THE TBX20-CASZ1 INTERACTION PROVIDES MECHANISTIC INSIGHT FOR DILATED CARDIOMYOPATHY PATHOGENESIS

Leslie M Kennedy

A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Cell and Developmental Biology in the Biology Department.

Chapel Hill 2017

Approved by:

Mark Peifer

Joan Taylor

Victoria Bautch

Steve Crews

Frank Conlon

© 2017 Leslie M Kennedy ALL RIGHTS RESERVED

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ABSTRACT

Leslie M Kennedy: The TBX20-CASZ1 Interaction Provides Mechanistic Insight for Dilated Cardiomyopathy Pathogenesis (Under the direction of Frank Conlon)

Heart disease is the leading cause of death in the U.S. and throughout the world. It claims more lives than all types of cancers combined, and constitutes a significant financial burden on the global society. Such grim statistics necessitate serious interventions and a better understanding of the genetic underpinnings that predispose to the cardiovascular pathologies that ultimately lead to heart failure and death. A primary method of combating the high rates of morbidity and mortality in those with heart disease, is early detection. In this thesis dissertation, I present the first account of an endogenous interaction between the essential cardiac transcription factors TBX20 and CASZ1. This is the first account of an unbiased affinity purification of TBX20 from cardiac tissue, with subsequent identification of TBX20 transcriptional network components by tandem mass spectrometry. I go on to show, that when this interaction is disrupted specifically in the cardiac environment, animals mutant for these two factors display cardiovascular defects that phenocopy the defects seen in human patients with the heart disease subtype dilated cardiomyopathy. I also found that a specific DCM mutation that maps to the T-box of TBX20 disrupts the interaction with CASZ1, and disruption in the combined transcriptional activity of TBX20 and CASZ1. As the leading type of cardiomyopathy in the young, and the leading cause of heart transplantation, dilated cardiomyopathy, it is critical that the field identifies treatable mechanisms that lead to DCM.

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ACKNOWLEDGEMENTS

First, thanks to my Heavenly Father for His grace and favor. In the midst of the struggles, His love has made all the difference.

Thanks to the Conlon lab for their support and training throughout this process. This has been an excellent lab for becoming a scientist, and learning to communicate science articulately and effectively. Special thanks to Frank for allowing me to join his lab family.

Frank has been a productive and insightful mentor from whom I’ve learned a lot over the years.

Thanks to Joan Taylor, Steve Crews, Mark Peifer, and Vicki Bautch for serving on my thesis committee. I cannot imagine how difficult it must be to balance functioning in so many different roles, while still giving students like me undivided attention and invaluable feedback during committee meetings. I understand their time is valuable, and I appreciate their lending a fraction of it to help me succeed.

I would not be in graduate school or completing my thesis dissertation without the guidance and support of the Biological and Biomedical Sciences Program, the Initiative for

Maximizing Student Diversity Program, the Initiative for Minority Excellence, and the

Postdoctoral Readiness to Obtain Professorial Success Program. I have had the great honor of working with Ashalla Freeman, Kathy Woods, Sibby Anderson-Thompkins, and countless more who have made it their goal to help me succeed and celebrate with me as I do. Their programs have made my tenure here at UNC an extraordinarily awesome adventure.

Huge thanks to my friends and family, who have supported me endlessly throughout my career. My family has been so incredibly helpful in so many ways. Just knowing that they were in my corner, calling to check on my daughter and I, praying for me, and routing for me

iv even when I didn’t realize it. Having a support system has been an incredible blessing for which I am incredibly thankful. I could not have hand-picked a better family of which to be a part.

Sincere appreciation goes to my big sister and brother who have been great role models in so many ways. My brother has traveled good part of the most interesting parts of the world, and is filled with passion and knowledge of things that are far beyond my understanding. My sister accomplished so many things in her career while raising my 3 beautiful niece and nephews with her husband. Both my sister and brother have conquered the world in my eyes, while staying humble and true to their family and friends. They are exemplary individuals I am truly honored to know and love.

Thanks to my incredible mother, always had a way of making me feel as if no person in the entire universe was as smart as I was or could do the things I did. That type of unwavering belief is incredibly impactful. Because of that example you set for me, I know how to show my daughter that I believe she can accomplish absolutely anything, without any doubt in my mind. Thanks to my mother for showing me what it means to truly believe in someone. To me, that is one of the most important lessons you taught me.

To my dear sweet daughter…who’s beautiful spirit encourages me to be better. My daughter, who is currently only 4 years old, will bring me a blanket if she even thinks I might be cold, tells me I should rest when my eyes look tired, shares her very last cookie if she sees that I have eaten all mine. My daughter is the kindest, sweetest, and most compassionate person I know – even to people she does not know. I’m so blessed to be her mother, and pray that I cultivate her to be the truest and best version of herself that she could possibly be.

Thus far, I am beyond impressed with the beautiful, intelligent, empathetic person that she already is.

Abundant gratitude to my ace. He has been present, and a lot of times having someone be truly present is everything. He has been an integral part of my life’s journey,

v especially the journey to completing my doctoral degree. His encouragement, his wisdom, his prayers, his unwillingness to allow me to settle or to quit, he has been such a critical part of my development as a scientist, as a professional, and as a person. The fact that I could never thank him certainly will not prevent me from trying to do so.

