathway Analysis

(IPA). Heterochromatic expansion in proximal promoter region was defined as at least two times increase in peak length during d0-d1 and d1-d2 transition. Initially sequencing and ChIP quality analysis were performed using FASTQC and Strand Cross Correlation analysis (Kharchenko et al., 2008).

2.5 DamID sequencing

Plasmid construction

pLgw V5-EcoDam (negative control), pLgw RFC1-V5-EcoDam, and pLgw CBX1-

V5-EcoDam (positive control) vectors were obtained from the Bas van Steensel

35 laboratory. We used Gataway Cloning system (D-TOPO cloning) (Invitrogen) to generate entry clone (pENTR) for human LMNA open reading frame (ORF) synthetized from RNA sample of a normal volunteer. LMNA entry clones harboring p.R453W and p.R482W mutations were generated using QuikChange Lightning Site-Directed Mutagenesis Kit

(Agilent Technologies). Gateway cloning system (LR clonase) (Invitrogen) was used to clone these three versions of LMNA ORF into pLgw RFC1-V5-EcoDam.

Lentivirus production and infection

HEK293T cells were grown in 10 cm dishes until 60-80% confluence. We used

Lipofectamine 2000 (Invitrogen) for lentiviral production. We mixed 67.7 μl of lipofectamine 2000 with 500 μl of OPTIMEM and added it to the following mixture: 3.5 μg pMD-G (envelope plasmid), 6.5 μg pCMV-ΔR8.2 (Packaging construct), 2.5 μg pRSV-

Rev (Rev-encoding plasmid), 10 μg of specific pLgw LMNA-V5-EcoDam, and 500 μl of

OPTIMEM. The mixture was incubated at room temperature for 20 min, and added drop wise to the HEK293T cells. After 8 hours, the medium was replaced with 6 ml fresh medium. Virus containing medium was harvested on 3 consecutive days and supernatants were harvested (18μl of infectious medium), filtered (0.45 um) and aliquoted (1ml aliquots). Virus aliquots were stored at -80⁰C.

Human skeletal myoblasts were plated on 6 well plates at high density.

Differentiation was induced the next day by serum starvation (DMEM with 10 μg/μl insulin and 50 μg/μl gentamycin). Myoblasts were differentiated for 24 hours, after which were infected with infectious medium diluted 2:1 in DMEM in the presence of 1.5 μl polybrene (10 mg/mL). The cells were then returned to 37°C for overnight incubation.

The following day, the media was replaced with 3 mL of fresh differentiating medium.

After 48 hours later, the cells were harvested and the genomic DNA (gDNA) was isolated

36 using the Qiagen DNA Micro Kit, “Isolation of gDNA from Small Volumes of Blood” protocol. The DNA was eluted in 200μl of Qiagen AE buffer (10 mM Tris-Cl and 0.5 mM

EDTA; pH 9.0).

DamID Library Preparation and Sequencing

The gDNA was ethanol precipitated overnight at -20⁰C, and resuspended in TE

(Tris EDTA) buffer pH 7.5 to a concentration of 1 μg/μl. 2.5 μl of gDNA were digested with 0.5μl of DpnI (NEB, 20 U/μl) at 37°C overnight. DpnI activity was inactivated by heating to 80°C for 20 min. DpnI-digested gDNA was ligated to the adaptor AdR (slowly annealed AdR-top (5’ CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG-

GA 3’) and AdR-bottom (5’ TCCTCGGCCG 3’)). The ligation reaction was performed at

16°C for 2 hrs using T4 Ligase (Roche, 5 U/μl). The enzyme was inactivated by heating to 65°C for 10 min. To remove the fragments that have unmethylated GATCs, we performed DpnII digestion at 37°C for 1 hr.

PCR was performed to amplify the regions flanked by adaptors. Reaction consisted of 10 μl DpnII digested DNA, 5 μl 10x cDNA PCR reaction buffer (Clontech),

0.625 μl primer bio-Adr-PCR (5’ bio-GGTCGCGGCCGAGGATC 3’, 100 uM), 1 μl dNTPs

(10mM), 1 μl PCR advantage enzyme mix (Clontech, 50X), 32.375 μl ddH2O. We used following amplification reaction program: 1 cycle of 68°C (10 min), 94°C (1 min), 65°C (5 min), 68°C (15 min); 3 cycles of 94°C (1 min), 65°C (1 min), 68°C (10 min); 17 cycles of

94°C (1 min), 65°C (1 min), 68°C (2 min). The PCR products were purified with the

Qiagen MinElute PCR Purification kit, and eluted in 20 μl ddH2O. 2 μl of the PCR products were separated by gel electrophoresis to verify successful digestion and amplification.

37 3 μg of each sample were diluted in 100 μl ddH2O and sonicated with Covaris using the following settings: 10% duty cycle, 175 Peak Incident Power, 200 cycles per burst, for

4.5 min total. Biotinylated ends of the PCR products were purified using MyOne

Streptavidin T1 beads Dynabeads (Invitrogen). 50 μl of these beads were washed three times with 1 ml of 1X Binding and Washing (B&W) buffer (5 mM Tris-HCl pH 7.5, 0.5mM

EDTA, 1M NaCl). Washed beads were resuspended in 75 μl of Covaris-sonicated DNA,

100 μl of 2X B&W buffer, and 25 μl H2O. This mixture was incubated at 4°C for 30min on a rotator. The beads were washed three times with 200 μl of 1X B&W buffer.

DNA was released off the beads by DpnII digestion at 37°C for 1 hour. The supernatant was saved and cleaned using the Qiagen MinElute Reaction Cleanup kit. DNA was eluted in 20 μl H2O and quantified with Qubit. qPCR analysis was performed to validate the DamID DNA before submission for sequencing (Figure 4).

39 Figure 4. DamID workflow. A schematic of the DamID assay that was used to identify genomic regions bound by the lamin A protein in human skeletal muscle cells.

Methylated DNA is first digested with DpnI and ligated with adaptors. The adaptors are used to amplify the enriched regions by PCR using a biotinylated primer (purple circle).

Amplified regions are purified using streptavidin beads (yellow circle), digested with

DpnII, and prepared for high-throughput (Illumina) sequencing.

40 2.6 Bioinformatics analysis

De-multiplexed .fastq files were aligned to reference genome (hg19) using Bowtie

(Langmead et al., 2009) and uniquely mapped reads were kept for further analysis.

Peaks were called using SICER algorithm (Zang et al., 2009) with parameters set as follows: window size 2000 bp,gap size 6000 bp and FDR1e-3. Distribution of peaks was determined using HOMER (annotatePeaks.pl –GenomeOntology and -Venn). Lamin A binding 2 kbp upstream/downstream of the TSS was queried using HOMER

(annotatePeaks.pl -ghist). Similar analysis was performed for genes induced by myogenic induction. To obtain genes induced by myogenic induction in human skeletal myotubes we downloaded ENCODE Affymetrix Exon 1.0 ST arrays data for skeletal myoblasts and myotubes (GSE15805). Data were normalized using Affymetrix

Expression console followed by Partek Genomic Suite analysis to obtain genes induced in myotubes (ANOVA one-way, p<0.01, Fold Change >1.2). Alignment files (.bam) for the histone peaks were downloaded from the ENCODE (H3K9me3 – GSM733730,

H3K4me3- GSM733637). Lamin A, H3K9me3 and H3K4me3 binding around TSS was determined using HOMER (annotatePeaks.pl -ghist).

2.7 Methylation analysis

DNA methylation analysis was performed on 3 time points in 2 different patient cell types (normal, EDMD) using Illumina 450K methylation bead array. Normalization was performed using SWAN algorithm in R Studio (Maksimovic et al., 2012). Methylation pattern of genes induced by myogenesis was plotted using Partek Genomic Suite.

41 2.8 Expression data

Human skeletal myotubes and myoblasts

Affymetrix expression data for human skeletal myotubes and myoblasts

(McDaniell et al., 2010; Thurman et al., 2012) were downloaded from GEO (accession number GSE15805).

Muscle biopsies

Affymetrix expression data for muscle tissue of Normal, EDMD-AD, EDMD-XR and FKRP patients (Bakay et al. 2006.) were obtained from GEO (accession number

GSE3307). Sets were normalized using Expression Console from Affymetrix using

PLIER algorithm. Annotation, fold change and p-values, as well as hierarchical clustering were calculated and plotted using Partek Genomic Suite.

2.9 Overexpression and myogenic differentiation experiments

Human immortalized myoblasts were transfected with SOX2 vector (EX-T2547-

M61; GeneCopoeia) and corresponding empty vector (EX-T2547-NEG; GeneCopoeia).

Both vectors carried information for GFP gene as well. Medium was changed the next day to induce myogenic differentiation. Cell were fixed 72 hours post differentiation induction and stained for myosin heavy chain (MF20, Developmental Studies Hybridoma

Bank) and DAPI (ProLong® Antifade Reagents with DAPI from Invitrogen). Cells that received the vector were scored based on GFP expression and myogenic potential was estimated as a ratio of MF20+ GFP + cells over the total number of GFP+ cells. Four image fields containing 300-500 cells were scored for each transfection experiment

42 3 Chapter: Results

3.1 Summary

Missense mutations of lamin A protein causing either muscular dystrophy

(p.R453W) or lipodystrophy (pR482W) were fused to bacterial adenine methyltransferase to define euchromatic-heterochromatin transitions at the nuclear envelope during myogenesis (Dam-ID-seq). Lamin A missense mutations disrupted appropriate formation of lamin A-associated heterochromatin domains in an allele specific manner, findings confirmed by ChIP-seq in murine cells and DNA methylation studies in patient cells carrying a distinct LMNA muscular dystrophy mutation (p.H222P).

Observed perturbations of epigenomic transitions included exit from pluripotency and cell cycle programs (euchromatin to heterochromatin), as well as induction of myogenic loci

(heterochromatin to euchromatin). Inappropriate loss of heterochromatin formation at the

Sox2 pluripotency locus in two muscular dystrophy phenocopies (gain/change of function lamin A; loss of function emerin) was associated with persistent mRNA expression of

Sox2 both in vitro and in vivo (patient muscle biopsies). Over-expression of Sox2 inhibited myogenic differentiation in vitro. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming due to altered

LAD formation.

43 3.2 Introduction

Genetic and biochemical perturbations of components of the nuclear envelope cause a broad range of clinical phenotypes, including muscular dystrophies, neuropathies, cardiomyopathies, and lipodystrophies (Maraldi et al., 2011). Dominant missense mutations of the lamin A/C (LMNA) intermediate filament protein cause the broadest range of phenotypes, where even neighboring mutations can show quite distinct tissue-restricted clinical disease. Skeletal muscle tissue appears particularly susceptible to abnormalities of the nuclear envelope, with LMNA mutations causing both

Emery-Dreifuss muscular dystrophy (EDMD) and limb-girdle muscular dystrophy.

Additional muscular dystrophy phenocopies seen with mutations of other nuclear envelope proteins include (emerin [EMD], nesprin 1 [SNYE1], nesprin 2 [SNE2], four and a half LIM domains [FHL1]) (Camozzi et al., 2014).

Myogenic differentiation requires the coordinated execution of three key cellular programs: inactivation of pluripotency programs (i.e., Oct4, Nanog, Sox2), exit from the cell cycle (CDK1, Rb1), and induction of myogenesis (MyoD, myogenin). In differentiating skeletal muscle cells, the LMNA gene is strongly induced at the onset of terminal differentiation of myoblasts into syncytial myotubes (Bakay et al., 2006). The

Lamin A/C protein has also been shown to play a key role in myoblast-myotube transition (Constantinescu et al., 2006; Röber et al., 1989). Missense mutations of the lamin A protein associated with muscular dystrophy cause impaired activation of Myog with inappropriate maintenance of heterochromatin on the myogenin promoter during differentiation in C2C12 cells (Håkelien et al., 2008). Importance of wild-type LMNA for

44 terminal differentiation was also seen in adipogenic cells, where a Lamin A protein with a

FPLD missense mutation disrupted adipocyte differentiation (Boguslavsky et al., 2006).

Taken together, these data suggest that laminopathies disrupt cell differentiation, but the mechanism of this disruption is not understood.

Some dominant gain- or change-of-function mutations in the lamin A/C proteins show similar muscular dystrophy phenotypes with loss-of-function of other nuclear envelop components (emerin, nesprin 1, FHL1), and it might be expected that molecular pathogenesis might also be shared between these genetically distinct nuclear envelopathies. Consistent with this, we previously showed shared mRNA expression fingerprints in human muscular dystrophy patient muscle biopsies with both LMNA (AD-

EDMD) and EMD mutations (XR-EDMD) suggesting a failure of appropriate induction of the myogenic terminal differentiation program (Bakay et al., 2006). Mechanistic studies bolstering these findings were carried out in a mouse knock out model of emerin deficiency, where the inappropriate timing of myogenic lineage-specific genes was confirmed (Melcon et al., 2006). This second study also showed evidence of failure to appropriately exit the cell cycle (delayed suppression of E2F pathways) (Melcon et al.,

2006). Thus, exit from the cell cycle and induction of the myogenic cell fate program; two of the three key programs that must be orchestrated during terminal differentiation and commitment to the myogenic lineage are perturbed in emerin deficiency.

There is accumulating evidence that lamin A protein is directly involved in epigenomic regulation of chromatin through heterochromatic lamina associated domains

(LADs). Genomic regions associated with nuclear lamina show enrichment in repressive heterochromatin marks and are associated with transcriptionally inactive domains (Kind and van Steensel, 2010). Transcriptional repositioning of silent genes to the nuclear

45 periphery (Reddy et al., 2008) together with sequestration of transcriptional factors by the interior (Yao et al., 2011) indicates that the nuclear lamina controls transcription, and likely cell fate. Consistent with this, murine LMNA null cells fail to acquire appropriate heterochromatin at the nuclear envelop during myogenesis, and show a loss of the myogenic program (Solovei et al., 2013). Also, a human EDMD LMNA missense mutation expressed as protein in C. elegans impaired tissue-specific reorganization of heterochromatin, with abnormal retention of a muscle-specific gene at the nuclear periphery (Mattout et al., 2011).

We hypothesized that different missense mutations of LMNA altered the euchromatin-heterochromatin transitions during terminal differentiation of cells mediated by the nuclear envelope in a tissue-restricted manner. In other words, that nuclear envelopathies are epigenetic disorders. We assessed this at a genome-wide level by fusing the lamin A protein carrying different missense mutations to the bacterial DNA adenine methyltransferase (Dam-ID) (Guelen et al., 2008; Rodriguez and Bjerling, 2013).

The resolution of the Dam-ID approach is about 1kb, or tens of nanometers from the tethered protein. In Drosophila, a Dam-ID/microarray approach was successfully used to assess euchromatin-heterochromatin transitions for the Escargot protein in intestinal cell development (Korzelius et al., 2014), and Hb9-lamina interactions in neuronal lineages

(Lacin et al., 2014). Here, we extended the Dam-ID method to study gain/change-of- function mutations of the lamin A protein, with nextgen sequencing as the read out

(Dam-ID-seq). Findings were validated with ChIP-seq and DNA methylation arrays.

46 3.3 Results

3.3.1 Lamin mutations alter genomic LADs of human muscle cells

We generated lentiviral constructs for wild-type (WT) lamin A, lamin A p.R453W mutation corresponding to Emery Dreifuss Muscular Dystrophy (EDMD; (Bonne et al.,

1999)) and lamin A p.R482W mutation observed in Familial Partial Lipodystrophy (FPLD;

(Shackleton et al., 2000)) fused to Dam (bacterial adenine methyltransferase) and transduced them into human myogenic cells. A Dam alone lentivirus was employed as control. The Dam-lamin A fusion proteins showed the expected molecular weights and subcellular localization restricted to the nuclear envelope (Figure 5). Infections were done 24 hrs after switching the cells to differentiation medium, and genomic material harvested 48 hours later (3 days post differentiation), when the majority of cells had differentiated into multinucleate myotubes. DamID-seq experiments were performed in duplicates for each Dam-LMNA construct and the negative control (DamOnly). Library sizes and sequencing read depth were comparable between samples (~30 million reads), and >80% of reads started with the expected GATC sequence of the Dam adenine methylase recognition site (Figure 6). Following sequencing, reads were aligned to reference human genome (hg19) and domains of enrichment were called using

SICER algorithm. Comparison of WT Dam-LMNA peaks with human myotube histone marks (ENCODE data) showed a strong positive correlation with the heterochromatic histone mark H3K9me3 and anti-correlation with euchromatic mark H3K4me3 (Figure 7).

These data are consistent with lamin A/C protein associating with heterochromatic regions, namely LADs (lamin-associated domains).

48 Figure 5. Transfection of DamLMNA constructs into HEK293T cells. Panel a:

Western blot analysis using antibody against V5 shows the product size corresponding to the fusion (Dam-V5-LMNA) and V5-Dam alone proteins. Panel b:

Immunofluorescence analysis shows classical nuclear lamina ring staining using antibody against V5, confirming that fusion protein localizes to the nuclear envelope.

50 Figure 6. Majority of DamID reads started with GATC sequence. FastQC reported that sequence and GC content across the reads showed sequence bias at the first four bases of each read where > 80% of reads GATC sequence at be beginning of the read

(DpnI recognition site).

52 Figure 7. LADs show enrichment for heterochromatin and show negative correlations with active marks. Panel a: Distribution of H3K9me3 (top), lamin A

(middle) and H3K4me3 (bottom) peaks around of transcription start site (2Kb +/-). Panel b: Scatter plot showing negative correlation between lamin A and H3K4me3 peaks.

Panel c: Percentage of LAD genes that show enrichment for other chromatin marks

(H3K4me3, H3K9me3, H3K27me3).

53 Genomic mapping of LADs showed WT lamin A to associate with ~11,000 genomic domains, with an average domain length of 32 kbp (~12% of the genome)

(Table 4). Both EDMD (p.R453W) and FPLD (p.R482W) lamin A mutations revealed a significant increase in the number of LADs, with nearly twice as many genomic loci labeled by Dam-ID compared to WT (p<0.001) (Table 4; Figure 8, Panel a).

Comparative analysis of genes associated with LADs in the three lamin A variants showed that the majority of LADs seen in the WT lamin A were shared with one or both mutant constructs (1239/1960; 63%) (Figure 8, Panel a). However, the promiscuous binding to de novo LADs by the mutant proteins led to a preponderance of mutation- specific LADs. For p.R453W, 69% of LADS (2397/3477) were not shared with WT, and

32% (1115/3477) were specific to this EDMD mutation. For p.R482W, 80% (3996/5011) were not shared with WT, and 54% (2715/5011) were specific to this FPLD mutation

(Figure 8; Panel a). WT LADs were larger (32.5 kbp mean size) than mutant LADs (22.3 kbp for lamin A-EDMD and 19.9 kbp for lamin A-FPLD; p value <0.001 vs. WT; Table 4).

These data showed that disease-associated missense mutations cause allele-specific alterations of chromatin association with the nuclear envelope.

54 Table 4. Genome wide DamID seq analysis of three lamin A proteins (WT, p.R453W LMNA causing EDMD and p.R482W LMNA causing FLPD). Properties of lamina-associated domains are shown in the table.

LADs Non-LAD Genome Min Max Mean Median genome coverage Number size size size size (kbp) coverage (%) (kbp) (kbp) (kbp) (%) DamLMNA 12.04 11,607 2 1070 26 32.5 87.96

DamLMNA-EDMD 13.88 19,502 2 245 17.9 22.3 86.12

DamLMNA-FPLD 14.6 22,947 2 194 15.9 19.9 85.4

55 3.3.2 EDMD mutations show allele-specific alteration of epigenomic programming of myogenic differentiation

To focus on the potential effects of lamin A mutations on gene transcription, we evaluated the lamin A – gene interactions occurring at regions encompassing the transcription start sites (TSS) of genes found in the LADs. Using 25 bp windows, WT lamin A showed a selective lack of binding ~200 bp around the TSS and strong enrichment 1-2 kb downstream of the TSS (Figure 8; Panel b). Mutant Lamin A LADs did not show the same loss of binding ~200 bp at the TSS, but did show enrichment 1-2 kbp downstream. This indicates that disease-causing lamin A/C mutations cause abnormal increased association of nuclear envelope – chromatin interactions specifically at the transcription start site.