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TABLE OF CONTENTS

LIST OF FIGURES………………………………….……………………………………………………...... ix

LIST OF TABLES..……………………………………………………………………………………………..…….....xi

LIST OF ABBREVIATIONS…………………………………………………………………………………………xii

CHAPTER 1: INTRODUCTION…………………………………………………………………………….……….1

Global Health………………………………………….……………………………………………….……….1

Heart Disease………………………………………….……………………………………………….……….2

Dilated Cardiomyopathy………………………….……………………………………………….………..3

Dilated Cardiomyopathy Causative Mutations…………………………………………….……….5

Essential Roles of TBX20 in Development…………………………………………….…………….7

Genetic and Molecular Interactions in Heart Disease Pathogenesis……………….……..10

Dissertation Goals…………………………………………….……………………………………………..12

References…………………………………………….…………………………………………………………18

CHAPTER 2: DUAL TBX20 AND CASZ1 HAPLOINSUFFICIENCY IS CAUSATIVE TO DILATED CARDIOMYOPATHY……………………………………………………..………………………26

Preface…………….……………………….……………..……………………….……………..……………..26

Introduction………………………………………….……………………………………………….….……26

Methods………………………………………….……………………………………………….……….…….28

Results………………………………………….……………………………………………….……….………33

Discussion………………………………………….……………………………………………….………….42

Supplemental Materials and Methods……………………………………………………………….60

References………………………………….……………………………………………….………………….84

CHAPTER 3: DISCUSSION………………………………………….……………………………………..………95

vii Developmental Requirement for Protein-Protein Interactions……..…….………………..95

Generating Pluripotent Material for Isolating Endogenous TBX20 Complexes………95

CASZ1 is a Novel TBX20 Interaction Partner……………..…….………………………………..98

Early Detection as a DCM Intervention……………………………………………………………..99

TBX20-CASZ1 Compound Heterozygosity Leads to Dilated Cardiomyopathy………102

Protein Changes in TBX20-CASZ1 Mutant Mice……………..…….……………………….…103

Compensatory and Decompensatory Cardiac Remodeling in Response to TBX20-CASZ1 Heterozygosity……………..…………….………….…………………………….104

References………………………………………….…………………………………..…….………………106

APPENDIX: A GRO/TLE-NURD COREPRESSOR COMPLEX FACILITATES TBX20-DEPENDENT TRANSCRIPTIONAL REPRESSION ……………………111

Preface………………………………………….……………………………………………….………………111

Introduction………………………………………….……………………………………………….………111

Methods………………………………………….……………………………………………….…………….114

Results………………………………………….……………………………………………….………………122

Conclusions………………………………………….……………………………………………….……….132

References………………………………………….……………………………………………….…..……148

viii LIST OF FIGURES

Figure 1.1 Myocardial effects of extensive concentric versus eccentric cardiomyocyte growth………………………………………………………………………………………..……….15

Figure 1.2 TBX20 mutations in human cardiovascular disease…………..………..………………….16

Figure 2.1 The TBX20Avi-BirA system for isolation of the TBX20 interactome………………..47

Figure 2.2. Endogenous TBX20 interactome…………………………………………………………………48

Figure 2.3 Double heterozygous hearts undergo pathological remodeling………………………..49

Figure 2.4. TBX20 and CASZ1 interact through their DNA binding domains…………………….51

Figure 2.5. TBX20F256I impairs the TBX20-CASZ1 interaction…………………………………………53

Figure 2.6. Proteomic analysis of mouse hearts with DCM reveals activation of complement cascades and decreased protein abundance involved in glycogen metabolic processes………………………………………………………………………………………….…………55

Figure S2.1 Construction of the Tbx20Avi allele…..…………………………………………………….……69

Figure S2.2 MS/MS identification of TBX20 and CASZ1…….………………………………………….70

Figure S2.3 The TBX20-CASZ1 interaction occurs with both the Xenopus laevis and Mus musculus protein isoforms. …………………………………………………….……………….…….71

Figure S2.4 TBX20 is highly conserved at p.F256, p.E258, and p.T259……………………………72

Figure S2.5. TBX20 and CASZ1 expression in the left ventricles of control and mutant hearts…………………………………………………………………………………………………….………75

Figure S2.6 Distribution of TMT ratios for biological duplicates……………………………….…….76

Figure S2.7 STRING network of differential proteins coded by Mut/CTL TMT abundance ratio………………………………………………………………………………………………….………77

Figure S2.8 Over-represented GO Biological Process Network for Differential Proteins…………………………………………………………………………………………………………….………78

Figure S2.9 Over-represented GO Cellular Component Network for Differential Proteins…………………………………………………………………………………………………………………….79

Figure A1. Tbx20-EGFP is nuclear-localized and transcriptionally active……………………….139

Figure A2. Shotgun proteomics of Tbx20-EGFP protein complexes reveals association with a chromatin remodeling and Groucho transcriptional protein network…………………………………………………………………………………………………………………...140

Figure A3. Tbx20 interacts with TLE1/3, HDAC2, and Tbx18………………………………………..141

ix Figure A4. Tbx20 assembles a Gro/TLE-NuRD repression complex via the eh1 binding motif. ……………………………………………………………………………………………..……..142

Figure A5. Endogenous Tbx20 interacts with TLE1/3 in mouse embryonic hearts…….…….143

Figure A6. RNA-sequencing reveals disrupted transcriptional output in Tbx20eh1mut -expressing embryos…………………………………………………………………………….144

Figure SA1. Venn diagram summarizing overlap of regulatory capabilities eh1mut between wild-type Tbx20 and Tbx20 ……………………………………………………………………146

x LIST OF TABLES

Table 1.1 TBX20 Disease Mutations.………………………………………………………………………..……17

Table 2.1. Lethality in mutant and control mice at 4, 8 and 16 weeks……………………….………57

Table 2.2. The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in young adult mice aged 4-7 weeks…..……………………………………………….……..58

Table 2.3 The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in mice aged 8-11 weeks…………………………………………………………………..……….59

Table S2.1. Proteins that interact in the endogenous TBX20 transcriptional network isolated from Day7 iCMS……..………………………………………………………………..……….80

Table S2.2 Cardiovascular physiology in young adult female and male mice…………………….81

Table S2.3 Cardiovascular physiology in mature adult female and male mice………….……….82

Table S2.4. Proteins showing significant changes in TBX20-CASZ1 mutant mouse hearts.………………………………………………………………………………….…………….……..……83