57 Figure 8. Promiscuous formation of shorter LADs when lamin A/C is mutated.

Interactions between lamin A nuclear envelope protein and chromatin domains were assessed by Dam- lamin A fusion protein using Dam-ID-seq (WT, p.R453W EDMD, p.R482W FPLD) in differentiating human myoblasts. Lamin-associated domains (LADs) were scored and compared between the three lamin A/C variants. Panel a: There was overlap in LADs (and genes found in LADs) between the three variants. However, mutant lamin A proteins showed promiscuous formation of novel LADs, of shorter length compared to WT (Table 4). Panel b: Wild-type Lamin A showed loss of LADs at transcription start sites, whereas mutant lamin A showed abnormal increased association with start sites. WT and mutations in lamin A showed most binding downstream of TSS.

58 We had previously shown delayed induction of some myogenesis-associated transcripts in EDMD cells (Melcon et al. 2006), and had hypothesized that this could be due to a relative failure in heterochromatin-euchromatin conversion at these loci during myogenic development. To test this using the DamID-seq data, mRNA transcripts significantly induced by the transition for myoblasts to myotubes were selected (ANOVA- one way p<0.01; FC>1.2), and then LADs mapped to these transcripts (n=322, Table 5).

As hypothesized, WT lamin A showed very low binding near the transcription start site of these myogenesis-related transcripts, consistent with euchromatization of these loci and high expression in myotubes (Figure 9, Panel a). In contrast, EDMD Lamin A showed pervasive heterochromatinization of these loci. This effect was allele-specific; FPLD lamin A association with myogenic genes was more similar to WT (Figure 9; Panel a).

These data are consistent with an allele-specific effect of LMNA gene mutations on developmental programs, and observed clinical involvement of muscle tissue in EDMD

(p.R453W) and not FPLD (p.R482W), despite the same Arg-Trp change at neighboring amino acids.

59 Table 5. Genes showing mRNA induction at myoblast–myotube transition

(Encode). Gene names, fold change and p values (FDR corrected) are shown in the table. Genes specific to skeletal and cardiac muscle development are shown in bold

(defined by IPA analysis). Genes are ranked by fold change.

Gene ID Fold change p value ATP1B4 66.71 2.00E-04 MYBPC1 62.04 8.20E-03 SLN 44.72 3.90E-03 A2M 44.09 2.30E-03 MYL1 28.66 3.10E-03 SGCG 27.31 1.60E-03 CD36 26.14 5.20E-03 MYOM1 24.49 2.80E-04 LRRC39 20.69 3.40E-04 ATP8A1 20.66 5.70E-03 RRAGD 18.29 5.80E-03 MYH7 17.04 9.60E-03 SOHLH2 16.67 7.80E-04 TMEM182 14.65 7.20E-03 CYSLTR1 14.35 2.00E-03 NRAP 14.11 8.30E-03 GPNMB 13.88 2.00E-04 MYOT 13.3 5.00E-03 REEP1 12.84 2.50E-03 MYOM2 11.89 8.00E-04 FILIP1 11.09 7.40E-03 APOBEC2 10.36 6.60E-03 CYP2J2 10.26 4.00E-03 KLHL31 10.1 6.70E-03 PRKG1 10.09 7.30E-03 FABP3 9.55 5.30E-03 HDAC9 9.48 2.80E-04 MYOM3 9.03 5.50E-03 CCDC141 9 5.20E-03 CKMT2 8.76 3.50E-03 CSRP3 8.6 4.70E-03 COL14A1 8.44 3.90E-03 ITGB1BP2 8.38 7.40E-03 PLN 8.32 9.80E-04 MYL4 8.3 1.00E-02 MAPRE3 8.22 8.40E-03 AGBL1 7.84 9.70E-03 JAM2 7.7 6.40E-03 TNNC2 7.63 5.80E-04 CACNA2D1 7.32 1.60E-03 ASB14 7.24 7.20E-04 TNNC1 7.23 9.80E-03 EGF 7.14 1.10E-03 PGM5 7.1 5.20E-03 LIMCH1 6.9 9.90E-03

60 ENO3 6.73 3.90E-03 PKHD1 6.56 2.30E-03 TMEM38B 6.53 2.50E-03 GALNT13 6.31 6.60E-03 SLCO5A1 6.28 3.30E-03 FADS2 6.23 3.50E-03 TPM3 6.15 5.80E-03 DPYSL5 5.94 1.90E-03 COL19A1 5.93 9.80E-03 FAM213A 5.57 3.90E-03 DNAJA4 5.48 4.00E-03 PPM1E 5.41 9.70E-03 NPY6R 5.37 8.40E-03 HRC 5.34 4.30E-03 LDB3 5.27 1.40E-03 SVIL 5.23 2.10E-03 TNNT1 5.11 9.70E-03 PPM1K 5.08 4.00E-03 CACNB1 5.01 9.60E-03 DCX 4.99 2.90E-03 DACT1 4.92 3.90E-03 MXRA5 4.89 2.20E-03 SCD 4.77 2.50E-03 FSD1L 4.7 3.10E-03 GPRC5B 4.64 2.30E-03 PPFIBP2 4.64 5.70E-03 MLLT11 4.62 8.60E-03 PARM1 4.55 5.50E-05 EXOC6 4.54 2.20E-03 TRIM54 4.53 8.20E-05 C1orf105 4.42 2.10E-03 LGR4 4.42 7.30E-03 TGFB3 4.39 3.00E-03 HMGCS1 4.33 5.20E-03 BHLHE40 4.25 3.20E-03 YIPF7 4.22 9.40E-03 SLC40A1 4.21 7.80E-03 AGL 4.18 6.00E-03 FBXO40 4.15 3.40E-03 PRKCQ 4.12 3.80E-04 NBEA 4.1 2.50E-04 FBXO16 4.02 3.10E-03 PIK3C2B 4.02 8.20E-03 F13A1 3.99 6.10E-03 VGLL2 3.99 6.50E-03 ZFP106 3.88 2.90E-03 LITAF 3.87 8.60E-04 MSMO1 3.8 5.20E-03 CXCL12 3.69 5.70E-03 DCLK2 3.6 7.70E-06 PLP1 3.58 1.60E-03 SLC15A2 3.58 1.80E-03 SLC4A4 3.58 8.50E-03 ELAVL2 3.54 2.30E-03 NUP50 3.54 5.70E-03 CPEB4 3.51 3.50E-04 TAL2 3.51 7.90E-03

61 DHCR7 3.44 9.50E-03 SLC41A2 3.42 8.10E-03 FRK 3.38 1.70E-03 HUNK 3.38 2.50E-03 NCALD 3.37 7.00E-03 SYNPO2L 3.34 3.00E-03 ZNF483 3.28 2.80E-03 POPDC2 3.26 8.80E-04 CDK18 3.23 3.50E-03 TPRG1 3.21 3.70E-03 RNF152 3.2 3.50E-03 SHISA4 3.2 4.50E-03 SORBS2 3.18 6.50E-03 FLRT2 3.16 7.90E-03 AIF1L 3.15 3.80E-03 ASB4 3.13 3.90E-03 CARNS1 3.11 6.40E-03 SEL1L3 3.07 2.20E-03 KBTBD5 3.05 3.20E-04 C7orf41 3.04 2.60E-04 LOC158696 3.04 8.10E-04 PCDHB12 3.04 5.30E-03 PDE7A 3.04 9.40E-03 FNDC5 3.02 5.20E-03 MAPK10 3.01 7.30E-03 MYL6B 2.99 7.60E-03 MEF2A 2.96 4.60E-03 ITGA1 2.94 3.40E-03 MED12L 2.94 6.70E-03 LDLR 2.91 1.60E-03 HMBOX1 2.88 6.30E-04 TRIM63 2.88 1.80E-03 KCNN3 2.87 1.80E-03 PKIA 2.85 7.70E-04 PKP2 2.85 2.00E-03 TNIK 2.85 9.00E-03 GRAMD3 2.83 8.00E-04 MYH6 2.81 6.50E-04 TSPAN9 2.81 4.80E-03 ZNF323 2.8 2.70E-03 VDR 2.77 8.10E-03 ARMC9 2.76 2.70E-04 CXCL13 2.76 1.90E-03 NAV3 2.76 3.20E-03 USP13 2.72 6.40E-03 PRUNE 2.71 3.10E-03 PDE2A 2.7 7.80E-03 AFAP1L1 2.69 8.60E-03 CPEB3 2.68 1.30E-03 ICAM1 2.67 8.20E-03 GPRC5C 2.66 5.80E-03 FRMPD1 2.65 8.00E-04 STK38L 2.65 1.90E-03 MOB1B 2.64 4.40E-03 PADI2 2.64 8.10E-03 40789 2.63 6.50E-03 AKD1 2.63 9.50E-03

62 PLD1 2.61 3.50E-04 POLB 2.61 6.60E-04 CDKN1A 2.57 1.90E-03 ICOSLG 2.56 1.40E-03 LRRN3 2.56 1.00E-02 RBFOX1 2.54 2.90E-03 NIPSNAP3B 2.49 2.40E-03 EGR2 2.48 1.60E-04 HSPA12A 2.48 3.90E-03 C12orf42 2.47 1.40E-03 ITGA6 2.47 1.60E-03 AK1 2.46 7.50E-03 OLFM2 2.44 5.70E-03 RAPGEF1 2.42 7.30E-03 CAP2 2.41 6.00E-04 MBP 2.41 9.50E-03 VASH1 2.4 5.60E-03 DHCR24 2.39 2.40E-04 TTC13 2.39 4.70E-03 PREPL 2.38 9.60E-03 DCP2 2.35 3.90E-03 FRMD7 2.35 6.10E-03 PCDHB11 2.35 6.80E-03 FAM189A2 2.34 6.00E-03 RBM24 2.33 1.00E-03 MAS1 2.28 9.40E-03 P2RX5-TAX1BP3 2.27 1.30E-03 NEO1 2.25 7.40E-04 INPP4B 2.24 3.50E-03 TMEM117 2.24 7.50E-03 CASQ1 2.23 6.80E-03 RAB15 2.23 8.40E-03 CYGB 2.21 7.70E-03 C10orf71 2.19 4.40E-04 EAF2 2.19 7.50E-04 SGCD 2.19 4.30E-03 GRASP 2.16 4.00E-03 TMEM62 2.14 3.70E-03 TRAF5 2.12 8.70E-03 ASAH1 2.11 8.30E-03 IL34 2.11 8.70E-03 ATXN1 2.1 2.00E-04 DYNC1I1 2.08 5.30E-03 LIMK2 2.06 7.00E-03 MICU1 2.06 9.30E-03 DTWD2 2.05 2.30E-03 PTPRB 2.05 8.80E-03 CYB5R1 2.03 5.60E-03 MYL3 2.03 8.30E-03 ABLIM3 2.02 5.40E-03 NEK7 2.02 5.40E-03 RNF115 2.01 4.80E-03 COLQ 2 7.50E-04 DENND1B 2 8.90E-04 FBXO30 2 9.70E-04 ARL4C 1.98 7.50E-03 SREBF2 1.98 8.50E-03

63 LRP8 1.97 2.30E-03 TRO 1.95 4.80E-03 PDE4DIP 1.94 9.10E-03 SLC36A1 1.94 9.70E-03 MACROD2 1.93 9.60E-04 PSEN2 1.91 2.90E-03 FGF13 1.9 5.70E-04 SAMD4A 1.9 4.60E-03 GPR98 1.89 1.30E-03 ATP1A1 1.88 3.30E-03 CDKN2B 1.88 5.00E-03 FAM69A 1.86 2.30E-03 RNF121 1.86 7.10E-03 CAMK2G 1.84 9.10E-04 PGAP1 1.84 6.50E-03 KCTD7 1.83 2.40E-03 KIAA0922 1.83 3.80E-03 ZNF610 1.83 9.10E-03 C13orf44 1.79 1.00E-03 SRPK3 1.79 6.00E-03 DENND1B 1.78 3.80E-03 RALGPS1 1.77 2.20E-04 PHLDB1 1.76 5.50E-03 SCAI 1.76 8.40E-03 TTC33 1.74 4.00E-03 ABCG2 1.73 5.40E-04 KIF2A 1.72 7.80E-04 RNF19B 1.72 2.80E-03 TBC1D1 1.72 9.40E-03 SQSTM1 1.71 6.00E-03 KIFAP3 1.7 3.40E-03 MOAP1 1.7 5.70E-03 GGA2 1.69 1.60E-03 AKNAD1 1.66 2.00E-03 BRF1 1.66 2.70E-03 C21orf91 1.66 5.00E-03 PLXDC1 1.66 8.50E-03 ASB1 1.64 5.10E-03 GXYLT2 1.64 9.80E-03 C5orf24 1.63 2.60E-03 CLCN6 1.63 6.30E-03 TRAK1 1.63 8.00E-03 CLIC5 1.62 5.60E-03 DNAJB6 1.62 5.60E-03 GFPT1 1.62 8.90E-03 NEURL 1.62 9.80E-03 SNX18 1.61 3.90E-03 NECAB3 1.6 3.80E-03 RRAGC 1.6 7.00E-03 DIP2B 1.59 6.20E-04 ZNF148 1.59 1.30E-03 KCNA10 1.58 1.10E-04 LOC646513 1.57 2.00E-03 PMEPA1 1.57 6.80E-03 OLFML1 1.55 2.90E-03 FAM190B 1.54 8.80E-03 KPNA1 1.53 4.80E-03

64 OR1D5 1.53 7.10E-03 TOM1L2 1.53 7.40E-03 ERBB2 1.51 1.70E-03 MAPK8 1.51 4.10E-03 ZNF643 1.51 4.80E-03 SPATA13 1.5 1.50E-03 ABHD4 1.49 2.90E-03 EFCAB7 1.49 4.20E-03 HRASLS2 1.49 4.40E-03 IMPAD1 1.49 5.90E-03 MGAT4A 1.49 8.20E-03 SLC1A2 1.49 9.80E-03 CLCN7 1.48 4.10E-03 JAZF1 1.48 7.30E-03 LEPROTL1 1.47 2.40E-03 CHRM4 1.46 3.80E-03 KCND3 1.46 5.90E-03 UAP1L1 1.46 9.80E-03 CELF1 1.45 8.60E-03 NCF2 1.44 3.70E-03 NUDT9 1.44 5.00E-03 SNN 1.44 6.50E-03 KLHDC10 1.43 7.40E-03 DPY19L2P4 1.42 8.90E-04 FAM212B 1.42 5.90E-03 SULF2 1.42 6.20E-03 ANKRD36BP1 1.41 7.00E-04 CACNB4 1.41 7.80E-03 PLCXD2 1.4 2.90E-03 ZSCAN16 1.4 5.30E-03 TMEM229B 1.39 1.60E-03 TSPAN18 1.38 6.50E-03 CDK20 1.37 7.40E-03 ANKRD50 1.36 2.40E-03 NFKB2 1.35 3.40E-03 PRAP1 1.35 4.10E-03 ENAM 1.34 5.70E-04 PPP1R15A 1.31 1.40E-03 SLCO2B1 1.31 2.70E-03 AFAP1 1.3 1.10E-03 CDKL1 1.3 5.60E-03 GSK3B 1.3 9.70E-03 UBE2D3 1.3 9.80E-03 C16orf54 1.29 5.70E-03 HAGH 1.27 9.80E-03 GNA14 1.26 2.90E-03 FAAH2 1.24 8.10E-03 AMZ2P1 1.23 5.80E-03 BAIAP2 1.23 6.10E-03 DMGDH 1.23 7.80E-03

66 Figure 9. Genes showing mRNA induction at the myoblast- myotube stage stay inadequately bound by EDMD lamin A and show abnormal DNA methylation patterns. Panel a: Restriction of analysis to transcript units associated with myogenic differentiation showed broad loss of lamin A binding with WT lamin A, consistent with euchromatinization of myogenic loci at the myotube stage. Lamin A containing an

EDMD mutation (p.R453W) showed persistent heterochromatization of myogenic loci, whereas p.R482W FPLD mutation retained appropriate loss of LADs similar to WT.

Panel b: DNA methylation status of these loci in patient MyoD converted human myoblasts showed expected loss in methylation in normal patients. In EDMD patient cells carrying different LMNA mutation (p.H222P), myogenic loci showed increase in methylation that was predicted by persistent heterochromatization and nuclear lamina attachment.

67 To extend our observations to patients affected by lamin A laminopathies, fibroblasts were obtained from an EDMD patient harboring a different, heterozygous

LMNA mutation (p.H222P), as well as control subjects. Cells were transfected with a

DOX-inducible MyoD construct to force conversion to the myogenic lineage and induced to differentiate into myotubes (Figure 10). EDMD and WT cells were harvested at 1, 3, and 5 days after differentiation, and genome-wide methylation patterns were detected by

Illumina arrays (Infinium Human Methylation450). The myogenesis-associated transcripts (n=322) were investigated for changes in methylation patterns as a function of myogenic differentiation, normalized to day 1 (Figure 9; Panel b, Table 6). This showed the predicted loss of DNA methylation as a function of myogenic differentiation in WT cells, consistent with the Dam-ID data presented above. In contrast, and similar to the Dam-ID data, the EDMD cells revealed persistent DNA methylation at many of these loci (Table 6) indicating a perturbation of the differentiation program despite formation of myotubes (Figure 9, Panel b; Table 6).

68 Table 6. DNA methylation of myogenesis associated genes of Normal and EDMD patient MyoD converted myoblasts normalized to day 1. Genes queried were those showing mRNA upregulation with human myoblast –myotube transition (Encode), and then ranked by decrease of methylation at day 3 in normal cells.

Day 3 Day 5 Gene ID Normal EDMD Normal EDMD DACT1 0.47 0.74 0.66 0.89 KCTD7 0.51 1.11 0.66 1.09 GSK3B 0.54 0.82 0.76 0.84 TPM3 0.56 0.57 0.68 0.73 JAM2 0.57 1.21 0.7 1.06 PADI2 0.57 0.93 0.78 0.88 SAMD4A 0.59 0.98 0.82 0.87 LIMCH1 0.6 1.01 1.05 0.92 GPRC5C 0.6 0.78 0.73 1.01 GNA14 0.61 0.84 0.52 1.17 FAAH2 0.62 0.97 0.6 0.94 PKP2 0.63 1.05 0.64 1.11 C10orf71 0.64 0.84 0.45 0.99 F13A1 0.65 1.14 0.74 1.02 HUNK 0.66 1.08 0.81 0.99 SC4MOL 0.66 0.74 0.88 0.83 FRMD7 0.67 0.54 1.3 0.79 ABHD4 0.68 0.99 0.64 1.36 PGAP1 0.69 0.68 0.85 0.96 CDK18 0.69 0.54 0.93 0.71 PPP1R15A 0.71 0.84 0.69 0.82 TTC13 0.71 1.39 0.85 1.29 SLCO5A1 0.72 1.07 0.71 1.06 PRKCQ 0.72 1.12 0.99 1.07 KLHDC10 0.73 0.91 1.03 0.93 ATP8A1 0.73 0.84 0.78 1.04 FABP3 0.74 1.19 0.92 1.11 CASQ1 0.74 0.94 0.63 0.87 TPRG1 0.76 0.98 0.98 0.97 TMEM182 0.77 0.87 0.99 0.89 PARM1 0.77 0.8 0.88 0.97 ANKRD50 0.77 0.65 0.52 1 HMGCS1 0.77 1.18 0.82 0.85 DYNC1I1 0.77 0.58 0.72 1.09 COL19A1 0.78 1.54 1 1.55 TOM1L2 0.79 0.93 0.65 0.92 CBARA1 0.79 0.9 0.95 1.05 OR1D5 0.8 0.57 0.85 0.96 YIPF7 0.8 0.83 0.88 1.1

69 SCD 0.81 0.85 1 0.84 CSRP3 0.81 0.98 0.73 1.59 TGFB3 0.81 1.08 0.91 1.05 FLJ32065 0.81 1.01 0.86 0.98 RAB15 0.82 0.88 0.95 0.54 CACNB4 0.82 0.87 0.9 0.99 TNNC1 0.82 0.81 0.98 0.96 NEURL 0.83 0.42 0.77 0.68 KIFAP3 0.83 0.9 0.9 0.89 LIMK2 0.83 0.74 0.94 0.77 MBP 0.84 0.98 0.79 1 ERBB2 0.85 0.9 0.91 0.8 NEO1 0.85 1.1 0.9 0.91 DHCR7 0.85 1.47 0.9 1.1 FBXO16 0.85 1.05 0.92 0.99 CYB5R1 0.85 1.81 1.15 1.28 CXCL12 0.86 1 0.68 1.11 LITAF 0.86 0.92 1.04 0.94 HDAC9 0.86 1.39 0.85 1.16 ZFP106 0.86 1.16 1.13 1 ZNF483 0.86 0.75 0.92 0.72 KPNA1 0.87 1.16 1.14 1.08 EFCAB7 0.87 1.05 0.97 0.96 ENAM 0.87 0.99 0.92 0.99 MYL1 0.88 0.78 1 0.7 ATXN1 0.88 0.95 0.94 0.95 HAGH 0.89 0.91 1 0.89 DPY19L2P4 0.89 0.89 0.98 0.94 SLC41A2 0.9 1.11 0.98 1.77 TAL2 0.9 0.98 0.98 0.96 SVIL 0.9 1.08 0.88 1.04 ATP1A1 0.9 1.08 1.06 0.97 ANKRD36BL1 0.9 1.03 0.92 1.1 FAM189A2 0.91 0.86 0.96 0.96 CACNB1 0.91 1.09 0.92 0.87 NECAB3 0.91 0.96 0.94 0.91 TRAK1 0.92 1.5 0.92 1.23 PTPRB 0.92 0.54 0.9 0.77 ATP1B4 0.92 0.46 0.94 0.62 MYL4 0.92 1.03 0.97 1.02 TNNT1 0.92 1.02 1.12 0.97 PDE4DIP 0.92 0.92 0.93 0.98 PIK3C2B 0.92 0.93 1.07 0.96 CXCL13 0.92 0.64 0.98 0.86 PPFIBP2 0.92 0.91 0.87 0.93 CHRM4 0.92 1 0.95 0.98 NRAP 0.93 1.09 0.87 1.04 ZNF610 0.93 1.24 1.04 1.24 MYH6 0.93 0.8 1.06 0.94 NEK7 0.93 0.99 0.97 0.96

70 RRAGD 0.93 0.87 0.95 0.99 DCP2 0.94 0.92 0.89 1.12 SOHLH2 0.94 0.97 0.95 1.03 IMPAD1 0.94 0.94 0.95 0.94 UAP1L1 0.94 0.89 1.14 0.98 LDLR 0.94 1.02 0.99 1.03 PGM5 0.94 0.72 0.9 1.03 FLRT2 0.94 0.83 1.13 0.93 FAM69A 0.95 0.98 0.96 0.99 SHISA4 0.95 1.23 1.04 1.06 KCNN3 0.95 0.99 1.14 0.98 GALNT13 0.95 0.89 0.99 0.94 OLFM2 0.95 1.06 1.01 1.04 ENO3 0.96 1.04 0.96 1.01 SGCD 0.96 0.96 0.99 0.96 APOBEC2 0.96 0.95 0.93 0.95 ARL4C 0.96 1.09 1.03 1.05 MXRA5 0.96 1.09 1.13 1 POLB 0.96 1.01 0.98 1 AKNAD1 0.96 0.99 1.02 0.94 MYOM3 0.96 1.04 1.05 0.93 ASB14 0.96 1.15 0.95 1.03 NAV3 0.97 1.14 1.04 1.13 PPM1K 0.97 0.99 0.99 0.99 KIF2A 0.97 0.85 1.7 0.86 SEL1L3 0.97 0.91 1.08 0.87 C1orf183 0.97 1.05 0.95 1.01 DNAJA4 0.97 1.04 0.95 1.04 TBC1D1 0.97 0.97 1 0.99 ITGA6 0.97 1.27 1.01 1.22 RNF19B 0.97 0.73 0.99 0.95 PCDHB12 0.98 1.05 1.05 0.93 LDB3 0.98 0.89 1 0.88 PKIA 0.98 0.98 0.91 0.98 SPATA13 0.98 1.07 1 1.04 SLC4A4 0.98 0.98 0.98 1.01 EGR2 0.98 0.8 1 0.95 CACNA2D1 0.98 0.99 1 0.97 CD36 0.98 0.93 0.98 0.91 CAP2 0.98 0.91 1.03 0.61 REEP1 0.98 1.29 0.98 1.25 LGR4 0.98 1.02 0.67 1.07 MYOM1 0.99 0.51 0.92 0.67 FBXO30 0.99 1.03 1 1.03 FRK 0.99 1 0.98 0.96 AK1 0.99 1 1.04 0.94 KCNA10 0.99 0.92 1.07 0.9 JAZF1 0.99 1.02 1.03 1.03 PPM1E 0.99 1.01 1.02 1 DMGDH 0.99 0.98 1 0.99

71 MGAT4A 0.99 0.89 1.06 0.94 TRIM63 0.99 0.86 0.95 0.82 GGA2 0.99 0.9 1.02 1.16 DTWD2 0.99 1.6 0.96 1.41 STK38L 0.99 1.01 0.98 0.98 TMEM62 1 1.02 1.02 0.99 RAPGEF1 1 0.93 1.03 1.02 TRIM54 1 0.99 1.02 0.99 TRAF5 1 1.08 0.99 1.12 PCDHB11 1 1.07 1.04 0.99 SLC36A1 1 1.01 0.98 0.97 RBM24 1 0.88 1.02 0.85 EGF 1 1 0.99 0.98 IL34 1.01 1.03 1.23 1.08 FAM190B 1.01 1 1.02 1.02 MAS1 1.01 1.09 1.06 1.31 TNNC2 1.01 1.09 1.02 1.08 FRMPD1 1.01 1 1.06 0.86 KLHL31 1.01 0.95 0.96 1.09 PLXDC1 1.01 0.82 1.02 0.94 HSPA12A 1.01 0.87 1.01 0.91 INPP4B 1.01 1.04 1.06 1 VDR 1.01 0.95 1.02 0.97 SYNPO2L 1.01 1.06 0.99 1.23 PDE7A 1.01 1.03 1.02 0.98 LRRN3 1.01 1.09 1 1.04 ASB4 1.02 0.96 1.04 0.98 LOC158696 1.02 0.9 1.16 0.89 CLIC5 1.02 0.95 0.99 0.98 GPNMB 1.02 0.99 0.98 1.01 HRASLS2 1.02 0.99 0.99 0.98 HRC 1.02 1.19 1.06 1.09 DHCR24 1.03 1.01 1.01 1.01 COL14A1 1.03 0.91 0.96 0.98 MYOM2 1.03 1.22 0.9 1.16 VASH1 1.03 1.01 1.04 1.06 NPY6R 1.04 0.97 1.06 0.94 TTC33 1.04 0.55 0.99 1.01 MED12L 1.04 1 1 0.98 CYGB 1.05 0.98 1.04 0.96 AFAP1 1.06 0.98 1 1.01 ATPGD1 1.06 1 1.04 1 PRUNE 1.06 0.78 1.13 0.86 ABLIM3 1.07 1.05 1.13 1.39 FSD1L 1.07 0.97 1.11 1.1 NBEA 1.09 1.04 1.01 1.02 AGL 1.09 1.02 1.29 1.09 KIAA0922 1.09 1.26 1.13 1.34 ZNF148 1.1 0.74 1.1 0.89 PLP1 1.12 0.46 1.22 0.67

72 CCDC141 1.14 0.75 1.17 1.14 C5orf24 1.14 1.16 0.76 0.87 FILIP1 1.16 0.91 0.98 0.84 SLCO2B1 1.16 0.45 1.18 0.7 SNN 1.19 1.31 1.49 1.2 AGBL1 1.19 0.85 0.75 0.76 SNX18 1.2 0.77 1.52 1.06 RNF121 1.21 1.09 0.93 0.89 PMEPA1 1.22 0.86 1.25 1.01 AFAP1L1 1.23 1.29 1.11 1.41 DCX 1.23 1.12 1.52 1.06 BAIAP2 1.23 0.83 1.17 0.9 PSEN2 1.29 1.33 1.37 1.53 DCLK2 1.33 0.96 1.36 1.31 PKHD1 1.36 1 1.41 1.23 ICAM1 1.41 0.99 1.16 1.08 EXOC6 1.52 1.33 1.62 1.4 GPR98 1.54 1.15 1.17 0.91 VGLL2 1.66 0.86 0.86 1.23 42262 1.9 1.13 1.58 1.52

74 Figure 10. Myogenic in vitro differentiation of MyoD-converted EDMD patient fibroblasts (p.H222P LMNA) into myoblasts. Immunostaining of EDMD patient fibroblasts show successful conversion into myogenic lineage capable of forming myotubes. Cells were stained for myogenic marker (myosin –MF20), MyoD and Hoechst

(nuclear marker).

75 3.3.3 Delayed E2F cell cycle suppression is predicted by lamin A mutation (EDMD-

AD)

The EDMD phenotype results from dominant missense mutations of LMNA

(above; EDMD-AD), as well as loss-of-function of the lamin-associated emerin protein

(X-linked recessive; EDMD-XR). We had previously shown similarities in mRNA expression profiles between EDMD-AD and EDMD-XR patient muscle biopsies, relative to other types of neuromuscular disease involving transition to myogenic terminal differentiation (Bakay et al. 2006). In emerin null cells, we showed relative inability to exit from the cell cycle via shut-off of E2F pathways (Bakay et al. 2006; Melcon et al.

2006). We queried the DamID-seq data from WT and mutant lamin A (EDMD, FPLD) proteins to determine if key E2F pathway members, CDK1 and Rb, showed abnormal regulation of chromatin (Figure 11; Panel a). Consistent with our previously published increased mRNA and protein data in emerin null cells (Melcon et al. 2006), EDMD lamin

A protein showed reduced association with the CDK1 and Rb loci; FPLD lamin A data were more variable.

77 Figure 11. Cell cycle loci show decreased association with EDMD lamin A. Lamina association of cell cycle loci assessed by DamIDseq showed reduction in binding of mutant lamin A. Panels a and b: Wig tracks of lamin A enrichment on two cell cycle –

CDK1 (a) and RB1 (b). Data are represented as log2 of DamLMNA/DamOnly counts.

Panel c: Normalized read counts of lamin A peaks on CDK1 and RB1 loci.

78 To integrate the lamin A protein data (DamID) with additional epigenetic marks, we carried out ChIP-seq for H3K9me3 at three time points during myogenic differentiation of WT and emerin null murine H2K cells. H3K9me3 peaks mapping relative to known start sites of transcription units showed similar distribution patterns in

WT and emerin null cells, with the majority of heterochromatic H3K9me3 peaks mapped to intergenic (non-coding) regions of the genome, 50-500 Mbp away from the nearest gene transcription unit start site (TSS). Analysis of loci near transcription start sites (± 10 kbp from TSS) showed all samples with a relative decrease in tag density compared to intergenic and intronic regions (Figure 12). We queried the Cdk1 and Rb1 loci to determine if the Dam-ID data correlated with heterochromatic marks. H3K9me3 was enriched in the vicinity of the Cdk1 and Rb1 loci in WT cells, but was absent in emerin null cells (vicinity of Cdk1) or failed to show increased enrichment as a function of myogenic differentiation (vicinity of Rb1) as seen in WT cells (Figure 13, Panels a and b). However, these heterochromatic marks were quite far downstream of the transcript unit (10 kbp from Cdk1; 200 kbp from Rb1). Thus, H3K9me3 and DamID marks were consistent, but not conclusive. To confirm these findings, ChIP-PCR was done in both

H2K and primary myoblast cells (Figure 13, Panels c and d). These data were consistent with delayed exit from the cell cycle in both EDMD-AD and EDMD-XR myogenic cells, and show that this is likely due to abnormal heterochromatinization of key loci.

80 Figure 12. Genome-wide analysis of H3K9me3 peaks in wild-type and emerin null

(emd) differentiating mouse myoblasts shows similar peak distributions. Panel a:

Distribution of H3K9me3 peaks in wild-type and emerin null myogenic cells. Panel b:

Sequence tag density coverage shows similar profiles for H3K9me3 in wild-type and emerin null cells. Heterochromatin enrichment shows drop in tag density in both cell types. Panel c: Properties of heterochromatic domains in wild-type and emerin null cells during differentiation are shown in the table.

82 Figure 13. Cell cycle loci show inadequate heterochromatin formation in differentiating EDMD myogenic cells. Panels a and b: Wig tracks of H3K9me3 enrichment on Cdk1 (a) and Rb1 (b) loci. Panel c and c: ChiP-qPCR validation of

H3K9me3 enrichment on Cdk1 in H2K cells (c) and primary myoblasts (d).

83 3.3.4 Genome-wide prioritization of ChIP-seq alterations identifies aberrant persistence of pluripotency programs in emerin null cells.

We then carried out a genome-wide assessment of changes in H3K9me3 heterochromatin marks during myogenic differentiation in murine H2K emerin null vs. WT cells. ChIP-seq experiment was done on at three time points of differentiation: myoblasts stage (d0), myotubes after 24 hrs of differentiation (d1) and 48 hrs post differentiation

(d2). SICER called peaks were intersected and annotated using Bedtools Suite to get 1) normalized read counts of each peak for both conditions at all time points; 2) cell type specific peaks and 3) expanded heterochromatic islands during d0-d1 and d1-d2 transition. Using Bedtools Suite we generated a list of peaks that showed heterochromatic decrease in enrichment in emerin null sample at day 0 and day 1

(myoblast-myotube transition). H3K9me3 ChIP-seq peaks were then analyzed using

GREAT web application for genomic annotation of cis regulatory elements. The analysis showed that loci involved in cell fate specification and transcriptional regulation of cell fate commitment, particularly Sox2 and its downstream targets (Gata6, Gbx2, Trim24,

Hoxb1, Pax6 and others) showed loss of heterochromatin marks in emerin null cells relative to WT (Figure 14; Table 7). Visualization of the enrichment around Sox2 locus in emerin null (pink) samples showed significant loss of heterochromatic H3K9me3 peaks at day 2 relative to both emerin null day 0 and wild-type samples (day 0- FC=-1.2,

FDR=0.04; day 1- FC= -1.3, FDR=0.02; day 2- FC=-1.6, FDR=1.72x10-7).

Table 7. Heterochromatin peaks (H3K9me3) showing decreased enrichment in emerin null cell during myogenic transition (day0-day1). Peaks for each condition were called using SICER (window size 1000 bp,gap size 3000 bp and FDR1e-

2) and merged to find union peaks. Union peak file was annotated with read counts and normalized to corresponding library sizes. Peaks showing decrease at day 0 and day 1 were annotated GREAT to identify genes associated with these peaks.

Chromosomal position Normalized reads counts Ratio GREAT annotation chr start end emd-d0 wt-d0 emd-d1 wt-d1 emd-d2 wt-d2 d0 d1 d2 Gene Symbol chr1 63957000 63972999 110.4 136.11 143.02 160.67 160.83 156.4 0.81 0.89 1.03 Fastkd2,Klf7 chr1 189262000 189276999 111.51 124.5 105.68 111.38 101.32 101.2 0.9 0.95 1 Esrrg , Gpatch2 chr1 35633000 35636999 31.19 38.43 32.25 34.24 37.53 41.2 0.81 0.94 0.91 Hs6st1 , Plekhb2 chr1 38371000 38374999 30.64 35.63 30.56 33.49 34.31 28.8 0.86 0.91 1.19 Rev1 , Aff3 chr1 58793000 58805999 160.08 236.98 193.95 229.15 194.6 181.2 0.68 0.85 1.07 Casp8 , Cflar chr1 91814000 91842999 237.36 270.21 286.05 307.04 315.76 275.6 0.88 0.93 1.15 Gbx2 , chr1 122188000 122199999 92.74 111.69 94.64 100.09 92.21 88 0.83 0.95 1.05 C1ql2 , Steap3 chr1 127283000 127288999 72.31 93.27 98.04 118.53 90.6 72 0.78 0.83 1.26 Actr3 , chr1 156843000 156847999 42.78 48.44 43.29 47.41 41.28 38 0.88 0.91 1.09 Cacna1e , Mr1 chr1 163118000 163123999 48.58 59.25 50.5 62.46 64.87 60 0.82 0.81 1.08 Ankrd45 , Prdx6 chr1 173421000 173430999 71.21 86.47 88.28 94.07 88.46 92 0.82 0.94 0.96 Refbp2 , F11r chr1 180678000 180687999 106.26 119.69 125.62 133.2 131.34 115.6 0.89 0.94 1.14 Kif26b , Smyd3 chr1 184636000 184659999 184.92 213.77 197.35 209.59 213.37 205.2 0.87 0.94 1.04 4922505E12Rik , Capn8 chr1 184610000 184631999 188.79 211.36 183.34 197.55 201.04 170.8 0.89 0.93 1.18 4922505E12Rik , Capn8 chr1 187316000 187322999 46.92 60.85 57.72 61.33 75.59 53.2 0.77 0.94 1.42 Slc30a10 , Lyplal1 chr1 193887000 193891999 52.16 72.86 87 92.19 81.49 90.4 0.72 0.94 0.9 Traf5 , Rd3 chr1 43077000 43081999 45.82 53.24 38.2 48.92 47.18 45.2 0.86 0.78 1.04 Gpr45 , Tgfbrap1 chr1 63244000 63247999 36.16 46.44 33.95 60.96 56.29 60.4 0.78 0.56 0.93 Gpr1 , Eef1b2 chr1 93019000 93025999 65.41 80.46 87 92.56 98.64 100.8 0.81 0.94 0.98 Rbm44 , Lrrfip1 chr1 168963000 168965999 25.12 32.43 24.62 35.37 25.2 23.6 0.77 0.7 1.07 Fam78b , Uck2 chr1 194099000 194099999 10.21 14.01 8.06 9.41 13.4 12.8 0.73 0.86 1.05 Kcnh1 , Hhat chr10 84013000 84025999 111.51 129.7 133.69 146.75 153.32 123.2 0.86 0.91 1.24 Ckap4 , Tcp11l2 chr10 120321000 120331999 78.94 112.09 95.07 105.73 120.09 109.2 0.7 0.9 1.1 Hmga2 , Msrb3