Table A1. Tbx20-Associated Proteins Identified by LC-MS/MS.…………………………………….145

xi LIST OF ABBREVIATIONS

AP-MS Affinity Purification- Mass Spectrometry

ANF Atrial Natriuretic Factor

ASD Atrial Septal Defects

Avi Avitag

CHD Congenital Heart Disease

CVD Cardiovascular Disease

DCM Dilated Cardiomyopathy

EBs Embryoid Bodies

E Embryonic day

ESC Embryonic Stem Cell eh1 Engrailed Homology

Gro Groucho

HDAC Histone Deacetylase

HEK Human Embryonic Kidney

HT Hypertension iCM Induced Cardiomyocyte

ICM Ischemic Cardiomyopathy

LEF Lymphoid Enhancer Binding Factor

MTA Metastasis-associated Protein

MBD3 Methyl-CpG Binding Domain Protein 3

NCL Nucleolin

NICM Non-ischemic Cardiomyopathy

NLS Nuclear Localization Signal

NPM1 Nucleophosmin

xii NuRD Nucleosome Remodeling and Deacetylase

PFO Patent Foramen Ovale

POF Pentalogy of Fallot

RV Right Ventricle

TAPVC Total Anomalous Pulmonary Venous Connection

Tbx T-box containing protein

TOF Tetralogy of Fallot

TLE Transducin-like enhancer of split

VSD Ventricular Septal Defects

Xbra Xenopus Brachyury

xiii

CHAPTER 1: INTRODUCTION

Global Health

From 1990 to 2013, the global life expectancy increased from 65.3 to 71.5 years.(Mortality and Causes of Death, 2015) This increase reflects changes in several factors that influence mortality, including infrastructure, resource stability, and access to disease intervention and treatment. Studies have shown that increased life expectancies are not due to equal improvements across all age groups. The largest improvements in life expectancy and the largest decreases in mortality rates between 1990 and 2013 were observed in children and infants less than 5 years of age (Mortality and Causes of Death, 2015). Mortality rates for individuals in this group were compared to mortality rates of individuals in 17 groups clustered by age (e.g. ages 5-9, 10-14…75-79, 80 and older). Decreases in mortality resulting from cardiovascular disease (CVD) was one of the factors that contributed to the increased life expectancy observed in children and infants less than 5 years of age.

CVD is the singular term for diseases of the heart and the blood vessels leading to and from the heart that affect the function of the heart. These include myocardial infarction

(heart attack), angina (chest pain), stroke, congenital heart defects, hypertension (high blood pressure), arrhythmia (abnormal heart rhythm), and heart failure (reduced cardiac output to dangerous levels). CVD creates a significant global burden as the leading cause of death throughout the world, with Russia and countries in Eastern Europe showing some of the highest death rates for both men and women (Writing Group et al., 2016). In the U.S., more than a third of the country is predicted to be living with at least 1 type of CVD (Writing

Group et al., 2016). Additionally, in 2013, CVD was responsible for more than a third of deaths globally (Mortality and Causes of Death, 2015). It leads to more than 17 million

1 deaths per year, more than those caused by all types of cancer combined (Roth et al., 2015).

Like most diseases, CVD arises due to pathological genetic mutations environmental factors, or a combination of the two. In a recent comparison of 12 CVDs in nearly 2 million CVD patients, heart failure predominated as the initial CVD diagnosis (George et al., 2015).

Heart Disease

Leading the U.S. and the world in death rates, heart disease, a major type of CVD, accounts for approximately 25% of U.S. deaths (Writing Group et al., 2016). Heart disease can either exist at birth and be identified in the neonate, or it can be absent or asymptomatic in the neonate and identified in adults. Structural and functional defects in the heart that are present at birth are termed congenital heart defects (CHDs), and are largely caused by inherited genetic mutations (Pierpont et al., 2007). CHD is the leading neonatal abnormality, presenting in approximately 1% of neonates (Marelli et al., 2007) and identified in 10% of still-borns (Hoffman, 1995a, b; Hoffman and Kaplan, 2002). With advancements in tools used to detect CHDs and with improvements in treatments, increasing numbers of CHD infants survive into adulthood. Of the infants that survive their first year of life, approximately 75% will reach adulthood (Gilboa et al., 2010; Pierpont et al.,

2007). Coincidentally, the percentage of CHD adults increases by nearly 5% each year

(Brickner et al., 2000a, b).

Neonatal and adult-onset heart disease is characterized by the presence of cardiovascular abnormalities that exist post-natally. There is speculation that cardiac functions reach a threshold beyond which disease mutations begin to manifest themselves.

In these cases, environmental factors may function as precipitating contributors to cardiovascular dysfunction, which often leads to heart failure and death. The most common cause of heart failure and most frequent abnormality leading to heart transplant is dilated cardiomyopathy (DCM) (Stehlik et al., 2012; Sugrue et al., 1992).

2

Dilated Cardiomyopathy

DCM is a leading myocardial disease characterized by enlargement of the cardiac ventricles, termed dilation, and systolic dysfunction. DCM is the leading cardiomyopathy in children less than 18 years of age (termed pediatric DCM), including in infants less than 1 year old, and has an annual incidence of 0.58 cases per 100,00 children (Wilkinson et al.,

2010). In only about a third of pediatric DCM cases are clinicians able to identify a cause of

DCM pathogenesis (Towbin et al., 2006). Although DCM can present in children, the incidence is 3.5 – 8.5 per 100,000 in adults (Manolio et al., 1992).

Due to the phenotypic overlap of DCM with cardiac abnormalities such as ischemic cardiomyopathy and hypertensive cardiomyopathy (Japp et al., 2016), the definition of DCM has recently been revised. Based on this revision DCM is now defined as ventricular dilation and systolic dysfunction in the absence of abnormally increased loading or coronary artery disease (Elliott et al., 2008; Lakdawala et al., 2013). In distinguishing DCM from ischemic cardiomyopathy (ICM), clinicians and researchers found that ICM occurs when inadequate blood is supplied to the heart as a result of myocardial infarction (heart attack) or coronary artery disease (Anversa et al., 1995). Additionally, ICM patients have a poorer survival rate than those with non-ischemic cardiomyopathy (NICM) such as DCM (Bart et al., 1997), and show at least 75% narrowing of the blood vessel lumen (termed stenosis) in one or more of the main vessels of the heart, including the left anterior descending coronary artery (Felker et al., 2002). As a NICM, DCM exhibits no vascular stenosis. DCM is distinct from hypertensive cardiomyopathy based on the absence presence of high blood pressure (termed hypertension), and concentric hypertrophy (Figure 1.1) (Japp et al., 2016; Lip et al., 2000).

Additionally, hypertensive cardiomyopathy is often diagnosed in older individuals (Devereux et al., 1994).

3 To characterize DCM etiology and disease progression, DCM can be grouped into subtypes of viral, autoimmune, familial, and idiopathic (Elliott et al., 2008; Richardson,

1996). Viral DCM results from viral infections that affect the heart. This was initially shown in studies of the effects of Coxsackie virus, where viral DNA was found in cardiac tissue, and

DCM developed as a result of viral infection (Mason et al., 1980; Orinius, 1968).

Autoimmune DCM was named based on the presence of auto-antibodies and immunological agents in the heart and blood in the absence of infection. In patients with DCM resulting from autoimmune diseases, immunoadsorption therapy was used to reduce immune- mediated cardiac damage and improve cardiac function (Wallukat et al., 1996). This therapy led to a drastic, but transient improvement in cardiac function that demonstrated the role of autoimmunity in DCM-related cardiac dysfunction. Patients with a family history of DCM that segregates with inheritance of a disease-associated mutation have familial DCM, and patients with DCM of an unknown etiology are diagnosed with idiopathic DCM.

Historically, DCM was studied as a disease that resulted mainly from viral infections

(Johnson and Palacios, 1982a, b), with some studies emphasizing the field’s general frustration with the phenomenon that unknown DCM etiologies led to more generalized medical treatment, rather than individualized care. Only in the mid- to late- 80’s did research groups begin to note that familial DCM had a higher incidence than initially thought (Michels et al., 1985; Schmidt et al., 1988). In patients initially diagnosed as having idiopathic DCM, their disease was potentially caused by mutations that had not yet been identified. Therefore, initial estimates found DCM to be caused by inherited mutations in less than 2% of cases (Michels et al., 1985; Michels et al., 1992), but more recent studies have discovered that nearly half of all DCM cases are familial, and are therefore caused by inherited mutations (Burkett and Hershberger, 2005; Mahon et al., 2005).

To understand mechanisms underlying DCM disease pathogenesis and to identify potential biomarkers that can be predictive of DCM progression, several groups have

4 performed gene expression analysis of hearts pooled from DCM patients. These hearts range from DCM to failing, which are stages identified based mainly on ejection fraction, which is defined as the volume of blood pumped from the heart with each contraction (systole) shown as a fraction of the total blood in the heart while the heart is at rest (diastole). The ejection fraction (EF) is shown as a percentage with a normal range of 55 – 70%. An EF less than 55% is abnormal, an EF less than 40% constitutes a clinical DCM diagnosis when combined with ventricular dilation, and an EF less than 35% can constitute heart failure. Overall, molecular changes that occur in DCM represent changes in energy metabolism, cell cytoskeleton, extracellular matrix composition, inter-cellular junctions, and cell-matrix junctions (Colak et al., 2016; Colak et al., 2009; Hwang et al., 2002; Shi et al., 2013).

Using mouse models to study the progression from genetic mutation to heart failure, a DCM mouse model expressing a mutant form of phospholamban, a critical factor in maintaining cardiomyocyte Ca2+ homeostasis, pre-DCM, DCM, and DCM-induced heart failure were distinguished based on histological and molecular profiles (Burke et al., 2016).

Molecular characterization at each stage in disease progression, mainly reflected alterations in energy metabolism, similar to those seen in adult human DCM. Results from these studies show some overlap in pathways disrupted in DCM patients, but very little overlap in the actual genes that are misregulated. This finding highlights the heterogeneity of DCM and the importance of defining a molecular signature that can be predictive of the clinical presentation in humans.

Causative DCM Mutations

Although familial and idiopathic DCM are 2 distinct DCM subtypes, there has long been speculation that a subset of idiopathic DCM cases involves mutations in genes that have not yet been linked to DCM. Between 1993 and 2002, the following 12 genes were identified as causal in human DCM. Each gene is shown with its protein product in

5 parentheses: DMD (Dystrophin), EMD (Emerin), ACTC (cardiac actin), LMNA (Lamin A/C),

DES (Desmin), G4.5 (Tafazzin), SGCD (δ-sarcoglycan), DSP (Desmoplakin), MYH7 (cardiac

β-myosin heavy chain), TNNT2 (cardiac Troponin T), TPM1 (α-Tropomyosin), and TTN

(cardiac Titin) (Moolman-Smook et al., 2003). Cardiac actin, Desmin, Cardiac β-myosin heavy chain, Cardiac Troponin T, α-Tropomyosin, and Cardiac Titin form the structural components of the sarcomere that play critical roles in synchronous cardiomyocyte contraction. Dystrophin, Emerin, and Lamin A/C function in cytoskeletal organization and protein scaffolding; while Desmoplakin and δ-sarcoglycan form structural components of cell-cell and cell-extracellular matrix (ECM) junctions, respectively. Mutations in sarcomere components led to severe post-natal dilated cardiomyopathy, heart failure, and death as early as 30-35 years of age (Muntoni et al., 1993). SGCD and LMNA mutations were also linked to DCM, with SGCD phenotypes presenting slightly earlier and appearing more severe than those observed in patients with DMD and LMNA mutations (Bonne et al., 1999;

Muntoni et al., 1993; Tsubata et al., 2000). Establishing a link between DCM and mutations in sarcomere and cytoskeletal components led to the classification of DCM as mainly a disease of cytoskeletal components, which we currently know to be a misnomer based on more recently identified gene mutations.

Over the next several years, several additional factors were added to the list of mutated genes found to be causal to DCM. These genes included the sarcomere component

TNNC1 (Troponin C); the cytoskeletal components ZASP (Cypher), CSRP3 (Cystein- and glycine-rich protein 3), DAG1 (α-/β-Dystroglycan) SGCG (γ-sarcoglycan), CAV3 (Caveolin

3), DTNA (Dystrobrevin), FKTN (Fukutin), FKRP (Fukutin-related protein); and 2 factors important for maintaining calcium homeostasis PLN (Phospholamban) SCN5A (Sodium channel), and transcriptional regulators. Although distinguishing between DCM subtypes has long been important for accurate diagnoses, DCM pathophysiology is also important in diagnosing and understanding disease progression (Johnson and Palacios, 1982a, b).