chr10 121234000 121247999 96.05 111.69 106.1 114.77 106.15 101.2 0.86 0.92 1.05 BC048403 , Srgap1 chr10 68434000 68450999 112.61 128.1 122.23 131.32 156 132 0.88 0.93 1.18 Rhobtb1 , Tmem26 chr10 69446000 69456999 141.87 178.94 157.88 167.44 172.62 187.6 0.79 0.94 0.92 Ccdc6 , Ank3 chr10 75609000 75613999 39.74 52.04 42.02 51.55 40.21 45.6 0.76 0.82 0.88 Slc5a4a , Prmt2 chr10 78520000 78535999 152.91 200.96 204.56 227.65 240.71 218.8 0.76 0.9 1.1 Olfr57 , 2610008E11Rik chr10 83122000 83132999 77.56 94.87 88.28 94.82 93.82 96.4 0.82 0.93 0.97 1500009L16Rik , Appl2 chr10 94343000 94369999 211.7 235.78 234.27 247.97 256.79 264.8 0.9 0.94 0.97 Plxnc1 , Ccdc41 chr10 120607000 120643999 278.49 311.44 277.13 302.15 312.01 297.2 0.89 0.92 1.05 Tbc1d30 , Wif1 chr10 121336000 121360999 175.54 216.97 197.77 214.1 220.34 177.2 0.81 0.92 1.24 Srgap1 , BC048403 chr10 126057000 126062999 54.1 60.85 47.53 56.07 35.38 45.2 0.89 0.85 0.78 Xrcc6bp1 , Lrig3 chr10 40250000 40253999 30.36 40.43 36.92 39.51 47.71 34.4 0.75 0.93 1.39 Slc22a16 , Cdk19 chr10 41064000 41064999 8.56 12.41 6.79 10.91 8.58 9.2 0.69 0.62 0.93 Zbtb24 , Fig4 chr10 42641000 42644999 40.3 45.24 35.65 43.65 46.64 50.4 0.89 0.82 0.93 Scml4 , Sobp chr10 63018000 63019999 20.7 26.82 23.34 24.83 33.77 18.4 0.77 0.94 1.84 Ctnna1 , Lrrtm3 chr10 67453000 67465999 132.76 148.11 138.35 183.62 132.95 140.8 0.9 0.75 0.94 Rtkn2 , Arid5b chr10 67470000 67482999 141.31 217.37 168.06 200.56 175.84 183.2 0.65 0.84 0.96 Rtkn2 , Arid5b chr10 67554000 67565999 123.37 154.52 140.9 158.79 128.66 140 0.8 0.89 0.92 Rtkn2 , Arid5b chr10 70306000 70311999 70.93 92.87 84.03 92.56 82.02 81.2 0.76 0.91 1.01 Phyhipl , Bicc1 chr10 75097000 75103999 61.83 69.25 69.6 80.15 69.69 68.8 0.89 0.87 1.01 Susd2 , Ggt5 chr10 75536000 75539999 43.88 52.44 47.11 50.42 53.61 50 0.84 0.93 1.07 Slc5a4b , Zfp280b chr10 78550000 78555999 49.4 61.65 46.68 58.7 60.58 60.8 0.8 0.8 1 2610008E11Rik , Olfr57 chr10 84692000 84696999 37.81 44.43 37.35 44.4 40.21 42.8 0.85 0.84 0.94 Btbd11 , Cry1 chr10 89211000 89215999 50.78 59.65 52.63 62.84 56.83 53.2 0.85 0.84 1.07 Anks1b , Uhrf1bp1l chr10 94443000 94450999 60.45 79.26 67.9 77.89 65.94 66.4 0.76 0.87 0.99 Plxnc1 , Cradd chr10 116910000 116912999 35.05 39.23 39.89 47.03 52.54 42 0.89 0.85 1.25 Cpm , Cpsf6 chr10 119440000 119447999 66.24 73.66 66.63 71.12 70.76 56.4 0.9 0.94 1.25 Helb , Grip1 chr10 119479000 119479999 8.28 9.21 8.06 10.16 10.72 8 0.9 0.79 1.34 Helb , Grip1 chr10 127410000 127415999 50.78 59.25 53.9 63.59 67.55 66.8 0.86 0.85 1.01 Hsd17b6 , Sdr9c7 chr10 128437000 128446999 84.18 110.49 122.65 129.44 135.63 150 0.76 0.95 0.9 Olfr763 , Olfr9 chr11 64486000 64492999 71.21 80.46 67.9 71.87 69.69 72 0.89 0.94 0.97 Elac2 , Hs3st3a1 chr11 49637000 49648999 96.88 107.68 100.16 114.39 91.14 84.4 0.9 0.88 1.08 Mapk9 , Gfpt2 chr11 59274000 59279999 65.97 92.47 96.76 103.1 124.91 116.8 0.71 0.94 1.07 Zfp867 , Jmjd4 chr11 94318000 94325999 58.24 66.45 60.27 66.6 64.33 52.8 0.88 0.9 1.22 Abcc3 , Cacna1g

chr11 96109000 96142999 277.66 316.25 344.19 370.26 324.88 369.6 0.88 0.93 0.88 Hoxb9 , AK078566 chr11 96221000 96237999 154.84 188.15 172.31 200.93 189.24 198.8 0.82 0.86 0.95 Skap1 , Hoxb1 chr11 108759000 108779999 176.37 198.55 187.16 203.94 211.22 197.2 0.89 0.92 1.07 Axin2 , Ccdc46 chr11 113258000 113263999 69.28 85.27 74.27 74.13 61.65 70 0.81 0.78 0.88 Slc39a11 , Sox9 chr11 120619000 120626999 66.24 86.87 98.04 112.13 107.22 94.8 0.76 0.87 1.13 Cbr2 , Rfng chr11 3000000 3004999 153.46 1921.89 150.66 1906.61 165.65 1698.4 0.08 0.08 0.1 Sfi1 , chr11 11897000 11897999 6.07 7.21 5.09 9.41 7.51 8 0.84 0.54 0.94 Ddc , Grb10 chr11 52537000 52539999 20.15 25.22 26.31 27.84 20.37 28.8 0.8 0.94 0.71 Fstl4 , 9530068E07Rik chr11 63652000 63657999 45.26 53.64 44.56 52.3 50.93 50 0.84 0.85 1.02 Hs3st3b1 , Pmp22 chr11 63937000 63942999 48.3 54.04 48.81 52.68 61.12 40.8 0.89 0.93 1.5 Hs3st3a1 , Cox10 chr11 64751000 64759999 58.79 66.05 59.42 66.23 59.51 49.2 0.89 0.9 1.21 Elac2 , Hs3st3a1 chr11 81555000 81555999 12.14 14.01 7.64 12.42 8.58 10 0.87 0.62 0.86 Ccl2 , 1700071K01Rik chr11 95787000 95789999 27.88 35.63 28.86 45.15 40.74 28 0.78 0.64 1.46 B4galnt2 , Igf2bp1 chr11 96213000 96216999 30.64 39.23 40.74 45.15 31.63 50 0.78 0.9 0.63 Hoxb1 , Hoxb2 chr11 96341000 96353999 87.49 105.28 99.31 111.38 113.12 114.8 0.83 0.89 0.99 Skap1 , Snx11 chr11 96358000 96358999 8 9.21 8.06 11.29 9.11 9.6 0.87 0.71 0.95 Skap1 , Snx11 chr11 96395000 96398999 24.84 34.03 28.43 30.48 27.34 37.2 0.73 0.93 0.73 Skap1 , Snx11 chr12 71489000 71499999 78.39 87.27 82.76 88.05 78.27 74.4 0.9 0.94 1.05 Tmx1 , Trim9 chr12 110848000 110856999 109.02 126.1 112.47 129.82 107.22 101.6 0.86 0.87 1.06 Dio3 , Rtl1 chr12 111034000 111047999 125.03 159.72 150.24 160.29 138.31 140.8 0.78 0.94 0.98 Dio3 , Rtl1 chr12 3109000 3110999 1206.69 1406.29 1214.64 1375.68 1313.44 1384 0.86 0.88 0.95 Rab10 , chr12 27837000 27837999 7.18 10.01 4.67 9.78 9.65 7.2 0.72 0.48 1.34 Sox11 , Cmpk2 chr12 57368000 57375999 60.72 68.05 77.67 82.03 65.4 62.8 0.89 0.95 1.04 Mbip , Brms1l chr12 75904000 75905999 245.92 274.61 235.12 274.31 267.51 279.2 0.9 0.86 0.96 Kcnh5 , Dbpht2 chr12 81645000 81648999 35.33 39.63 37.35 39.51 27.34 36.8 0.89 0.95 0.74 Galntl1 , Erh chr12 85525000 85527999 55.2 68.05 78.51 84.66 80.41 85.2 0.81 0.93 0.94 Pnma1 , C130039O16Rik chr13 43150000 43163999 87.22 100.08 95.49 101.97 92.74 98.4 0.87 0.94 0.94 Tbc1d7 , Phactr1 chr13 45187000 45187999 11.32 12.81 8.06 9.41 17.16 9.6 0.88 0.86 1.79 Mylip , Dtnbp1 chr13 47095000 47109999 154.29 206.56 178.67 191.53 204.25 191.6 0.75 0.93 1.07 Kif13a , Nhlrc1 chr13 54531000 54545999 115.92 131.3 123.5 140.73 152.79 141.2 0.88 0.88 1.08 Thoc3 , Cplx2 chr13 54848000 54854999 56.31 63.25 61.96 76.76 67.55 72 0.89 0.81 0.94 Gprin1 , chr13 56246000 56250999 37.54 46.84 42.44 54.56 51.47 50.8 0.8 0.78 1.01 H2afy , Tifab chr13 60305000 60326999 177.75 200.56 236.82 249.85 264.83 236.4 0.89 0.95 1.12 Dapk1 , Gas1

chr13 67174000 67178999 33.12 43.63 38.62 41.77 41.82 47.2 0.76 0.92 0.89 Zfp712 , Zfp708 chr13 83855000 83872999 115.37 133.7 152.78 177.6 165.12 182.4 0.86 0.86 0.91 Tmem161b , Mef2c chr13 84375000 84377999 30.64 42.03 35.23 37.25 52 43.6 0.73 0.95 1.19 Ccnh , Tmem161b chr13 13018000 13024999 102.95 148.92 108.22 146.37 109.36 133.2 0.69 0.74 0.82 Prl2c3 , Prl2c2 chr13 38241000 38247999 60.45 71.66 58.14 75.63 73.98 73.6 0.84 0.77 1.01 Snrnp48 , Dsp chr13 45171000 45177999 51.89 64.05 58.14 61.33 75.05 55.2 0.81 0.95 1.36 Mylip , Dtnbp1 chr13 45714000 45721999 115.92 144.51 129.87 151.64 137.24 136 0.8 0.86 1.01 Gmpr , Atxn1 chr13 46129000 46130999 14.9 18.01 16.98 18.06 13.4 16.8 0.83 0.94 0.8 Gm1574 , Atxn1 chr13 48580000 48593999 122.82 148.52 138.35 153.15 157.61 146.4 0.83 0.9 1.08 Zfp169 , Id4 chr13 53760000 53767999 77.01 87.27 77.67 84.29 75.05 66.8 0.88 0.92 1.12 Msx2 , Drd1a chr13 67591000 67597999 49.96 61.65 57.29 64.34 65.4 62.4 0.81 0.89 1.05 Zfp874b , Zfp58 chr13 99238000 99249999 96.6 126.5 114.16 123.04 134.02 117.6 0.76 0.93 1.14 Foxd1 , Tmem174 chr13 99570000 99570999 17.39 19.62 14.43 18.06 23.05 24.8 0.89 0.8 0.93 Tmem171 , Fcho2 chr13 107942000 107947999 43.61 49.24 39.47 46.28 40.74 42 0.89 0.85 0.97 Kif2a , Zswim6 chr13 112396000 112406999 75.63 89.27 78.94 89.18 80.41 78 0.85 0.89 1.03 Gpbp1 , Mier3 chr13 112464000 112473999 72.04 97.28 83.61 88.05 99.18 81.6 0.74 0.95 1.22 Gpbp1 , Mier3 chr13 115165000 115167999 26.22 34.83 30.98 33.11 33.24 31.2 0.75 0.94 1.07 Ndufs4 , Arl15 chr14 48641000 48647999 50.51 58.85 52.2 60.2 65.4 51.6 0.86 0.87 1.27 Peli2 , Ktn1 chr14 76053000 76054999 19.04 24.82 16.98 19.94 15.55 18.4 0.77 0.85 0.84 4930564B18Rik , Cog3 chr14 104870000 104873999 42.5 57.64 49.66 58.32 50.93 50.8 0.74 0.85 1 Pou4f1 , chr14 118831000 118833999 24.29 32.83 24.19 30.48 24.12 25.2 0.74 0.79 0.96 Sox21 , Abcc4 chr14 25419000 25423999 43.33 51.24 44.56 47.41 52.54 45.2 0.85 0.94 1.16 Zmiz1 , Rps24 chr14 28238000 28241999 36.71 54.44 50.93 54.94 56.29 61.2 0.67 0.93 0.92 D14Abb1e , chr14 37859000 37861999 16.56 22.82 19.1 21.45 22.52 20.4 0.73 0.89 1.1 Fam190b , Rgr chr14 46685000 46687999 23.74 28.42 29.28 31.61 23.05 28 0.84 0.93 0.82 Ddhd1 , Gm15217 chr14 55826000 55827999 30.91 34.43 30.98 36.88 42.89 40 0.9 0.84 1.07 Jph4 , Dhrs2 chr14 58952000 58962999 86.67 130.1 91.67 100.47 105.08 87.2 0.67 0.91 1.2 Fgf9 , 1700129C05Rik chr14 61823000 61831999 93.29 120.89 84.88 107.24 92.74 125.2 0.77 0.79 0.74 Sacs , Sgcg chr14 64691000 64699999 62.65 72.06 70.03 73.75 71.3 77.6 0.87 0.95 0.92 Prss55 , Rp1l1 chr14 65198000 65199999 24.29 27.62 18.67 24.83 20.37 24.8 0.88 0.75 0.82 Msra , Kif13b chr14 73401000 73403999 34.22 41.63 37.35 42.9 46.1 35.2 0.82 0.87 1.31 Fndc3a , Cysltr2 chr14 75476000 75478999 19.32 26.02 22.07 28.22 24.66 21.2 0.74 0.78 1.16 Lrrc63 , 5031414D18Rik chr14 79057000 79061999 58.24 75.26 58.57 66.23 77.2 60.4 0.77 0.88 1.28 Akap11 , Dgkh

chr14 120835000 120839999 43.88 52.44 55.17 59.83 53.07 56.4 0.84 0.92 0.94 Rap2a , Mbnl2 chr14 121347000 121351999 46.64 52.44 52.2 63.21 58.97 46.8 0.89 0.83 1.26 Farp1 , Ipo5 chr14 122229000 122240999 109.57 137.71 113.74 123.42 134.02 124.4 0.8 0.92 1.08 Dock9 , Ubac2 chr14 122737000 122739999 21.25 28.42 23.77 27.09 20.37 19.2 0.75 0.88 1.06 Zic5 , Clybl chr15 79690000 79708999 112.89 134.5 131.99 142.61 149.04 134.8 0.84 0.93 1.11 Npcd , Apobec3 chr15 25744000 25758999 120.34 158.52 132.41 154.65 149.57 144.8 0.76 0.86 1.03 Fam134b , Myo10 chr15 56075000 56082999 52.72 60.45 68.33 75.26 68.62 78 0.87 0.91 0.88 Sntb1 , Has2 chr15 96446000 96455999 104.05 116.89 105.25 127.56 134.02 119.6 0.89 0.83 1.12 Scaf11 , Slc38a1 chr15 97466000 97476999 107.37 125.7 105.68 114.39 112.04 107.6 0.85 0.92 1.04 Amigo2 , Rpap3 chr15 25763000 25770999 81.42 106.08 87.43 108.74 119.55 107.2 0.77 0.8 1.12 Fam134b , Myo10 chr15 25775000 25786999 115.65 140.91 128.59 142.99 128.13 135.2 0.82 0.9 0.95 Zfp622 , Fam134b chr15 38644000 38648999 59.89 78.06 61.11 65.85 69.69 70 0.77 0.93 1 Baalc , Atp6v1c1 chr15 58447000 58457999 76.45 86.87 78.94 93.69 94.89 90.8 0.88 0.84 1.05 Tmem65 , D15Ertd621e chr15 86625000 86625999 11.04 12.41 8.06 13.17 11.26 12 0.89 0.61 0.94 Fam19a5 , Tbc1d22a chr16 56095000 56100999 44.99 58.85 61.96 69.24 82.02 59.6 0.76 0.89 1.38 Impg2 , Senp7 chr16 44448000 44454999 56.58 63.65 63.24 69.24 63.8 60.4 0.89 0.91 1.06 Wdr52 , Boc chr16 48297000 48303999 43.06 53.64 54.75 64.72 58.43 57.2 0.8 0.85 1.02 Dppa2 , Dppa4 chr16 87587000 87601999 120.06 144.11 141.75 162.93 134.02 159.2 0.83 0.87 0.84 Bach1 , ORF63 chr16 90353000 90363999 73.42 86.47 83.18 90.68 112.04 100 0.85 0.92 1.12 Scaf4 , Hunk chr16 90616000 90619999 30.08 35.23 22.49 28.6 29.49 29.2 0.85 0.79 1.01 2610039C10Rik , Hunk chr16 92473000 92509999 308.85 346.27 369.23 390.2 393.5 367.2 0.89 0.95 1.07 Rcan1 , Clic6 chr16 17270000 17271999 24.29 30.02 31.83 35.75 40.21 34 0.81 0.89 1.18 Tmem191c , Hic2 chr16 22184000 22195999 79.49 90.47 87 93.32 107.76 89.2 0.88 0.93 1.21 Igf2bp2 , Tra2b chr16 23178000 23179999 18.77 21.62 20.8 24.83 19.3 14.8 0.87 0.84 1.3 St6gal1 , Adipoq chr16 38220000 38221999 26.5 46.44 35.65 39.51 30.56 30.4 0.57 0.9 1.01 Nr1i2 , Gsk3b chr16 91165000 91166999 10.76 14.81 12.73 14.3 16.62 11.2 0.73 0.89 1.48 4932438H23Rik , Olig2 chr17 4961000 4969999 56.03 65.25 64.93 73.37 77.2 59.2 0.86 0.88 1.3 Arid1b , chr17 8575000 8585999 85.84 95.67 96.34 103.1 109.36 106.8 0.9 0.93 1.02 T , Prr18 chr17 12644000 12654999 76.73 88.07 87.85 94.82 90.06 91.2 0.87 0.93 0.99 Slc22a3 , Plg chr17 23754000 23760999 45.82 64.05 50.5 57.19 55.75 57.2 0.72 0.88 0.97 Zscan10 , Mmp25 chr17 28518000 28526999 81.97 98.48 96.34 115.52 102.39 108.4 0.83 0.83 0.94 Tulp1 , Fkbp5 chr17 33471000 33475999 35.6 42.03 31.41 46.66 41.28 39.6 0.85 0.67 1.04 Zfp81 , Zfp955b chr17 47980000 47996999 126.69 148.11 151.94 168.95 171.55 168 0.86 0.9 1.02 Mdfi , Foxp4

chr17 84000000 84011999 81.15 91.67 78.09 89.18 81.49 82 0.89 0.88 0.99 Cox7a2l , Kcng3 chr17 7821000 7825999 60.17 76.06 84.03 89.93 120.62 117.2 0.79 0.93 1.03 Gm9992 , Fndc1 chr17 30533000 30536999 37.26 51.64 36.5 39.51 40.21 39.2 0.72 0.92 1.03 Btbd9 , Zfand3 chr17 50432000 50433999 20.7 32.83 27.16 38.38 24.12 30.4 0.63 0.71 0.79 Dazl , chr17 66307000 66313999 77.01 120.49 117.98 141.86 116.33 120.8 0.64 0.83 0.96 Twsg1 , Ankrd12 chr17 69182000 69182999 9.94 14.81 10.61 12.42 9.65 12.8 0.67 0.85 0.75 Epb4.1l3 , L3mbtl4 chr17 71066000 71068999 32.57 46.44 38.62 43.65 45.03 44.4 0.7 0.88 1.01 Tgif1 , Dlgap1 chr17 78989000 78995999 63.48 72.46 72.15 76.01 73.45 63.6 0.88 0.95 1.15 Vit , Strn chr17 79823000 79826999 35.6 48.84 47.96 54.18 49.32 46.4 0.73 0.89 1.06 Fam82a1 , Cdc42ep3 chr17 80364000 80370999 56.86 75.66 57.72 71.49 79.34 60.4 0.75 0.81 1.31 Atl2 , Hnrpll chr17 85595000 85599999 45.26 50.44 47.11 55.31 60.58 49.6 0.9 0.85 1.22 Six3 , 1700106N22Rik chr17 88241000 88243999 30.64 36.43 33.53 38.38 38.6 40 0.84 0.87 0.96 Msh6 , Kcnk12 chr18 24322000 24330999 74.8 87.67 83.18 89.55 102.93 86 0.85 0.93 1.2 Ino80c , Galnt1 chr18 34130000 34156999 186.58 209.36 196.07 211.85 216.05 197.2 0.89 0.93 1.1 D0H4S114 , Epb4.1l4a chr18 60454000 60461999 51.34 61.25 65.36 73 64.33 73.2 0.84 0.9 0.88 Iigp1 , Gm4841 chr18 65930000 65948999 165.33 203.76 176.98 187.76 191.92 177.2 0.81 0.94 1.08 Sec11c , 5330437I02Rik chr18 74670000 74679999 100.19 112.09 98.04 105.36 85.24 92.4 0.89 0.93 0.92 Acaa2 , Myo5b chr18 76503000 76521999 137.45 156.52 142.17 157.28 155.47 149.2 0.88 0.9 1.04 Skor2 , Smad2 chr18 78969000 78976999 64.86 74.86 64.08 68.86 83.63 74.8 0.87 0.93 1.12 Slc14a2 , Setbp1 chr18 85076000 85083999 61.27 70.05 61.54 70.74 64.33 62 0.87 0.87 1.04 Fbxo15 , Cyb5 chr18 10856000 10860999 51.89 64.05 58.99 60.58 70.76 61.2 0.81 0.94 1.16 Gata6 , Mib1 chr18 13127000 13132999 45.82 68.85 47.96 56.44 67.01 67.2 0.67 0.85 1 Impact , chr18 37678000 37682999 42.23 47.24 37.35 44.02 44.5 44.8 0.89 0.85 0.99 Pcdhb22 , Slc25a2 chr18 61760000 61764999 67.9 90.47 84.03 89.55 91.14 86.8 0.75 0.94 1.05 Il17b , Csnk1a1 chr18 75949000 75953999 44.71 62.85 51.35 63.21 64.87 58 0.71 0.81 1.12 Gm672 , Zbtb7c chr18 81601000 81601999 8.28 10.81 6.37 8.28 6.97 8 0.77 0.77 0.87 Sall3 , Galr1 chr18 83219000 83220999 23.74 29.22 20.8 23.33 19.3 27.2 0.81 0.89 0.71 Zfp516 , chr19 30260000 30266999 50.23 58.05 56.87 66.6 64.87 52 0.87 0.85 1.25 Mbl2 , Gldc chr19 37780000 37784999 47.47 58.05 62.39 68.11 77.2 68.4 0.82 0.92 1.13 Cyp26a1 , Myof chr19 55242000 55247999 46.92 61.25 59.42 63.59 64.33 54.4 0.77 0.93 1.18 Gpam , Tectb chr19 37760000 37762999 52.72 65.65 55.6 66.98 83.1 75.2 0.8 0.83 1.1 Cyp26a1 , Cyp26c1 chr19 56242000 56242999 11.59 14.01 14.43 15.43 23.05 8.4 0.83 0.94 2.74 Habp2 , Ppnr chr2 71038000 71042999 42.78 52.04 41.17 48.54 51.47 50.8 0.82 0.85 1.01 Dync1i2 , Cybrd1

chr2 92693000 92804999 927.65 1065.63 1128.06 1217.64 1164.94 1168 0.87 0.93 1 Syt13 , Chst1 chr2 143911000 143932999 208.38 265.01 240.21 267.53 274.48 282 0.79 0.9 0.97 Banf2 , Snx5 chr2 147061000 147065999 48.85 54.84 41.59 44.78 30.56 40.8 0.89 0.93 0.75 Pax1 , Nkx2-2 chr2 166035000 166068999 299.74 335.46 345.46 369.88 327.02 318.4 0.89 0.93 1.03 Sulf2 , Prex1 chr2 5137000 5158999 169.19 190.15 182.07 194.54 202.65 180 0.89 0.94 1.13 Ccdc3 , Camk1d chr2 52264000 52266999 42.5 60.85 65.78 71.49 62.19 89.6 0.7 0.92 0.69 Neb , Arl5a chr2 70464000 70477999 113.99 129.7 124.77 136.21 151.18 121.2 0.88 0.92 1.25 Gorasp2 , Gad1 chr2 74873000 74875999 28.7 32.83 23.34 26.34 23.05 33.6 0.87 0.89 0.69 Hnrnpa3 , Mtx2 chr2 120960000 120965999 42.78 52.44 50.5 54.18 46.1 64 0.82 0.93 0.72 Adal , Lcmt2 chr2 127722000 127765999 329.83 375.09 362.44 420.3 380.09 386.8 0.88 0.86 0.98 Bcl2l11 , Acoxl chr2 152337000 152351999 119.51 135.71 136.66 147.5 143.14 142.4 0.88 0.93 1.01 Defb28 , chr2 157377000 157389999 138.83 179.74 155.76 181.37 202.65 204.8 0.77 0.86 0.99 Peg5 , Nnat chr2 165155000 165168999 98.53 125.3 97.61 112.51 117.41 121.6 0.79 0.87 0.97 Elmo2 , Zfp663 chr2 5654000 5658999 49.96 57.64 58.99 73.37 65.4 65.2 0.87 0.8 1 Camk1d , Cdc123 chr2 9803000 9813999 101.29 125.7 125.62 133.58 131.34 135.2 0.81 0.94 0.97 Gata3 , Taf3 chr2 49824000 49835999 81.42 90.87 95.49 102.72 96.5 90 0.9 0.93 1.07 Lypd6 , Lypd6b chr2 52271000 52274999 38.92 51.64 54.32 58.32 62.72 56.4 0.75 0.93 1.11 Neb , Arl5a chr2 59156000 59157999 23.74 44.03 31.41 33.11 33.77 40.8 0.54 0.95 0.83 Dapl1 , Pkp4 chr2 61641000 61650999 87.22 97.68 115.44 121.91 119.55 110.8 0.89 0.95 1.08 Slc4a10 , Tbr1 chr2 70256000 70265999 79.49 88.47 85.73 97.83 84.17 78.8 0.9 0.88 1.07 Sp5 , Myo3b chr2 73784000 73784999 9.38 11.61 8.49 11.29 10.19 7.2 0.81 0.75 1.41 Atp5g3 , Lnp chr2 74944000 74944999 8.56 10.81 8.06 8.65 6.43 10 0.79 0.93 0.64 Hnrnpa3 , Mtx2 chr2 77816000 77820999 51.34 58.85 49.66 61.71 60.04 51.6 0.87 0.8 1.16 Ube2e3 , Cwc22 chr2 80283000 80284999 25.12 30.82 23.77 32.74 27.88 31.2 0.81 0.73 0.89 Frzb , Dnajc10 chr2 93423000 93427999 32.57 40.03 32.25 35.75 39.14 39.2 0.81 0.9 1 Cd82 , Alx4 chr2 103811000 103813999 26.5 31.22 36.5 39.89 38.06 36.8 0.85 0.92 1.03 Fbxo3 , Lmo2 chr2 104990000 104990999 9.11 13.21 8.06 11.29 11.26 11.6 0.69 0.71 0.97 0610012H03Rik , Wt1 4833422F24Rik , chr2 165243000 165243999 8.56 10.41 10.61 11.66 8.58 12.8 0.82 0.91 0.67 Slc13a3 chr2 170353000 170357999 48.3 57.24 42.44 45.91 50.39 53.2 0.84 0.92 0.95 Dok5 , Pfdn4 chr3 35141000 35150999 60.45 70.85 66.21 77.89 58.43 70.4 0.85 0.85 0.83 Atp11b , Sox2 chr3 36251000 36258999 55.48 77.26 64.51 68.11 68.08 69.6 0.72 0.95 0.98 Qrfpr , Anxa5 chr3 51004000 51023999 168.09 207.36 209.65 243.83 204.25 219.6 0.81 0.86 0.93 Slc7a11 , Ccrn4l

chr3 57705000 57730999 182.44 202.96 197.35 223.89 195.68 190.4 0.9 0.88 1.03 Tsc22d2 , Pfn2 chr3 57741000 57754999 88.87 103.68 99.31 109.12 124.37 116 0.86 0.91 1.07 Tsc22d2 , Pfn2 chr3 82900000 82919999 158.15 192.55 167.21 179.49 166.19 205.6 0.82 0.93 0.81 Sfrp2 , Plrg1 chr3 87055000 87066999 97.43 109.28 97.61 107.24 96.5 91.6 0.89 0.91 1.05 Kirrel , Fcrls chr3 87383000 87393999 85.29 98.88 79.36 98.21 93.82 79.6 0.86 0.81 1.18 Arhgef11 , Etv3 chr3 98081000 98100999 147.66 171.33 155.76 164.81 171.55 158.4 0.86 0.95 1.08 Hmgcs2 , Phgdh chr3 104005000 104005999 18.77 23.62 18.25 25.21 25.2 20.8 0.79 0.72 1.21 Magi3 , Phtf1 chr3 131415000 131416999 28.43 34.83 28.86 32.74 38.6 30.8 0.82 0.88 1.25 Dkk2 , Papss1 chr3 138305000 138325999 166.16 202.56 204.56 230.66 229.45 213.2 0.82 0.89 1.08 Tspan5 , Eif4e chr3 152533000 152540999 81.42 95.67 63.66 77.51 67.55 66 0.85 0.82 1.02 St6galnac5 , Pigk chr3 8639000 8646999 66.24 78.06 73 91.44 72.91 67.6 0.85 0.8 1.08 Hey1 , Stmn2 chr3 37989000 37991999 30.08 42.03 36.92 39.89 44.5 46 0.72 0.93 0.97 Gm5148 , Ankrd50 chr3 52221000 52226999 48.85 66.45 56.02 60.96 78.27 67.6 0.74 0.92 1.16 Foxo1 , Cog6 chr3 66774000 66778999 42.23 60.85 52.63 63.97 51.47 54 0.69 0.82 0.95 Veph1 , Shox2 chr3 88549000 88553999 61.83 68.85 66.21 85.42 71.3 73.6 0.9 0.78 0.97 Syt11 , Rit1 chr3 98223000 98234999 108.19 184.94 151.94 167.82 180.13 179.2 0.59 0.91 1.01 Zfp697 , Hsd3b4 chr3 105658000 105658999 7.45 8.81 4.67 7.53 6.97 8 0.85 0.62 0.87 Rap1a , Adora3 chr3 129174000 129178999 46.09 66.45 50.93 59.83 57.9 40.8 0.69 0.85 1.42 Enpep , Elovl6 B930007M17Rik , chr3 138749000 138750999 21.8 29.22 24.19 29.73 27.88 28 0.75 0.81 1 Rap1gds1 chr3 146541000 146546999 48.85 64.85 58.99 67.35 80.95 67.6 0.75 0.88 1.2 Ttll7 , chr4 129879000 129894999 138 154.92 132.84 159.54 122.23 145.6 0.89 0.83 0.84 Tinagl1 , Gm853 chr4 150062000 150080999 204.52 244.19 230.45 250.23 247.68 221.6 0.84 0.92 1.12 Errfi1 , Slc45a1 chr4 33040000 33086999 338.38 389.5 402.33 452.66 457.83 471.6 0.87 0.89 0.97 Ankrd6 , Rragd chr4 46847000 46863999 141.59 164.13 151.09 168.2 159.22 156.4 0.86 0.9 1.02 Tbc1d2 , Gabbr2 chr4 57863000 57873999 143.25 168.93 144.72 188.89 159.22 159.6 0.85 0.77 1 Akap2 , D630039A03Rik chr4 117079000 117135999 456.24 514.8 491.03 529.05 567.19 484.8 0.89 0.93 1.17 Rnf220 , Tmem53 chr4 117981000 118006999 229.91 261.8 305.57 325.11 355.97 345.6 0.88 0.94 1.03 Hyi , Ptprf chr4 119074000 119083999 72.59 102.48 80.64 91.06 92.74 97.2 0.71 0.89 0.95 Ppih , Ccdc30 chr4 128550000 128567999 135.52 152.92 168.91 185.88 166.19 162.8 0.89 0.91 1.02 Trim62 , chr4 140812000 140826999 123.93 160.12 145.15 170.83 169.94 173.2 0.77 0.85 0.98 Epha2 , Arhgef19 chr4 145661000 145677999 154.56 181.34 150.24 161.8 150.11 154.8 0.85 0.93 0.97 Gm13251 , Gm13242 chr4 33953000 33955999 32.84 45.24 30.13 37.25 41.28 33.6 0.73 0.81 1.23 Cnr1 , Rngtt

chr4 43632000 43639999 65.41 88.47 73.85 80.9 98.11 76 0.74 0.91 1.29 Msmp , Npr2 chr4 47111000 47111999 6.62 11.61 10.19 12.04 8.58 13.2 0.57 0.85 0.65 Col15a1 , Galnt12 chr4 53217000 53226999 66.24 77.26 65.78 76.76 64.87 75.2 0.86 0.86 0.86 Slc44a1 , Abca1 6430704M03Rik , chr4 57041000 57044999 33.67 38.83 34.38 43.65 41.28 33.2 0.87 0.79 1.24 Epb4.1l4b 6430704M03Rik , chr4 57035000 57036999 13.8 21.22 19.95 23.71 19.84 20.4 0.65 0.84 0.97 Epb4.1l4b chr4 58159000 58161999 29.53 47.64 42.44 45.15 47.71 34.4 0.62 0.94 1.39 Txndc8 , Svep1 chr4 98644000 98646999 33.95 48.84 51.78 55.31 47.71 38.8 0.7 0.94 1.23 Angptl3 , Usp1 chr4 111166000 111171999 53.27 59.25 44.14 50.42 45.57 55.2 0.9 0.88 0.83 Spata6 , Bend5 9530002B09Rik , chr4 121689000 121696999 87.22 98.08 84.88 92.56 64.87 77.2 0.89 0.92 0.84 Gm12887 chr5 21495000 21497999 31.46 37.23 30.98 32.74 35.92 32 0.85 0.95 1.12 Slc26a5 , Reln Srpk2 , chr5 23195000 23215999 158.43 194.15 167.64 192.28 167.26 161.2 0.82 0.87 1.04 ENSMUSG00000073777 chr5 100532000 100535999 28.7 33.63 24.62 30.48 25.73 28.8 0.85 0.81 0.89 Tmem150c , Enoph1 chr5 144834000 144847999 94.39 117.69 104.83 111 114.19 107.6 0.8 0.94 1.06 Ocm , Lmtk2 chr5 20460000 20471999 88.32 110.09 98.89 112.51 130.27 116.4 0.8 0.88 1.12 Rsbn1l , Ptpn14 chr5 27735000 27741999 46.64 53.24 54.32 57.95 65.94 59.6 0.88 0.94 1.11 Speer4b , Dpp6 chr5 33387000 33392999 51.89 60.05 57.29 60.96 69.69 63.6 0.86 0.94 1.1 Slc5a1 , Ywhah chr5 65288000 65293999 51.34 66.85 54.75 78.27 68.62 74 0.77 0.7 0.93 Tlr1 , Klf3 chr5 67272000 67283999 94.95 112.09 100.16 103.48 105.61 103.2 0.85 0.95 1.02 Limch1 , Phox2b chr5 72957000 72961999 45.26 57.24 42.86 50.42 52 44.8 0.79 0.85 1.16 Nfxl1 , Zar1 chr5 110556000 110562999 63.48 89.67 75.97 85.04 102.39 70 0.71 0.89 1.46 Chfr , Zfp605 chr5 120104000 120117999 136.35 154.12 157.45 168.57 136.71 136 0.88 0.93 1.01 Tbx3 , chr5 127171000 127175999 37.54 48.04 39.47 44.78 28.95 31.2 0.78 0.88 0.93 Tmem132c , chr5 137283000 137312999 237.92 269.81 288.59 320.97 330.24 335.2 0.88 0.9 0.99 Emid2 , Myl10 chr5 144718000 144722999 55.75 62.85 47.11 59.08 55.22 51.6 0.89 0.8 1.07 Rsph10b2 , Ccz1 chr5 150355000 150362999 77.28 91.67 75.12 80.52 87.92 78.8 0.84 0.93 1.12 Wdr95 , Hsph1 chr5 75383000 75393999 73.97 92.47 83.18 95.95 117.41 87.2 0.8 0.87 1.35 Lnx1 , Chic2 chr5 104602000 104606999 38.36 47.24 41.17 46.28 48.78 49.2 0.81 0.89 0.99 Dmp1 , Dspp chr5 104943000 104949999 58.51 75.66 69.18 76.76 100.79 76 0.77 0.9 1.33 BC005561 , Zfp951 chr5 106431000 106437999 61.83 68.85 54.75 61.71 54.15 55.6 0.9 0.89 0.97 Zfp326 , Barhl2

chr5 106974000 106981999 72.04 80.86 60.69 70.74 48.25 60 0.89 0.86 0.8 Barhl2 , Zfp644 chr5 119312000 119321999 109.85 125.7 114.59 126.81 120.62 121.2 0.87 0.9 1 Tbx3 , Med13l chr5 126310000 126318999 81.42 95.27 76.82 81.65 61.65 71.2 0.85 0.94 0.87 Tmem132b , chr5 126425000 126433999 87.77 100.88 86.58 94.45 69.16 76.4 0.87 0.92 0.91 Tmem132b , A330070K13Rik , chr5 131336000 131339999 40.85 45.64 39.89 44.78 37.53 36 0.9 0.89 1.04 Wbscr17 chr5 135730000 135732999 35.33 41.63 34.38 39.13 53.61 47.6 0.85 0.88 1.13 Fzd9 , chr5 148538000 148551999 123.65 146.91 145.57 158.41 136.71 132.8 0.84 0.92 1.03 Pomp , Flt1 chr5 150702000 150702999 6.35 8.01 7.64 12.42 9.65 7.2 0.79 0.62 1.34 Rxfp2 , B3galtl chr6 145613000 145630999 150.42 180.14 165.94 175.72 185.49 183.2 0.84 0.94 1.01 Rassf8 , Tuba3b chr6 37630000 37638999 76.73 88.47 81.49 90.31 98.64 85.2 0.87 0.9 1.16 Atp6v0c , Akr1d1 chr6 37822000 37830999 98.26 126.9 139.2 147.5 137.78 118.8 0.77 0.94 1.16 Trim24 , Svopl chr6 87389000 87405999 115.37 135.71 125.62 139.6 133.49 148.4 0.85 0.9 0.9 Bmp10 , Arhgap25 chr6 87638000 87668999 240.68 273.01 247.43 298.77 292.17 276 0.88 0.83 1.06 Aplf , Ccdc48 chr6 117257000 117270999 108.19 132.9 137.51 154.27 127.59 136 0.81 0.89 0.94 Zfp637 , Cxcl12 chr6 144816000 144819999 35.88 42.43 30.56 34.99 38.06 42.4 0.85 0.87 0.9 Sox5 , Bcat1 chr6 8570000 8572999 29.81 36.83 36.5 44.02 47.18 44.4 0.81 0.83 1.06 Glcci1 , Ica1 chr6 28887000 28892999 37.26 47.64 42.86 46.28 56.29 44 0.78 0.93 1.28 Lep , Lrrc4 chr6 31593000 31594999 14.35 17.21 17.4 18.44 16.62 19.2 0.83 0.94 0.87 1700012A03Rik , Podxl chr6 37685000 37690999 41.4 51.24 55.17 59.83 42.89 42 0.81 0.92 1.02 Atp6v0c , Akr1d1 chr6 52152000 52156999 87.49 107.68 102.28 114.01 85.78 92 0.81 0.9 0.93 Hoxa4 , chr6 85074000 85077999 47.75 56.84 47.53 54.18 55.75 44.8 0.84 0.88 1.24 Gm5878 , chr6 118379000 118380999 26.5 40.03 40.74 43.27 58.97 55.2 0.66 0.94 1.07 Bms1 , Zfp248 chr6 136391000 136393999 30.91 41.23 29.28 40.64 41.28 37.6 0.75 0.72 1.1 Atf7ip , LOC100502936 chr6 142830000 142833999 27.05 35.63 25.89 37.25 40.21 22.8 0.76 0.69 1.76 Gm766 , St8sia1 chr6 143314000 143316999 40.02 50.04 41.59 46.28 43.96 36 0.8 0.9 1.22 Etnk1 , Sox5 chr6 144433000 144437999 40.85 48.44 39.89 49.29 50.93 39.2 0.84 0.81 1.3 Sox5 , Bcat1 chr6 144452000 144452999 8.56 10.41 7.64 9.41 8.04 7.6 0.82 0.81 1.06 Sox5 , Bcat1 chr7 122550000 122572999 191.27 212.97 179.95 198.3 154.93 169.2 0.9 0.91 0.92 Sox6 , Insc chr7 19447000 19453999 65.41 73.26 65.36 69.24 83.63 60.4 0.89 0.94 1.38 Pglyrp1 , Mill2 chr7 28766000 28776999 118.41 166.53 140.05 155.03 173.16 162 0.71 0.9 1.07 Zfp780b , Psmc4 chr7 29047000 29052999 49.96 68.45 65.36 75.26 60.04 58 0.73 0.87 1.04 Eid2 , chr7 29457000 29462999 43.06 59.65 53.47 63.97 69.69 71.2 0.72 0.84 0.98 C330005M16Rik ,