6 Noticeably absent from one of the earlier composite lists of causal DCM mutant genes were transcription factors. Some of the first transcriptional regulators implicated in

DCM disease pathogenesis were the transcriptional co-activator EYA4 (Schonberger et al.,

2005), and the cardiac transcription factor TBX20 (Kirk et al., 2007; Qian et al., 2008).

These findings shifted the understanding of the proposed pathophysiology underlying DCM, and exposed the potential for tight pathway regulation to be involved in DCM pathogenesis upstream of simply mutating components of the sarcomere and cell cytoskeleton.

Essential Myocardial Roles of TBX20

TBX20 is a member of the T-box family of transcription factors. Each member of the

T-box family contains a highly conserved DNA binding domain, termed the T-box, which is named for the founding member of the T-box family, Brachyury/T (Kispert and Herrmann,

1993) mediates sequence-specific binding to DNA. The T-box is in the center of TBX20, and is flanked by a nuclear localization signal in the amino terminus, and both transactivation and repression domains in the carboxy terminus (Figure 1.1). The Tbx20 gene encodes 3 isoforms of Tbx20, with the smallest isoform, Tbx20c, which is 301 amino acids (aa’s), being perfectly identical to the first 301 amino acids of the longer isoforms (Stennard et al., 2003).

Tbx20c is truncated, so that it lacks the repression domain and the majority of the 130 aa transactivation domain. The next smallest isoform, Tbx20b, is comparable in size to Tbx20c, but is 4 aa’s longer and therefore has more of the transactivation domain (Meins et al.,

2000; Stennard et al., 2003). The largest isoform, Tbx20a, encodes a 445 aa protein in mouse and houses both the transactivation and repression domains. Both Tbx20a and

Tbx20b are expressed in the heart (Kraus et al., 2001; Stennard et al., 2003); however,

Tbx20a is the dominant Tbx20 isoform in the heart (Stennard et al., 2003). As the predominant cardiac Tbx20 isoform, the remainder of this thesis will refer solely to Tbx20a.

7 Tbx20 is expressed in the eyes, cranial motor neurons, neural tube, limbs, and heart, and its expression in cardiac tissue is highly conserved as shown in Caenorhabditis elegans

(worm) (Agulnik et al., 1997), Drosophila melanogaster (fly) (Brown et al., 2003), Danio rerio (zebrafish) (Ahn et al., 2000), Xenopus laevis (frog) (Brown et al., 2003), Gallus gallus

(chick) (Iio et al., 2001), and Mus musculus (mouse) (Kraus et al., 2001). Supporting the highly conserved expression of Tbx20 in cardiac tissue, embryonic Tbx20 depletion impairs cardiac looping morphogenesis in zebrafish, frog, and mouse and leads to embryonic lethality (Brown et al., 2005; Cai et al., 2005; Singh et al., 2005; Stennard et al., 2005; Szeto et al., 2002; Takeuchi et al., 2005).

In zebrafish, Tbx20 is required for proliferation of cardiac progenitor cells, which will coalesce to form the sheet of cells that will proliferate, migrate, and differentiate to form the

4-chambered heart. Conflicting results in zebrafish show that Tbx20 overexpression can lead to either drastically increased on drastically reduced cardiac cell numbers (Just et al., 2016;

Lu et al., 2017). Although variable, these results indicate Tbx20 expression must be regulated within a narrow window to maintain homeostasis and avoid dysfunction. The requirement for tight Tbx20 regulation is also evident when Tbx20 is overexpressed in frog

(Stennard et al., 2003).

In addition to its requirement for regulating expansion of cardiac progenitors, Tbx20 is also required for looping of the linear heart tube, differentiation of the chamber myocardium, and lumenizing main blood vessels such as the dorsal aorta during vasculogenesis (Szeto et al., 2002). Lumen formation is the process by which groups of cells forming a tube, hollow along the length of the tube to form the vascular lumen. Chamber differentiation describes the process by which molecular changes in the cardiac precursors that form the linear heart tube distinguish cells of the atrial myocardium from the ventricular myocardium. Chamber differentiation begins just prior to looping of the linear heart tube.

8 In Xenopus, morpholino-mediated Tbx20 depletion arrests cardiac development at the cardiac looping stage with death occurring shortly thereafter (Brown et al., 2005).

Cardiac looping defects were accompanied by cardiac hypoplasia and pericardial edema.

These defects are highly similar to those seen when Tbx20 is depleted in zebrafish. No vascular defects were reported when Tbx20 was depleted in Xenopus.

Genetic ablation of Tbx20 in mice was independently characterized by 4 research groups. Concurrently, these groups found that loss of Tbx20 led to expansion of the Tbx2 expression domain, cardiomyocyte hypoplasia, decreased proliferation, aberrant looping of the linear heart tube, and mid-gestational embryonic lethality. Cells in the myocardium of

Tbx20 mutants also displayed levels of cell death that were comparable to wildtype animals, indicating no change in cell survival. Variable among the findings were requirements for

Tbx20 in motor neuron differentiation (Takeuchi et al., 2005), patterning of the linear heart tube (Takeuchi et al., 2005), spatial confinement of the Tbx5 expression domain (Cai et al.,

2005; Stennard et al., 2005; Takeuchi et al., 2005), and trabeculae and endocardial cushion formation (Boogerd et al., 2016; Takeuchi et al., 2005). Trabeculae are finger-like structures that project into the lumenal space of the cardiac ventricles. The specific function of the trabeculae are not well known, but disrupting trabeculae formation can lead to defects in both conduction and contractility, and can also lead to death (Liu et al., 2010; Samsa et al.,

2013). Endocardial cushions are the structural precursors to cardiac valves, and disruption in these precursor structures can lead to severe valve defects including reversed blood flow.