Fbxo27 chr7 31539000 31540999 29.53 35.23 25.04 30.85 29.49 31.2 0.84 0.81 0.95 Dmkn , Sbsn chr7 89436000 89440999 50.51 62.45 52.63 58.32 57.9 54.8 0.81 0.9 1.06 Adamtsl3 , Sh3gl3 chr7 108962000 108969999 66.52 87.67 64.51 81.65 80.95 89.2 0.76 0.79 0.91 Phox2a , chr7 145161000 145171999 103.78 116.09 84.03 102.35 71.3 86.4 0.89 0.82 0.83 Tcerg1l , Glrx3 chr7 145307000 145320999 138.28 158.92 105.68 125.68 99.71 116.4 0.87 0.84 0.86 Tcerg1l , Glrx3 chr7 145420000 145426999 61.55 71.66 59.42 74.88 49.86 66 0.86 0.79 0.76 Tcerg1l , Glrx3 chr7 149089000 149095999 46.64 53.64 54.75 61.71 61.12 58 0.87 0.89 1.05 Tollip , chr7 6264000 6269999 41.95 49.24 50.93 55.69 66.48 62.4 0.85 0.91 1.07 Zfp583 , Zfp667 chr7 16539000 16543999 68.45 84.47 71.3 87.3 71.3 75.2 0.81 0.82 0.95 Gltscr2 , Ehd2 chr7 26486000 26487999 25.94 29.62 32.68 31.61 35.92 38.8 0.88 0.89 0.93 Tgfb2 , Ccdc97 chr7 28813000 28816999 44.99 53.64 46.68 56.07 54.68 49.2 0.84 0.83 1.11 Zfp780b , Psmc4 chr7 29475000 29487999 118.13 138.11 152.78 165.94 157.08 190.8 0.86 0.92 0.82 Fbxo17 , Fbxo27 chr7 31257000 31259999 27.32 30.42 25.89 27.84 40.74 32.4 0.9 0.93 1.26 Prodh2 , Nphs1 chr7 31837000 31837999 10.21 11.61 10.19 11.66 12.33 10.4 0.88 0.87 1.19 Fxyd7 , chr7 73502000 73509999 87.49 126.5 121.38 132.45 105.08 107.6 0.69 0.92 0.98 Lrrk1 , Chsy1 chr7 74206000 74209999 32.84 41.23 25.89 28.97 36.99 38 0.8 0.89 0.97 Lysmd4 , Adamts17 chr7 89446000 89451999 60.72 68.05 54.75 71.12 81.49 64 0.89 0.77 1.27 Adamtsl3 , Sh3gl3 chr7 118360000 118367999 60.72 72.46 75.97 83.53 90.06 81.2 0.84 0.91 1.11 Eif4g2 , Galntl4 chr7 120827000 120827999 8 10.01 4.67 7.53 11.79 5.6 0.8 0.62 2.11 Spon1 , Far1 chr7 122532000 122533999 16.01 20.42 14.43 16.56 11.26 21.2 0.78 0.87 0.53 Sox6 , Insc chr7 124398000 124398999 11.32 14.41 10.19 12.04 8.04 6.4 0.79 0.85 1.26 Xylt1 , Nucb2 chr7 143368000 143372999 46.37 51.64 44.14 50.8 47.71 50.8 0.9 0.87 0.94 Mgmt , Mki67 chr7 149296000 149305999 70.38 84.07 66.21 77.51 75.59 62 0.84 0.85 1.22 Dusp8 , Krtap5-2 chr8 10479000 10488999 61.55 77.26 58.57 65.47 79.88 67.2 0.8 0.89 1.19 Myo16 , Irs2 chr8 127007000 127014999 58.51 67.25 64.08 70.36 66.48 63.6 0.87 0.91 1.05 Pgbd5 , Cog2 chr8 27363000 27384999 151.25 186.95 165.52 182.5 169.41 173.6 0.81 0.91 0.98 Zfp703 , Thap1 chr8 27432000 27454999 211.14 248.19 227.48 249.47 205.86 208.8 0.85 0.91 0.99 Zfp703 , Thap1 chr8 46093000 46115999 237.09 292.63 224.93 293.87 239.64 229.6 0.81 0.77 1.04 Mtnr1a , Fat1 chr8 46120000 46129999 95.5 108.48 75.97 106.49 82.56 88.4 0.88 0.71 0.93 Mtnr1a , Fat1 chr8 47361000 47366999 49.13 56.04 50.08 53.06 63.26 58 0.88 0.94 1.09 Slc25a4 , Helt chr8 63913000 63923999 80.04 96.47 89.97 98.59 107.22 100 0.83 0.91 1.07 Cbr4 , Sh3rf1 chr8 85553000 85561999 72.87 93.27 70.88 77.14 69.16 65.6 0.78 0.92 1.05 Tbc1d9 , Rnf150

chr8 85917000 85918999 10.21 14.81 10.61 17.31 14.47 17.6 0.69 0.61 0.82 Clgn , Scoc chr8 112797000 112803999 59.89 74.46 56.45 63.59 50.93 58 0.8 0.89 0.88 Vac14 , Hydin chr8 14592000 14597999 59.62 68.85 61.54 67.73 71.3 71.2 0.87 0.91 1 Cln8 , Dlgap2 chr8 27007000 27016999 67.62 79.26 76.39 85.04 85.78 76 0.85 0.9 1.13 Kcnu1 , Hgsnat chr8 37488000 37490999 21.8 24.42 27.59 31.61 33.77 23.6 0.89 0.87 1.43 Lonrf1 , 6430573F11Rik chr8 48121000 48125999 40.57 46.84 36.92 46.66 43.96 37.2 0.87 0.79 1.18 Enpp6 , Stox2 chr8 80617000 80619999 26.5 31.22 25.89 31.23 28.41 22.4 0.85 0.83 1.27 Ednra , Ttc29 chr8 83478000 83479999 20.98 26.02 17.82 20.7 23.05 16.8 0.81 0.86 1.37 Gab1 , Usp38 chr8 87070000 87074999 42.5 47.24 40.32 45.91 42.35 46 0.9 0.88 0.92 Ier2 , Cacna1a chr8 94883000 94891999 137.73 198.55 154.48 182.5 136.71 180.8 0.69 0.85 0.76 Irx6 , Irx5 chr8 111316000 111319999 63.48 73.26 58.57 66.98 86.31 82.4 0.87 0.87 1.05 Pmfbp1 , Zfhx3 chr8 114169000 114178999 71.76 93.67 87 91.81 114.19 116.8 0.77 0.95 0.98 Zfp1 , Ctrb1 chr8 128184000 128185999 14.08 19.21 15.28 17.69 20.91 18.4 0.73 0.86 1.14 Sipa1l2 , 4933403G14Rik chr9 27069000 27085999 117.3 134.9 118.41 126.81 113.12 111.2 0.87 0.93 1.02 Jam3 , Igsf9b 9030425E11Rik , chr9 40330000 40342999 114.82 136.51 124.77 137.34 122.23 124 0.84 0.91 0.99 Gramd1b chr9 121392000 121407999 125.03 139.71 129.87 158.41 158.69 146 0.89 0.82 1.09 Cck , Trak1 chr9 20241000 20243999 50.23 60.85 63.24 67.35 57.9 53.2 0.83 0.94 1.09 Zfp560 , Zfp26 chr9 22010000 22010999 10.49 14.41 9.76 11.29 17.16 13.2 0.73 0.86 1.3 Zfp809 , Zfp872 chr9 34625000 34625999 7.45 8.81 8.49 9.78 6.97 8 0.85 0.87 0.87 St3gal4 , Kirrel3 chr9 40589000 40590999 28.43 36.43 29.71 39.51 30.02 33.6 0.78 0.75 0.89 Hspa8 , 9030425E11Rik chr9 41832000 41835999 28.43 36.03 32.68 34.99 31.09 30.4 0.79 0.93 1.02 Ubash3b , Sorl1 chr9 42747000 42747999 6.9 8.41 7.21 7.9 7.51 7.6 0.82 0.91 0.99 Tbcel , Grik4 chr9 51708000 51715999 66.79 75.26 66.63 71.12 88.99 65.2 0.89 0.94 1.36 Fdx1 , Arhgap20 chr9 60003000 60003999 10.49 12.01 8.06 8.65 8.58 7.2 0.87 0.93 1.19 Thsd4 , Thsd4 chr9 69016000 69020999 33.95 38.83 33.1 38.76 33.77 36 0.87 0.85 0.94 Narg2 , Rora chr9 77518000 77520999 32.29 37.23 33.95 36.12 45.57 42.8 0.87 0.94 1.06 Gclc , Klhl31 7420426K07Rik , chr9 98784000 98785999 30.36 40.43 33.53 39.51 20.37 31.2 0.75 0.85 0.65 2410012M07Rik chr9 100994000 101000999 48.02 62.45 51.78 61.71 61.65 64.8 0.77 0.84 0.95 Msl2 , Ppp2r3a chr9 103777000 103783999 48.85 62.45 47.96 57.57 57.9 47.6 0.78 0.83 1.22 Nphp3 , Tmem108 chr9 108726000 108737999 122.55 154.52 151.09 174.59 171.55 160.8 0.79 0.87 1.07 Slc26a6 , Celsr3 chr9 110578000 110582999 67.62 78.06 78.94 96.7 91.67 75.6 0.87 0.82 1.21 Ccdc12 , Pth1r

chr9 114019000 114024999 54.93 65.25 68.33 84.29 68.08 80 0.84 0.81 0.85 Bcl2a1c , Fbxl2 chr9 116400000 116404999 44.16 49.24 53.47 45.91 55.75 42 0.9 0.9 1.33 Tgfbr2 , chr9 119761000 119761999 10.49 12.41 8.06 10.16 11.79 6.4 0.85 0.79 1.84 Wdr48 , Scn11a chr9 122493000 122499999 64.59 74.86 51.35 65.85 54.15 68.8 0.86 0.78 0.79 Gm9524 , Abhd5 chr9 123259000 123261999 37.54 50.04 40.32 51.55 64.33 48.4 0.75 0.78 1.33 Tmem158 , Lars2 chrY 1635000 1637999 63.48 70.85 44.56 53.81 40.21 47.2 0.9 0.83 0.85 Gm6026 , Zfy2

Figure 14. Decreased and absent H3K9m3 enrichment in emerin null cells is associated with genes involved in cell fate regulation and transcriptional regulation of embryonic stem cells – Oct4-NANOG-Sox2 signaling pathway. Top panel shows top 3 GO biological processes associated with decreased H3K9me3 enrichment in emerin null samples (GREAT web application). Middle panel shows top canonical pathway generated using genes associated with decrease (purple) or absence

(green) of H3K9me3 enrichment in emerin null samples (Ingenuity Pathway Analysis).

Bottom panel shows wig tracks of ChIPseq data around SOX2 locus.

Heterochromatic areas are increasingly recognized to expand along DNA during terminal differentiation of cells (Hawkins et al., 2010). We hypothesized that the nuclear envelope may serve a role in heterochromatic expansion along chromatin, and that the process of expansion of loci could be perturbed in EDMD cells. To test this, we analyzed the expansion of heterochromatic loci as a function of myogenic differentiation (d0, d1, d2) using ChIP-seq data (Figure 15). Proximal promoter analysis (-1000 bp, +200kb bp) identified 1163 peaks in wild-type, 474 in emerin null and 390 peaks shared between conditions. Next, we sought to identify regions where heterochromatin islands increased in length during d0-d1 or d1-d2 transition (2 times or more). In wild type cells, 20% of heterochromatic peaks at gene promoters showed heterochromatic expansion from day

0 to day 1 while, only 7% of emerin null peaks showed this expansion (Figure 15, Panel a). Comparing d1 to d2 data, the expansion of heterochromatic loci in wild-type cells showed a dramatic slowing (5-fold), from 20% to 4% of loci. On the other hand, emerin null cells maintained a relatively constant rate of 7% of loci with expansions.

An example of this differential heterochromatic expansion is visualized in Figure

15, Panel b. Wild-type cells showed a 12 kbp H3K9me3 expansion of the locus during the first day of differentiation, whereas emerin null cells showed only a 2kb expansion.

We extracted the differentially expanded loci, and entered the corresponding genes into

Ingenuity Pathway Analysis. This showed that the differentially expanded heterochromatic loci were predominantly at gene loci involved in embryonic development

(Figure 15, Panel c).

100

101

Figure 15. Emerin null cells show aberrant heterochromatin spreading during myogenic differentiation. Heterochromatic expansion was studied by comparing

ChIP-seq data between time points during myogenic differentiation in both wild-type and emerin null H2K cells. Panel a: The percentage of heterochromatic gene promoter loci showing expansion was calculated between d0 to d1, and d1 to d2. In wild-type cells

20% of loci showed expansion early in differentiation (d0-d1), however this rate slowed quickly to 4% (d1-d2). Emerin null cells showed a loss of these differentiation-specified transitions in heterochromatic expansion. Panel b: Shown is a visualization of ChIP-seq data for an exemplar locus. Emerin null cells (emd) show only marginal expansion (2 kbp) between days 0 and 1, whereas wild-type cells show a 12 kbp expansion. Panel c.

Gene ontology analysis of differentially expanded loci in wild-type vs. emerin null showed enrichment for genes involved in embryonic development.

102

3.3.5 Targeted validation shows inappropriate transcriptional up-regulation of Sox2 pluripotency pathways in emerin null myogenic cells in vitro and in vivo.

The genome-wide H3K9me3 ChIP-seq data suggested an abnormal gain-of- function of cell fate pathways in emerin null cells, due to lack of heterochromatin initiation and/or heterochromatin expansion. One key pathway appeared to be Sox2-Oct4-

NANOG (Figure 14), a well-studied cell fate pathway induced in self-renewal and pluripotency (Loh et al., 2006). ChIP-seq results were validated in vitro using differentiating wild-type and emerin null myogenic cells using qRT-PCR (d0, d1, d4).

Sox2 gene expression showed a dramatic drop from d0 to d1 in wild-type cells, consistent with greater heterochromatin at the respective gene promoter (Figure 16,

Panel a). In contrast, emerin null cells showed smaller decrease in Sox2 mRNA expression from d0 to d1, with a 4-fold increase from d1 to d4. We also studied two downstream targets of Sox2, Gata6 and Hoxb1 (Figure 16, Panels b, c). Gene expression data similarly confirmed ChIP-seq data, where both Gata6 and Hoxb1 showed inappropriate high expression in emerin null cells, and abnormal patterns of expression change during myogenic differentiation (although not at exactly the same time point).

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Figure 16. Sox2 and downstream targets show abnormal gain-of-function in differentiating emerin null myogenic cells. Differentiating wild-type and emerin null

(emd) cells were studied at d0, d1 and d4 following induction of myogenic differentiation. qRT-PCR of Sox2 and two downstream transcriptional targets (Gata6 and Hoxb1) showed down-regulation of Sox2 and its two targets in wild type cells, consistent with the induction of heterochromatic marks at these loci (Figure 14). Emerin null cells show abnormal high expression of Sox2 and targets, consistent with the failure of the heterochromatin marks shown above. Two-way ANOVA with repeated measures, followed by Bonferroni's multiple comparisons test; *** p<0.0005 d1 emd vs wild-type, ** p<0.005, d2 emd vs wild-type n=3. Error bars indicate +\-SEM.

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Our data in emerin null murine myogenic cells suggested that human patients with EDMD would also show aberrant regulation of SOX2 in their muscle. To test this, previously published 125 human muscle biopsy mRNA profiling data set was assessed, including muscle biopsies from EDMD (both emerin null and lamin A/C dominant mutations) (Table 8). qRT-PCR of patient muscle biopsy mRNA was used to compare the steady state mRNA levels of SOX2 in the EDMD biopsies, relative to normal volunteer muscle, and a different muscular dystrophy used as disease control (LGMD2I;

FKRP missense mutations) (Figure 17, Panel a). SOX2 mRNA showed similar steady state levels between normal volunteers and disease control (LGMD2I), but significantly elevated mRNA levels in both lamin (EDMD-AD) and emerin (EDMD-XR) patient muscle.

Unsupervised hierarchical clustering of expression data to test for association of additional transcripts associated with decreased heterochromatin (ChIP-seq data) in the human muscle biopsy data set was used, comparing autosomal dominant (lamin A/C gain of function) forms, with the FKRP/LGMD2I disease control (Figure 17, Panel b;

Table 9). This showed that significant upregulation of multiple SOX2 pathway members together with other ChIP-seq targets was specific for autosomal dominant lamin A/C patients. These data suggest that the gain-of-function of SOX2 pathways seen in murine emerin null myogenic cells are likely shared with human EDMD patients.

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Figure 17. Sox2 and target genes are abnormally expressed in Emery-Dreifuss muscular dystrophy human patient muscle biopsies. mRNA profiles of human muscle biopsies were accessed (Bakay et al. 2006). Panel a: Shown is query of Sox2 mRNAs in muscle biopsies from normal volunteers (Normal; n=13), Emery Dreifuss muscular dystrophy patients (emerin null; n=4 and lamin A/C mutations; n=3), and a muscular dystrophy disease control (FKRP; LGMD2I; n=7). Panel b: Emery Dreifuss muscular dystrophy patient muscle showed disease-specific upregulation of specific gene loci marked by decreased H3K9me3 enrichment in emerin null ChIP-seq data.

Hierarchical clustering of mRNA profiling data was done using Partek Genomic Suite and was limited to genes showing differential expression in EDMD-AD patient biopsies.

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Table 8. Characteristics of the patient muscle biopsies used for mRNA profiling.

EDMD-AD (n=3), EDMD-XR (n=4) and FPLD (n=7) muscle biopsies are shown in the

table. Genes affected and the specific mutation for each patient are shown in last two

columns.

Sample ID Diagnosis File Name [Profile] Sex Age Gene Mutation ED-L-2 EDMD-AD HUD-EDMD-2aUA-s2 F 2 LMNA c.IVS7-1G>T (c.1381G>T) ED-E-1 EDMD-XR HUD-EDMD-3aUA-s2 M 16 EMD exon 4; 18 bp intronic duplication ED-L-3 EDMD-AD HUD-EDMD-4aUA-s2 M 2 LMNA R249Y/+ ED-L-4 EDMD-AD HUD-EDMD-5aUA-s2 M 23 LMNA exon 4/+ ED-E-2 EDMD-XR HUD-EDMD-6aUA-s2 M 35 EMD exon 6; g.1675-1678delTCCG ED-E-3 EDMD-XR HUD-EDMD-7aUA-s2 M 37 EMD exon 6; g.1675-1678delTCCG ED-E-4 EDMD-XR HUD-EDMD-8aUA-s2 M 42 EMD exon 6; g.1675-1678delTCCG FKRP-1a LGMD2I (FKRP) HUD-FKRP-1aUA-s2 F 14 FKRP L276I/L276I FKRP-1b LGMD2I (FKRP) HUD-FKRP-1bUA-s2 F 14 FKRP L276I/L276I FKRP-3 LGMD2I (FKRP) HUD-FKRP-3aUA-s2 F 12 FKRP L276I/L276I FKRP-6a LGMD2I (FKRP) HUD-FKRP-6aUA-s2 M 45 FKRP L276I/L276I FKRP-6b LGMD2I (FKRP) HUD-FKRP-6bUA-s2 M 45 FKRP L276I/L276I FKRP-7a LGMD2I (FKRP) HUD-FKRP-7aUA-s2 M 55 FKRP L276I/L276I FKRP-7b LGMD2I (FKRP) HUD-FKRP-7bUA-s2 M 55 FKRP L276I/L276I

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Table 9. ChIP-seq targets show upregulation in muscle biopsies from EDMD patients. Shown in bold are the transcripts that show disease specific upregulation in

EDMD. Red marked transcripts are shared between EDMD-XR and EDMD-AD.

EDMD-XR FKRP EDMD-AD FKRP gene ID FC FDR FC FDR gene ID FC FDR FC FDR ACTR3 1.617 3.1E-03 1.368 ns ACTR3 2.302 5.2E-05 1.368 ns ADIPOQ 7.255 1.0E-03 3.777 ns ADIPOQ 10.788 1.8E-03 3.777 ns ANXA5 1.723 1.8E-03 1.745 ns ANXA5 2.694 1.9E-04 1.745 ns CHIC2 1.651 1.4E-05 1.043 ns CHIC2 1.812 5.3E-05 1.043 ns EHD2 2.210 3.3E-04 1.242 ns EHD2 3.431 3.1E-04 1.242 ns GCLC 1.607 1.1E-03 -1.077 ns GCLC 1.587 5.2E-03 -1.077 ns ID4 1.532 2.1E-03 1.297 ns ID4 1.678 6.5E-04 1.297 ns KIF2A 1.432 2.4E-03 1.124 ns KIF2A 1.687 5.3E-05 1.124 ns MBIP 1.384 5.6E-04 -1.105 ns MBIP 1.980 7.9E-05 -1.105 ns SOX2 1.519 7.0E-05 -1.070 ns SOX2 1.329 0.04 -1.070 ns STRN 1.517 2.3E-04 -1.007 ns STRN 1.424 2.6E-03 -1.007 ns TGFB1 1.952 4.9E-04 1.911 6.92E-05 TGFB1 3.090 1.4E-04 1.911 ns TGIF1 1.665 1.0E-03 1.880 2.01E-06 TGIF1 2.512 1.6E-08 1.880 5.7E-07 TMEM158 2.560 1.9E-04 1.812 9.46E-03 TMEM158 3.010 7.5E-05 1.812 ns ATP6V1C1 1.396 2.9E-03 -1.222 ns ADAMTSL3 2.067 1.7E-03 1.015 ns ATXN1 1.533 9.0E-04 -1.021 ns ARID5B 1.673 2.8E-03 1.145 ns CASP8 1.513 8.9E-05 1.109 ns ATP11B 1.902 6.2E-03 1.019 ns CHFR 1.454 6.3E-05 1.410 1.40E-05 BACH1 1.577 1.5E-03 1.050 ns DAPK1 1.523 1.1E-03 -1.200 ns BEND5 -1.670 5.4E-03 -1.229 ns DYNC1I2 1.422 3.1E-03 1.252 ns CCL2 7.725 2.2E-03 3.098 ns FBXO17 1.872 8.6E-04 -1.026 ns CDC42EP3 1.920 2.8E-03 1.172 ns FGF9 1.644 2.7E-03 1.109 ns CDK19 1.598 4.2E-03 -1.196 ns GFPT2 1.331 2.2E-03 -1.078 ns CHST1 -1.226 9.5E-04 1.067 ns GPATCH2 1.433 4.9E-04 1.031 ns CHSY1 2.022 8.4E-06 1.112 ns GSK3B 1.507 4.1E-03 -1.075 ns DAZL -1.718 5.2E-03 -1.614 6.3E-04 HOXA5 1.462 8.3E-05 -1.316 5.89E-03 DNAJC10 1.506 4.7E-03 -1.044 ns HSD17B6 1.415 1.5E-03 1.161 ns ELOVL6 -1.312 6.5E-03 -1.092 ns KIF13A 1.436 3.7E-03 -1.218 ns ERH 1.448 5.9E-04 1.261 3.63E-03 MSRA 1.434 1.8E-03 1.113 ns FAT1 3.878 2.0E-04 1.686 ns

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PHACTR1 -1.801 2.3E-03 -1.260 ns FOXO1 2.086 1.8E-03 1.128 ns RAP2A 2.221 2.7E-04 1.162 ns GAS1 3.551 3.4E-04 1.781 ns RIT1 1.334 3.2E-03 1.015 ns HGSNAT 1.944 1.3E-03 1.048 ns SLC30A10 1.501 3.5E-03 -1.374 ns IMPG2 -1.388 4.0E-03 -1.199 ns SLC44A1 1.914 1.8E-04 -1.136 ns JAM3 3.226 6.7E-05 1.759 ns SNTB1 -1.857 1.4E-03 -1.243 ns LRRK1 1.818 2.4E-03 1.424 ns TBC1D2 3.800 4.1E-04 -5.939 ns MSX2 1.461 3.5E-03 1.055 ns THAP1 1.435 4.1E-03 -1.119 ns PAPSS1 1.724 2.1E-03 1.013 ns TINAGL1 -1.617 3.1E-03 1.263 ns PELI2 2.346 1.1E-03 -1.064 ns TRAK1 1.412 2.4E-03 1.116 ns PMP22 2.077 5.7E-03 1.353 ns TSPAN5 -1.342 4.1E-03 -1.179 ns PNMA1 2.184 1.8E-03 1.453 ns ZFHX3 1.789 7.2E-04 1.017 ns RAP1GDS1 1.572 4.3E-03 -1.079 ns SH3GL3 1.754 2.7E-03 -1.657 ns SHOX2 2.320 3.9E-04 1.130 ns SLC38A1 3.180 1.3E-05 -1.812 ns SLC7A11 1.360 4.4E-03 -1.473 6.1E-04 SPON1 5.373 7.8E-04 1.077 ns TBX3 2.088 1.9E-03 -1.563 ns TGFBR2 2.639 6.8E-04 1.966 3.7E-03 TMX1 1.783 3.2E-03 1.035 ns TRIM9 3.543 1.1E-04 1.682 ns VAC14 1.588 1.6E-04 -1.209 ns XYLT1 2.006 1.2E-03 -1.071 ns ZMIZ1 2.597 1.2E-05 1.193 ns

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3.3.6 Gain-of-function of Sox2 cell fate perturbations are specific to the disease-type of lamin A/C mutation.

We used the same patient-derived MyoD-converted cells (WT, EDMD LMNA p.H222P and FPLD LMNA p.R482W mutation) described above to carry out targeted validations of SOX2 pluripotency pathway persistence four days after triggering myogenesis in vitro. SOX2, GATA6, and HOXB1 loci were studied using H3K9me3

ChIP to assess heterochromatin levels at gene promoters, as well as mRNA qRT-PCR for gene expression (Figure 18). Cells carrying the EDMD LMNA p.H222P missense mutation showed reductions in heterochromatin at GATA6 and HOXB1 relative to normal control (Figure 18; Panel a and B). SOX2 was not tested (failure of qPCR). mRNA transcript levels showed upregulation relative to normal control as well as FPLD cells

(Figure 18; Panel c, D, E). Data suggest that both EDMD-AD (LMNA missense gain-of- function) and EDMD-XR (emerin loss-of-function) share persistence of the SOX2- associated pluripotency pathways due to alterations of epigenetic programming. Finally, these data are consistent with specific mutations of lamin A/C causing a differential gain of function of abnormal initiation and spreading of heterochromatin marks, thus altering cell fate in a mutation-specific manner.

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Figure 18. Sox2 and downstream targets show abnormal gain-of-function in differentiating emerin null myogenic cells and in Emery-Dreifuss muscular dystrophy human patient muscle biopsies. Panels a, b, c: Differentiating wild-type and emerin null (XR-EDMD) cells were studied at d0, d1 and d4 following induction of myogenic differentiation. qRT-PCR of Sox2 (Panel a) and two downstream transcriptional targets [Gata6 (b) and Hoxb1(c)] showed down-regulation of Sox2 and its two targets in wild type cells while emerin null cells show abnormal high expression of

Sox2 and targets. Two-way ANOVA with repeated measures, followed by Bonferroni's multiple comparisons test; *** p<0.0005, ** p<0.005; n=3. Error bars indicate +\-SEM.

Panels d, e: Muscle biopsy mRNA profiles were accessed (Bakay et al., 2006) (Normal, n=13; EDMD-XR, n=4; EDMD-AD, n=3; FKRP, n=7). Panel d shows query of the Sox2 mRNA showed abnormal upregulation in both EDMD-XR and EDMD-AD patient muscle.

Panel e shows hierarchical clustering of mRNA transcripts corresponding to H3K9me3

ChIP-seq loci that showed specific decreases in heterochromatin in emerin null H2K myogenic cells (n=42). The large majority of these murine ChIP-seq loci showed upregulation of the corresponding mRNA transcripts in EDMD patient muscle biopsies.

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3.3.7 Wild-type lamin A protein directly interacts with SOX2 locus and this interaction is altered by LMNA missense mutation

The ChIPseq and ChIP-qPCR imply that lamin A directly interacts with the SOX2 locus and that mutations of lamin A or emerin disrupt this interaction, but this interaction could be mediated via other proteins. To test for direct protein/DNA interactions between wild-type and mutant lamin A proteins with the SOX2 genomic locus, we returned to the

DamID method. First, we queried the DamID-seq data presented earlier for associations of lamin A protein with the SOX2 gene locus (Figure 19, Panel a). This revealed WT lamin A to show association with the SOX2 upstream region, and relative loss of this association with the p.R453W mutation. We then used the Dam-ID methyl-adenine chromatin from the same myogenic time points to carry out targeted analysis of SOX2 locus enrichment (Figure 19, Panel b). This confirmed the genome-wide data, with relative loss of lamin A protein association with the SOX2 locus for both the EDMD

(p.R453W) and FPLD (p.R482W) LMNA mutations. Collectively, these data show that tethering of SOX2 genomic region to the nuclear lamina is impaired when LMNA is mutated, as well as in emerin null cells.

3.3.8 Overexpression of SOX2 perturbs myogenesis

The epigenetic studies above suggest that three pathways are perturbed by both

EDMD LMNA mutations and emerin loss-of-function: exit from pluripotency programs

(SOX2), exit from cell cycle (E2F, Rb1), and induction of myogenesis. These three pathways are likely interrelated and coordinated during myogenic differentiation. To determine if over-expression of SOX2 was sufficient to perturb myogenesis, SOX2

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overexpression in normal human myoblasts was driven by transfection with a CMV- driven SOX2 expression construct, and a marker for commitment to the myogenic lineage, MF20+ (myosin), scored. This showed that cells transfected with the SOX2 vector failed to enter the myogenic lineage compared to those transfected with empty vector (Figure 19; Panel c).

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Figure 19. Allele-specific perturbation of direct interaction of LMNA with the SOX2 genomic locus and inhibitory effects of SOX2 overexpression on muscle differentiation. The direct link between lamin A mutations and SOX2 locus was studied by DamID-seq and DamID-qPCR using primers specific for SOX2 locus. Effects of SOX2 on myogenic differentiation were assessed by overexpression experiments. Panel a: Wig tracks of lamin A enrichment on SOX2 locus showing reduction in EDMD lamin A-SOX2 association. Panel b: Validation experiment of lamin A enrichment on SOX2 locus using

DamID-qPCR. The analysis compared binding abilities of WT and two mutations of lamin

A and showed decreased enrichment potential for the mutated lamins. Panel c: SOX2 overexpression significantly (p=0.02) reduced differentiation potential of human immortalized myoblasts. Differentiation potential was assessed by the ability of transfected cells (GFP+) to form myosin heavy chain (MF20) positive myotubes and was calculated as ratio of MF20+/total number of transfected myoblasts (GFP+ DAPI cells).

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4 Chapter: Data interpretation and integration into models of pathogenesis

We report the genome-wide assessment of physical interactions between the lamin A protein and regions of the genome during myogenesis using the DamID-seq approach. The mapping of LADs was done for WT lamin A protein and two human disease causing missense mutations; p.R453W causing Emery-Dreifuss Muscular

Dystrophy, and p.R482W causing Familial Partial Lipodystrophy. The data showed gain-of-function properties of mutant lamins, consistent with the observed dominant inheritance pattern of these mutations (and most laminopathies). WT lamin A showed direct interactions with about 11,000 regions of the genome, whereas EDMD p.R453W increased interactions to 19,000 and FPLD p.R482W to 21,000 loci. The mutant lamins also showed dominant-negative features, with loss of the euchromatin transition of myogenic loci (genes showing de novo expression in the developmental transition to myotubes), and this finding was mutation-specific (seen by the EDMD mutation, but not

FPLD) (Fig. 9).

Disease-specific disruptions of both the cell cycle and myogenesis pathways at the mRNA and protein levels had been previously observed in EDMD patient muscle biopsies (carrying either lamin A/C missense or emerin null mutations) (Bakay et al.,

2006), and had been validated in emerin null mice by studies of in vivo myogenesis

(Melcon et al., 2006). Data presented here found that these perturbations were likely the result of mutation specific altered LADs, and downstream consequences on DNA

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methylation for at the genomic loci for members of these pathways (Fig. 9). For example, the genomic loci of both CDK1 and RB1 showed LAD sites in WT consistent with exit from cell cycle, but these LADs were lost with EDMD p.R453W lamin A.

Myogenic loci should show increasing euchromatin formation and mRNA expression during myogenesis, and this was seen by both DamID-seq and DNA methylation data.

In contrast, there was broad loss of this transition in EDMD by both DamID-seq

(p.R453W) and in genome-wide methylation studies from cells of a EDMD patient with a second mutation (p.H222P).

EDMD has features of a syndrome, with muscular dystrophy and marked muscle wasting, as well as cardiac conduction block. The disease shows genetic heterogeneity, caused by both dominant LMNA mutations, and X-linked recessive EMD mutations, where the encoded lamin A and emerin are interacting biochemical partners at the nuclear envelope. To integrate the epigenetic model across both disorders, genome- wide H3K9me3 (heterochromatin mark) ChIP-seq was carried out at three time points during the in vitro myogenic differentiation of emerin null and wild-type murine myogenic cells. This showed similar alterations of the epigenetic transitions of both cell cycle and myogenesis loci, as seen with the gain-of-function lamin A DamID data. The increased time series permitted an additional assessment of epigenetic function, namely spreading of heterochromatin laterally as a function of time (Fig. 15). For example, the initiation of heterochromatin at the Cdk1 locus appeared to fail entirely in emerin null cells, whereas

Rb1 heterochromatin initiated but showed relative inability to spread laterally (Fig. 13;

Fig. 15).

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In carrying out a genome-wide prioritization of cellular functions altered in the emerin null ChIP-seq time series, we noted significant alterations of Sox2-associated pluripotency pathways that we had not recognized in our previous studies (Bakay et al.,

2006; Melcon et al., 2006). Sox2 loci showed progressive gain of heterochromatin during myogenesis by both DamID-seq and ChIP-seq, but lamin A mutations and emerin loss of function both caused persistent expression of Sox2 pathway loci due to alterations of epigenetic programming. This added abnormal persistence of pluripotency programs to the previously discuss myogenesis and cell cycle functions, all driven by alterations of

LADs, histone associations, and DNA methylation patterns caused by lamin A missense and emerin loss of function. Overexpression of Sox2 protein in differentiating myogenic cells delayed their differentiation (Fig. 19c), suggesting that persistence of pluripotency may be upstream of the myogenesis loci alterations. This would require further experiments to validate.

We believe that the epigenetic effects of nuclear envelope disorders provide a unifying molecular model able to explain the dramatic range of clinical phenotypes seen with different lamin A/C missense mutations, as well as the clinical phenocopies seen with other nuclear envelope proteins, including emerin deficiency. Specific lamin A mutations have gain-of-function (promiscuous LADs) and dominant-negative (inability to initiate and spread of LADs) consequences during the development of cell fate. Different mutations affect different cell lineages and cell-specific LADs. We provided evidence for allele-specificity, where the epigenetics of myogenic loci were altered by a muscular

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dystrophy lamin A mutation (EDMD p.R453W), but not an adipose tissue disorder (FPLD p.R482W).

While lineage-committed cells share an identical genome with embryonic stem cells (ESCs), the epigenomic landscape is dramatically different with most changes arising from redistribution of H3K9me3 and H3K27me3 marks (Hawkins et al., 2010).

Our model suggests that many of these changes are driven by the increased expression of lamin A seen during terminal differentiation of myoblasts and other cells seen by us and others (Bakay et al., 2006; Constantinescu et al., 2006). This model is consistent with more general findings in pluripotent stem cells, where epigenetic changes appear to drive cell fate memory, with large fractions of the genome associated with heterochromatin (H3K9m2), facilitated by chromatin nuclear lamina re-association after mitosis in lineage-committed cells (Wen et al., 2009). This is also supported by the findings of Towbin at al. showing step-wise formation of H3K9me3 heterochromatic foci at the nuclear periphery using H3k9me1/2 as substrate (Towbin et al., 2012). Taken together, our data suggest that in the presence of LMNA and EMD mutations, and by extension all nuclear envelope disorders, there is inappropriate nuclear lamina- heterochromatin association upon differentiation. This results in three parallel events that may have a cumulative effect: slowing of exit from cell cycle (heterochromatinizatoin of

Cdk1), slowing of exit from pluripotency programs (heterochromatinization of Sox2) and poorly coordinated induction of terminal differentiation myogenic programs.

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5 Chapter: Discussion

5.1 Choice of techniques: DamID vs. ChIP

DNA adenine methyltransferase identification (Dam-ID) assay is a technique that has been successfully used to study in vivo genome binding sites of a variety of DNA- binding and chromatin proteins (Braunschweig et al., 2009; Pickersgill et al., 2006; van

Steensel and Henikoff, 2000). DamID relies on prokaryotic adenine methyltransferase

Dam that methylates adenines within GATC sequence that are specifically recognized by

DpnI enzyme (van Steensel and Henikoff, 2000; Vogel et al., 2007). When Dam is fused to a protein of interest, direct interaction of that protein and DNA brings the bacterial

Dam enzyme in close proximity of the eukaryotic genome leading to methylation of the

GATC sites nearby (a modification not normally seen in eukaryotic DNA). Thus, by identifying the newly methylated GATC sites, DamID provides a direct method for studying protein-DNA interactions.