TBX20 is a known transcription factor, that based on the defects that result in its absence, exerts critical and conserved functions in the myocardium. Examining both the levels and localization of factors known to be expressed in cardiac precursors revealed ectopic expression of Tbx2, a transcription factor which marks ventricular myocardium, but not atrial myocardium; decreased expression of Nkx2-5, a transcription factor required for expression of several critical genes in the cardiogenic program; decreased expression of

9 NPPA, which marks differentiated myocardium; and decreased expression BMP2, which marks the developing outflow tract (which will become the pulmonary artery and aorta)

(Yamada et al., 2000), These defects demonstrate an early requirement for TBX20 in regulating developmental processes including cardiac development.

Complementing these requirements for TBX20 in the embryo, TBX20 was also shown to be critical for adult cardiac function. Conditional loss of TBX20 in the adult mouse heart causes significant thinning of the myocardial wall, dilation of the ventricles, increased cell death, systolic dysfunction, and lethality within weeks of TBX20 loss (Shen et al., 2011).

Compared to wildtype mice, TBX20 mutants exhibited up-regulation of Desmin and NPPA, and down-regulation of potassium channels, sodium channels, regulators of calcium homeostasis, gap junction proteins, and cardiac transcription factors. Together, these findings demonstrate an essential role for TBX20 in regulating key factors in modulating ion homeostasis in terminally differentiated cardiomyocytes.

With several groups having independently established a requirement for TBX20 in the developing and adult myocardium, it became crucial to determine the clinical relevance of TBX20 in patients with cardiac anomalies (CHD and DCM mutations in TBX20 listed in

Table 1.1 and shown in Figure 1.2.) The Harvey research group screened 352 unrelated CHD probands for mutations in TBX20, and identified 1 missense mutation, I152M, and 1 nonsense mutation, Q195X, both of which map to the T-box. These mutations were absent from healthy controls, and sequencing of TBX20 in related individuals showed that these mutations segregated with cardiac defects, such as ventricular septal defects (VSDs), atrial septal defects (ASDs), and DCM (Kirk et al., 2007). Since then, TBX20 mutations associated with CHD and other cardiac defects have been found throughout the TBX20 coding region.

Genetic and Molecular Interactions in Heart Disease Pathogenesis

10 Mounting evidence from groups across the country has established monogenic mutations in essential cardiac factors as causal in several cardiac disease phenotypes.

However, monogenic mutations can also constitute a sensitized environment, where additional mutations in other genes and environmental risk factors synergize with the primary mutation to produce disease phenotypes. In 331 CHD patients that were screened for mutations in NKX2-5, GATA4, and TBX5, which encode essential cardiac transcription factors, the NKX2-5 mutation L122P was identified in a patient with secundum ASD

(Granados-Riveron et al., 2012). This mutation was also identified in the patient’s unaffected father. However, a mutation was also identified in MYH6, which encodes a sarcomere component, in the patient, but not in the patient’s unaffected father. This supports a mechanism whereby one mutation functioned to sensitize the individual, so that additional mutations in essential cardiac genes, produced cardiac abnormalities. A similar mechanism can also be deduced from the V380M mutation in GATA4, which was pathogenic to CHD in one study (Tang et al., 2006), but was present in nearly 10% of more than 300 control subjects in another study (Schluterman et al., 2007).

In addition to polygenic mutations creating a sensitized environment, there is also evidence that disrupting protein-protein interactions plays a role in disease pathogenesis. In

Xenopus laevis, morpholino-mediated TBX20/TBX5 depletion led to cardiac hypoplasia, cardiac looping defects, premature termination of cardiac morphogenesis, and embryonic lethality (Brown et al., 2005). Germline ablation of Nkx2-5 and Mef2c in mice led to hypoplasia, aberrant cardiomyocyte specification, failed chamber differentiation, and defective cardiac looping (Vincentz et al., 2008). In adult mice, Tbx20-Nkx2-5 compound heterozygosity led to mild ventricular dilatation accompanied by systolic dysfunction

(Stennard et al., 2005). For these examples, the phenotypes of the animals in which both factors were depleted was far more severe than the phenotype of depleting each factor alone.

11 This further establishes the critical functions of cardiac transcriptional networks in heart development and adult heart function.

Here, we explore the potential for cardiac interaction networks to modulate adult heart function by first identifying CASZ1 as a member of a novel TBX20 transcriptional complex. The human CASZ1 gene encodes a short isoform of CASZ1, CASZ1b which is 1166 amino acids, and a longer isoform, CASZ1a which is 1759 amino acids. CASZ1b (the shorter isoform) retains high identity to the first 1166 amino acids of CASZ1a, and is also more highly conserved than CASZ1a. CASZ1b is a zinc finger transcription factor with a nuclear localization signal in its amino terminus, 4 highly conserved zinc fingers in the central part of the protein, and 1 zinc finger in the carboxy terminus. It’s expressed in the eyes, heart, limbs, and central nervous system (Dorr et al., 2015); and in the heart, CASZ1 regulates cell cycle progression, proliferation, differentiation, and ventricular septation (Charpentier et al.,

2013; Dorr et al., 2015; Liu et al., 2014). Supporting a requirement for CASZ1 in the human heart, recent independent studies focused on CASZ1 mutations in patients with cardiac anomalies have identified the CASZ1 mutant L38P, which segregates with CHD (Huang et al., 2016), and K351X which segregates with DCM (Qiu et al., 2017). Both mutations were absent from healthy control subjects, and both showed little to no activity in in vitro transcriptional assays. Considering both TBX20 and CASZ1 are essential for viability in mice, and mutated forms of each lead to embryonic and adult cardiac anomalies in humans, we investigated a Tbx20-Casz1 genetic interaction by generating mutant mice in which cardiac-specific heterozygosity of Tbx20 and Casz1 was mediated by Nkx2.5Cre.

Dissertation Goals

Heart disease occurs due to a complex interplay between genetic and environmental factors. Once a threshold level is reached by each contributing factor, a threshold that varies from one individual to the next, cardiac defects arise either in the developing fetus or in the

12 adult. Extensive work has created a wealth of knowledge on the environmental risk factors that precipitate cardiac dysfunction. However, novel gene mutations and their disease pathogenesis are still being identified.

Based on the need for identifying new candidates for cardiovascular disease pathogenesis, I have focused my analysis on the well-described cardiac transcription factor

TBX20. TBX20 has been shown to play essential roles both in establishing the myocardium during development and in maintaining cardiomyocyte function in the adult. These functions involve both molecular and morphological changes that, together, create a functional myocardium. The process of heart development involves several critical gene products performing tightly-regulated functions. However, cardiac phenotypes resulting from compound depletion of multiple cardiac factors simultaneously demonstrates roles for transcription factor interactions in cardiac function.

In understanding how protein-protein interactions function in the cardiovascular environment, we created a strategy for isolating endogenous TBX20 transcriptional complexes from cardiac tissue. This strategy employed the use of a novel Tbx20 allele in which Tbx20 was tagged with the Biotin accepter peptide, Avi. TBX20AVI expressing cells were differentiated into cardiomyocytes in culture, and use as source material for isolating endogenous TBX20 transcriptional network. Using this method, we identified the cardiac transcription factor Castor (CASZ1) as a member of a TBX20 transcriptional network.

Upon identifying and validating the interaction between TBX20 and CASZ1, we brought both the Tbx20 and Casz1 alleles to heterozygosity in the heart, and assessed mutants for defects. Cardiac-specific Tbx20/Casz1 heterozygosity led to lethality in adult mice, preceded by ventricular dilatation, systolic dysfunction, interstitial fibrosis, and mild cardiomyocyte hypertrophy. We also related the Tbx20/Casz1 mutant phenotype back to human disease by showing the TBX20 DCM mutant TBX20F256I disrupted the interaction with CASZ1, and disrupted TBX20-CASZ1-mediated transcriptional activity. Together, our

13 results provide a new mechanism for DCM pathogenesis whereby disruptions in essential protein-protein interactions cause misregulation of essential cardiac programs that ultimately lead to cardiomyocyte dysfunction, heart failure, and death.

14 Figure 1.1 Myocardial effects of extensive concentric versus eccentric cardiomyocyte growth.

Right ventricle is crescent shaped, while the left ventricle is elliptical. The ventricular wall is shown in pink, with the diameter indicated as a blue horizontal line. The ventricular lumen is the white space in inside the ventricular wall, with the diameter indicated as the red horizontal line.

15 Figure 1.2 TBX20 mutations in human cardiovascular disease. Mis-sense and non-sense mutations that map to TBX20 are shown in the region of TBX20 to which they map.

Mutations that lead to DCM are shown along the top of the domain map, while mutations that lead to other cardiac defects are shown along the bottom of the domain map. Mutations shown along the top and bottom, lead to DCM in some patients, and to other cardiac defects in other patients.

16 Table 1.1. TBX20 CHD and DCM Disease Mutations. ASD - Atrial septal defect, DCM -

Dilated cardiomyopathy, HT – Hypertension, PFO - Patent foramen ovale, POF - Pentalogy of Fallot, RV - Right ventricle, TAPVC - Total anomalous pulmonary venous connection ,

TOF Tetralogy of Fallot.

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Writing Group, M., Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M., Das, S.R., de Ferranti, S., Despres, J.P., Fullerton, H.J., Howard, V.J., Huffman, M.D., Isasi, C.R., Jimenez, M.C., Judd, S.E., Kissela, B.M., Lichtman, J.H., Lisabeth, L.D., Liu, S., Mackey, R.H., Magid, D.J., McGuire, D.K., Mohler, E.R., 3rd, Moy, C.S., Muntner, P., Mussolino, M.E., Nasir, K., Neumar, R.W., Nichol, G., Palaniappan, L., Pandey, D.K., Reeves, M.J., Rodriguez, C.J., Rosamond, W., Sorlie, P.D., Stein, J., Towfighi, A., Turan, T.N., Virani, S.S., Woo, D., Yeh, R.W., Turner, M.B., American Heart Association Statistics, C., Stroke Statistics, S., 2016. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation 133, e38-360.

Yamada, M., Revelli, J.P., Eichele, G., Barron, M., Schwartz, R.J., 2000. Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev Biol 228, 95-105.

25

CHAPTER 2: DUAL TBX20 AND CASZ1 HAPLOINSUFFICIENCY IS CAUSATIVE TO DILATED CARDIOMYOPATHY1

PREFACE

Chapter two describes an endogenous cardiac TBX20 transcriptional network which includes the cardiac transcription factor CASZ1. The TBX20-CASZ1 interaction is required to maintain adult cardiac function in mice, and has an implied function in human dilated cardiomyopathy pathophysiology. This was the first work in which an endogenous cardiac

TBX20 transcriptional network was isolated and ascribed physiological relevance. This work was completed in collaboration with Erin Kaltenbrun, Todd Greco, Laura Herring, Brenda

Temple, and Ileana Cristea. My contribution to the work was generating and characterizing the compound heterozygous mice; performing histological and gene/protein expression analysis; co-immunoprecipitations validating the interaction between TBX28 and TBX20 by performing co-immunoprecipitation experiments from HEK293 cells transiently expressing tagged versions of both proteins.

INTRODUCTION

Heart failure is a major cause of morbidity in the United States with more than 5 million people in the US living with this disease (Go et al., 2013). A major risk factor for developing heart failure is dilated cardiomyopathy (DCM). Clinically recognized as systolic dysfunction accompanied by dilation of one or both ventricles, DCM is a predominating cardiomyopathy and the most common disease requiring heart transplantation in the US

(Boucek et al., 2001; Hunt et al., 2002); however, nearly half of DCM cases are of unknown etiology (Mozaffarian et al., 2015).