Chromatin immunoprecipitation (ChIP) is a more commonly utilized method of defining protein/DNA interactions. The major difference between ChIP and DamID is in the way sample is prepared and genomic regions of interest identified. ChIP requires formaldehyde crosslinking of the sample to introduce covalent bonds between the target protein and bound DNA, thus identifying more stable interactions. DamID is an enzymatic modification of DNA that is more capable of detecting transient protein/DNA interactions as well as stable ones. The second major difference is in the way these two techniques identify genomic domains that are enriched for the protein of interest. ChIP

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requires the use of antibodies against of protein of interest, with pull down of the cross- linked DNA/protein complexes. The procedure is dependent on the quality of the antibody used, and the ability to pull down low solubility heterochromatic domains. The cross-linking procedure in ChIP can also induce sequencing artifacts (Zhou, 2012).

DamID avoids these artefacts by direct methylation of the genomic DNA. Furthermore, since DamID is not dependent on an antibody, it is more broadly applicable to proteins where adequate antibodies may not exist (van Bemmel et al., 2013). DamID is also useful for testing the effects of mutations on DNA binding, or for studying different isoforms of the same protein (Greil et al., 2006). For our study, we were particularly interested in the ability of DamID to analyze genome binding of different disease causing mutant forms of lamin A/C protein (Normal, p.R453W-EDMD, p.R482W-FPLD).

The DamID method also has limitations. First, the fusion of the bacterial enzyme to the protein of interest may alter the activity and/or DNA binding properties of the resulting fusion protein. Second, DamID is not suitable for studying posttranslational modifications (PTMs) of the target protein. This is in contrast to to ChIP that often uses

PTM-specific antibodies to identify genomic regions of enrichment. DamID is an enzymatic modification of DNA that occurs over the entire time period of the experiment, making DamID less ideal for dissecting changes at a specific point in time. Additionally, during replication Dam methylation events get erased, limiting DamID applications in fast replicating cells (early embryonic development) (Askjaer et al., 2014). Comparative studies have shown that both DamID and ChIP techniques produce comparable genome wide binding profiles, suggesting that many of these potential limitations may not have a

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large impact on the results and data interpretation (Moorman et al., 2006; Nègre et al.,

2006).

5.2 Lamin A mutations show tissue specificity in chromatin tethering to nuclear lamina

The nuclear envelope is the defining membrane bound structure that sets eukaryotic cells apart from prokaryotic cells (Hetzer, 2010). Vertebrate nuclei are highly organized structures in which genetic material is arranged in a way that enables diverse biological processes such as DNA replication, transcription and RNA processing to occur

(Dechat et al., 2008). The nuclear envelope is increasingly recognized as a site for transcriptional regulation of chromatin and developmental programming of cells

(Anderson et al., 2009). Proper spatial- and time-dependent association of transcriptional regulators with the nuclear envelope and with associated chromatin ensures normal cell functioning. Indeed, the evolution of the nuclear envelope seems to have enabled more complex developmental programs, and thus more complex species.

The major components of the nuclear envelope are the lamin intermediate filament proteins type V. Two main intermediate filaments are type A (lamin A and lamin

C) encoded by LMNA gene (Fisher et al., 1986; McKeon et al., 1986) and type B (B1 and B2) encoded by LMNB1 and LMNB2 (Lin and Worman, 1993; Peter et al., 1989;

Vorburger et al., 1989). All lamins but lamin C are subjected to post-translational modification process to produces mature forms. Mature forms polymerize into separate networks parallel to the inner nuclear membrane (Dechat et al., 2008). Lamin A/C and lamin B are differently regulated during development. While B type lamins show constitutive expression in all cells, lamin A/C expression is developmentally regulated

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and it shows upregulation with differentiation (Constantinescu et al., 2006; Röber et al.,

1989).

Identification of mutations in the LMNA gene and other nuclear envelope components have provided a new means of studying the importance and biochemical role of the nuclear envelope. The first laminopathy defined at the molecular level was an

X-linked recessive muscular dystrophy with cardiac conduction defect called Emery-

Dreifuss Muscular Dystrophy (EDMD), caused by loss-of-function mutations of the EMD gene encoding emerin (Bione et al., 1994; Emery and Dreifuss, 1966). Since then more than 13 different phenotypes have been described as consequence of mutations in genes encoding 10 nuclear envelope components (emerin, lamin A/C, Lamin B1, LBR,

MAN1, nesprins (SYNE1, SYNE1, SYNE2), TMEM43, TMPO, FHL1) (Capell and Collins,

2006; Maraldi et al., 2011; Schreiber and Kennedy, 2013; Worman and Bonne, 2007).

LMNA mutations causing skeletal muscle phenotypes share clinical features with gene mutations of multiple other nuclear envelope components (emerin, nesprin 1 and 2,

FHL1), and it might be expected that molecular pathogenesis might also be shared between these genetically distinct disorders. Consistent with this, we previously showed shared mRNA expression fingerprints in muscle biopsies from muscular dystrophy patients with both LMNA (AD-EDMD) and EMD mutations (XR-EDMD) suggesting a failure of appropriate induction of the myogenic terminal differentiation program (Bakay et al. 2006). Mechanistic studies bolstering these findings were carried out in a mouse knock out model of emerin deficiency, where the inappropriate timing of myogenic lineage-specific genes was confirmed (Melcon et al. 2006). This second study also showed evidence of failure to appropriately exit the cell cycle (delayed suppression of

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E2F pathways) (Melcon et al 2006). Thus, two of the three key programs that must be orchestrated during terminal differentiation and commitment to the myogenic lineage were perturbed; induction of the myogenic cell fate program, and exit from the cell cycle.

To our knowledge, no study of the third key program, inactivation of pluripotency programs, has been reported. Also, no genome-wide study of allele-specific and lineage-specific programs has been reported.

Accumulating evidence indicates that lamin A/C protein is involved in euchromatin/heterochromatin transitions at the nuclear envelope during development.

This is consistent with the abundance and timing of LMNA gene expression: the lamin

A/C is a prominent component of nuclear envelope and expression of the LMNA gene appears tied to transition to terminal differentiation state. The actual point of interaction between chromatin and the nuclear lamina (envelope) is through lamina-associated domains (LADs) that contain discrete DNA regions responsible for these interactions –

LAS (lamina associated sequences). LADs are very large domains (on average hundreds kbp) covering nearly 35% of the genome. Genes within LADs are generally, but not limited to, transcriptionally inactive domains. Peric-Hupkes et al. compared mouse embryonic stem (ES) cells with differentiated cell types and showed that genes interact with the nuclear lamina in a cell-type specific fashion. Upon differentiation, cell type specific genes lose the nuclear lamina association while stem cell-specific loci move to nuclear periphery and establish new nuclear lamina interactions (Peric-Hupkes et al.,

2010).

Genomic regions associated with the nuclear lamina show enrichment in repressive heterochromatin marks and are associated with transcriptionally inactive

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domains (Kind and van Steensel, 2010). Transcriptional repositioning of silent genes to the nuclear periphery (Reddy et al., 2008) together with sequestration of transcriptional factors by the interior (Yao et al., 2011) indicates that the nuclear lamina controls transcription, and likely cell fate. As such, the nuclear lamina appears important for heterochromatin formation. Towbin et al., showed that through a self-enforcing mechanism H3K9me3 heterochromatin is established at the periphery in a step-wise fashion that requires H3K9me1/2 as a substrate and the action of peripherally (nuclear envelope) localized SET-25 (Towbin et al., 2012). Lamin A/C and LBR are necessary for heterochromatin tethering to nuclear lamina and the absence of these two proteins leads to loss of peripheral heterochromatin and inversion of chromatin structure (Solovei et al.,

2013). Finally, a human EDMD LMNA mutation expressed in C. elegans impairs tissue- specific reorganization of heterochromatin and abnormal retention of integrated gene arrays bearing a muscle-specific gene at the nuclear periphery (Mattout et al., 2011). It should be noted that release from the nuclear lamina is not sufficient for transcriptional activation of the genomic region (Lund et al., 2013). Down-regulation of lamin A/C remodels repressive and permissive chromatin and promotes transcriptional potential but requires additional factors for activation (Lund et al., 2013).

In this dissertation we carry out a genome-wide analysis of interactions between lamin A protein and chromatin domains during human myogenic cells differentiation via

DamID sequencing. Here, we mapped genomic interactions of WT lamin A and two lamin A mutations that are known to cause human disease (p.R453W causing Emery-

Dreifuss Muscular Dystrophy, and p.R482W causing Familial Partial Lipodystrophy). We showed that these missense mutations in the lamin A protein caused promiscuous

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formation of novel, but shorter lamina associated domains (LADs). The mutations in lamin A showed interactions with approximately twice as many genomic domains relative to WT (WT ~11,000, EDMD p.R453W ~19,000 and FPLD p.R482W ~21,000 loci). These novel LADs formed by mutations in lamin A were much shorter on average when compared to WT (Table 4). On the other hand, the mutations in lamin A causing EDMD specifically failed to release the myogenic loci during myoblast-myotube transition causing inadequate chromatin remodeling during muscle differentiation (Figure 9).

Finally, these data suggest that different missense mutations interact with different lineage-specific networks involved in chromatin remodeling and transcriptional regulation that drive tissue specificity observed with laminopathies.

We have previously shown that disease-specific mutations of lamin A/C show perturbations in cell cycle and myogenesis pathways at the level of mRNA in EDMD patient muscle biopsies (EDMD-AD caused by mutations in lamin A and EDMD-XR caused by loss of function mutation in emerin), as well as emerin null mice (Bakay et al.,

2006; Melcon et al., 2006). Here, we extended this model by showing that perturbations seen at the mRNA level were likely downstream of the failed chromatin remodeling at the nuclear periphery caused by mutations in lamin A. We validated this further, by showing that a different EDMD lamin A mutation (p.H222P) changed the normal DNA methylation profile of myogenic loci during myogenic induction. Finally, we showed that cell cycle pathways were perturbed as well. Using examples of RB1 and CDK1 (cell cycle genes), we show that while these loci form lamina associations with WT lamin A upon myogenic induction, these interactions are lost when with EDMD p.R453W lamin A.

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5.3 Failed epigenetic remodeling in emerin null myogenic cells

Dominant missense mutations of LMNA cause EDMD, as do loss-of-function mutations in the emerin gene (EMD). Above, we discussed perturbations of chromatin- nuclear envelope interactions due to LMNA mutations. Here, we also describe studies of emerin null cells during early myogenic differentiation, at the point of exiting the cell cycle and commitment to the myogenic lineage. We show that the process of heterochromatin spreading is perturbed by loss of emerin at the nuclear envelope, as was also seen with

LMNA mutations. The perturbation of epigenetic silencing led to an inappropriate persistent expression of the SOX2 locus, and downstream perturbations of normal cell fate evidenced by broad yet inappropriate activation of the Sox2 pathways. We hypothesized that the locus-specific perturbations of heterochromatin formation we found in emerin null cells could be extended to lamin A/C mutations, where lamin A/C missense mutations might lead to mutation-specific abnormalities of cell fate. To test this, cells from patients with dominant lamin A/C mutations causing Emery-Dreifuss muscular dystrophy (p.H222P) and familial partial lipodystrophy (p.R482W) were studied for inappropriate regulation of the Sox2 pathway in the emerin null cells. Consistent with our model, the p.H222P EDMD mutation cells showed abnormal activation of Sox2 pathways, while the p.R482W FPLD cells did not. We believe that this provides an explanation for clinical variation among laminopathies where mutation-specific activation of tissue specific transcriptional networks is disrupted but LMNA mutations.

This mutation-specific perturbation of the Sox2 cell fate pathways was further confirmed in patient muscle biopsies having distinct EDMD lamin A/C mutations.

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Namely, the perturbations of heterochromatin formation at specific gene loci seen in emerin null ChIP-seq data showed disease-specific transcriptional activation in autosomal dominant EDMD (LMNA) patient muscle biopsies. Direct interactions between wild-type LMNA protein and the SOX2 gene locus were shown by Dam-ID methods, and disease-causing LMNA mutations perturbed these interactions.

Furthermore, via overexpression experiments, we showed that increased expression of

Sox2 protein in differentiating myogenic cells delays differentiation (Figure 19). These data suggest that persistence of pluripotency programs may be upstream of the perturbation of myogenic and cell cycle pathways.

5.4 Building an integrated model of nuclear envelope molecular pathogenesis

The data presented here may answer some previously unresolved questions regarding nuclear envelope disorders. First, how do the different lamin A/C missense mutations cause tissue-restricted gain-of-function? Our model suggests that specific missense mutations perturb gene-silencing programs during terminal differentiation, leading to persistent expression of inappropriate cell fate programs. Our finding that the

EDMD LMNA p.H222P mutation caused persistent expression of Sox2 pathways during myogenesis, whereas FLPD p.R482W cells did not, provides the proof of principle for this model. By extension, the p.R482W mutation likely causes abnormalities of silencing of specific key adipogenesis loci during development of fat tissue that are irrelevant to the myogenic lineage. In support of this, the effects of EDMD versus FPLD mutations on the three dimensional structure of lamin A/C protein have been predicted, where perturbations in the 3D structure may disrupt different tissue-specific chromatin-lamina

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interactions (Krimm et al., 2002). Furthermore, Oldeburg et al. show that p.R482W

LMNA mutation could inhibit adipocyte differentiation but also stimulate a myogenic program in adipose precursors through altered interaction with FXR1P (Oldenburg et al.,

2014). This could explain both lipoatrophy and muscle hypertrophy in FPLD patients, but also explain why these cells were not affected when differentiated into myogenic lineage

(Oldenburg et al., 2014). This model is also attractive in providing a ‘gain-of-function’ mechanism – Sox2 expression becomes the gain-of-function downstream of the p.H222P mutation during myogenesis in EDMD muscle development or regeneration.

Our data also suggest a molecular basis for the phenocopies that result from loss-of-function of emerin and certain gain-of-function missense mutations of lamin A/C – both causing the tissue-restricted phenotype of EDMD. Our data suggest that inappropriate silencing, and hence persistent expression, of Sox2 pathways and other key loci detected by ChIP-seq could be unifying molecular features (Figure 20).

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Figure 20. Heterochromatin formation and cell fate maintenance during muscle differentiation in normal and EDMD muscle. During normal myogenic differentiation heterochromatin is formed and expanded on cell fate loci causing heterochromatinization of these genomic domains (top panel. In EDMD myoblasts, perturbations in nuclear envelope components (loss of emerin or mutation in lamin A/C) change chromatin remodeling on cells fate loci leading to decrease heterochromatin enrichment and reduced heterochromatin spreading (lower panel).

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Genome wide ChIP-seq analysis of emerin null myogenic cells showed reduced heterochromatin (H3K9me3) enrichment on genomic regions associated with regulation of development and maintenance of cell fate. Our results provide validation for signaling pathways already implicated in EDMD pathology like Rb/MyoD (Bakay et al., 2006), but more importantly identify novel regulatory networks (Oct4-NANOG-Sox2) which members show inappropriate heterochromatin enrichment and consequent transcriptional upregulation (Sox2, Gata6, Hoxb1). Our study shows that alterations in epigenomic signaling include both loss-of-function of E2F and myogenic transitions, but also gain-of-function (new induction) of inappropriate cell pathways (e.g. Sox2). This model likely explains the dominant nature of different LMNA mutations: combination of both inappropriate loss and gain of cell commitment at the point of terminal differentiation.

5.5 Conclusion

This study shows that mutations in nuclear envelope genes and proteins cause mutation-specific changes in chromatin remodeling at the nuclear periphery. Further, it shows that this has downstream effects on inactivation of pluripotency programs and appropriate induction of differentiation. Mutations in lamin A/C that cause muscle and adipose phenotypes affect its binding to chromatin at the loci of muscle expressed genes. While muscle-expressed genes showed an expected drop in DNA methylation during myogenic differentiation in Normal control samples (WT), both EDMD and FPLD patient samples showed a failure of the expected heterochromatin-euchromatin transitions.

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We showed that LADs (Lamin A/C-chromatin binding domains) were enriched for heterochromatin (H3K9me3 and H3K27me3). To further investigate the effects of EDMD mutations on chromatin remodeling during muscle differentiation, we performed

H3K9me3 ChIP on differentiating emerin null and wild type myogenic cells. The data showed that genomic domains that show loss of heterochromatin in emerin null cells are involved in cell fate maintenance and pluripotency regulation. Ingenuity pathway analysis showed that top canonical pathway pointed to perturbations of the signaling downstream of SOX2-Nanog-Oct4. These predictions were validate by showing that SOX2 and its downstream targets (GATA6 and HOXb2) were abnormally regulated during myogenic differentiation in both murine (emerin null) and human (LMNA patients) cells as well as in patient muscle biopsies. Together, these results suggested that mutations in both emerin and lamin A/C change chromatin remodeling at the nuclear periphery that specifically impair inactivation of pluripotency programs, exit from the cell cycle and myogenic induction.

To show direct effects of lamin A/C mutations on the peripheral localization of the

SOX2 locus during myogenic differentiation, DamID-seq and DamID-qPCR were used.

The data showed that SOX2 locus interacts with wild type LMNA in myogenic cells and that this interaction was decreased by mutations in LMNA (DamID-qPCR). These results were confirmed by DamID-seq. Finally, overexpression of SOX2 significantly reduces differentiation potential of normal human myoblasts. This provided further evidence that increased SOX2 expression seen in EDMD patient muscle and myogenic cells affects both inactivation of pluripotency programs and myogenic induction. Together, LMNA mutations show decreased affinity towards the pluripotency loci predicting their loss of

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heterochromatin and transcriptional upregulation that in turn has dramatic effects on myogenic differentiation and development.

5.6 Future directions

In this dissertation, various molecular and bioinformatics techniques were employed to study the role of nuclear envelope mutations in chromatin remodeling during myogenic differentiation. This study showed that disease specific lamin A mutations exhibit differential enrichment in the vicinity of genes induced by myogenic differentiation. In the future, it would be valuable to perform the same DamID experiment in adipogenic cell lines to validate the disease specific nature of LMNA mutations in non- muscle tissues. Our predicted result is that LMNA mutations causing FPLD (fat restricted) phenotypes would show perturbations of adipogenesis developmental pathways.

Another line of future research could identify the specific histone protein ‘marks’ that are correlated with chromatin-LMNA nuclear interactions. It was expect that these would be a subset of previously characterized heterochromatic marks, but such research might identify new marks, or tissue-specific marks.

There are additional genetic loci that cause EDMD phenotypes, yet have not been studied at the protein level. Particularly interesting are the nesprins – proteins that link the inner nuclear membrane to outer nuclear membrane. Dominant mutations of nesprin 1 and nesprin 2 (SYNE1, SYNE2) cause EDMD. It is important to determine if

DamID fusions to WT nesprins show association with similar chromatin loci as those we have defined for WT lamin A/C. Likewise, it would be interesting if dominant missense

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mutations of nesprins led to persistent expression of Sox2, and similar failure in euchromatin-heterochromatin transitions during myogenesis.

Finally, visualization of differentially enriched loci relative to nuclear lamina and lamin A staining using 3D fluorescent in situ hybridization (FISH) would provide an important information regarding the localization of these loci in wild-type and EDMD

(emerin and lamin mutation) myogenic cells. This has been reported for studies of C. elegans but not in higher vertebrates. The effect of LMNA gain-of-function mutations, or loss of function of the emerin protein on 3D FISH would also be instructive.

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