1 Chapter 2 is currently under review at a peer-reviewed journal.

26 In efforts to understand the etiology of idiopathic DCM, mutations in over 50 genes including components of the contractile apparatus and cell cytoskeleton, as well as in factors involved in excitation-conduction coupling, have been identified as causative in DCM (Japp et al., 2016; Kimura, 2016). However, few studies have explored the potential for aberrant transcriptional regulation of these factors to contribute to disease pathogenesis. In exception to this, recent studies have identified mutations in the T-box transcription factor TBX20 associated with DCM (Liu et al., 2008; Qian et al., 2008; Zhao et al., 2016).

Results of genetic analysis and protein depletion studies are consistent with an essential role for TBX20 during the early stages of vertebrate heart development (Ahn et al.,

2000; Brown et al., 2003; Brown et al., 2005; Griffin et al., 2000; Iio et al., 2001; Kraus et al., 2001; Meins et al., 2000; Yamagishi et al., 2004). Hearts lacking Tbx20 show progressive loss of cardiomyocytes, failure of the heart to undergo looping and chamber formation, and defects in cardiomyocyte maturation (Brown et al., 2005; Cai et al., 2005;

Singh et al., 2005; Stennard et al., 2005; Takeuchi et al., 2005). In humans, loss-of-function mutations in TBX20 can cause dilated cardiomyopathy, atrial septal defects, or mitral valve disease, while gain-of-function mutations in TBX20 have been reported in patients with

Tetralogy of Fallot (i.e., pulmonary outflow tract obstruction, ventricular septal defect, overriding aortic root and right ventricular hypertrophy) (Hammer et al., 2008; Kirk et al.,

2007; Liu et al., 2008; Posch et al., 2010; Qian et al., 2008). It has been further demonstrated that ablation of Tbx20 in adult mouse cardiomyocytes leads to the onset of severe cardiomyopathy leading to death within 1-2 weeks after Tbx20 loss (Shen et al., 2011).

While TBX20 is an essential transcription factor for heart development and its disease relevance is well established, many fundamental questions remain about the mechanism of TBX20 function. Principle among these is how TBX20 mutations associated with DCM circumvent the essential embryonic cardiac requirement for TBX20.

27 To elucidate the mechanisms by which mutations in TBX20 lead to human adult pathological states, we identified endogenous TBX20 cardiac protein-protein interactions by coupling a genetically tagged endogenous allele of Tbx20 with unbiased proteomic analysis.

Results from these studies revealed TBX20 interacts with the essential cardiac transcription factor Castor (CASZ1), a gene recently linked to DCM (Qiu et al., 2017). We confirmed that

TBX20 and CASZ1 interact biochemically and genetically and show mice haploinsufficient for Tbx20 or Casz1 are asymptomatic, while mice heterozygous for both Tbx20 and Casz1 die beginning at 4 to 8 weeks post birth and exhibit cardiomyocyte hypertrophy, interstitial fibrosis, and severe DCM. Interestingly, in vitro analyses show the DCM mutant TBX20F256I bypasses the early essential requirement for TBX20 but leads to DCM. We report here that

TBX20F256I disrupts the TBX20:CASZ1 interaction, ascribing clinical relevance to this protein complex. Further, by using quantitative proteomics we have identified the molecular pathways altered in TBX20:CASZ1-mediated DCM. Together, these results identify a novel interaction between TBX20 and CASZ1 essential for maintaining cardiac homeostasis. These findings imply that DCM can be inherited through a digenic mechanism.

METHODS

Generation of Tbx20Avi allele

The Tbx20Avi allele was created by introducing the biotin acceptor peptide (Avi) targeting cassette (Waldron et al., 2016) in-frame to the terminal exon of Tbx20 in collaboration with the UNC Animal Models Core and the UNC BAC Core (Chapel Hill).

Generation of Tbx20Avi; BirA cell line

The Tbx20Avi; BirA cell line was generated by targeting the Avitag sequence followed by a loxP-flanked neo cassette was inserted into the stop codon of exon 8 of a Tbx20a genomic fragment derived from a 129 Sv genomic BAC library. The targeting construct was

28 linearized and electroporated into ESCs of E14TG2a.4 origin. Targeted ESCs were placed under 250 µg/mL G418 selection for 7-10 days and G418-resistant ESC clones (n=384) were screened for homologous recombination by Southern blot analysis. Three ESC clones were correctly targeted, and one of these clones was subsequently used to derive the Tbx20Avi/+;

BirA cell line. Briefly, Tbx20Avi/+ ESCs were grown to approximately 40% confluence and transduced with 5 MOI Lenti-BirA for 8 hrs. Twenty-four hours following transduction, cells were placed under 200 µg/mL hygromycin selection for 4-5 days. Hygro-resistant Tbx20Avi/+ cells were subsequently used for cardiomyocyte differentiations.

ESC differentiation

Tbx20Avi/+; BirA ESCs were maintained on gelatin-coated dishes in a feeder-free culture system and differentiated (Kattman et al., 2011) in serum-free (SF) media according to the Keller protocol (Gadue et al., 2006). Briefly, ESCs were trypsinized and cultured at

75,000 cells/mL on uncoated petri dishes in SF medium without additional growth factors for 48 hrs. Two-day-old aggregated embryoid bodies (EBs) were dissociated and the cells reaggregated for 48 hr in SF medium containing 5 ng/mL human Activin A, 0.1 ng/mL human BMP4, and 5 ng/mL human VEGF (all growth factors purchased from R&D

Systems). Four-day-old EBs were dissociated and 2 x 106 cells were seeded into individual gelatin-coated wells of a 6-well dish in StemPro-34 SF medium (Invitrogen) supplemented with 2 mM L-glutamine, 1 mM ascorbic acid, 5 ng/mL human VEGF, 20 ng/mL human bFGF, and 50 ng/mL human FGF10 (R&D Systems). Cardiomyocyte monolayers were maintained in this media for 4-5 additional days with cells typically beginning to beat 2 days after seeding onto gelatin (total of 7-8 days of differentiation).

Immunofluorescence

29 For immunofluorescence of cardiomyocytes, four-day-old ES cell-derived embryoid bodies were dissociated and seeded into 8-well chamber slides precoated with 0.1% gelatin.

Induced cardiomyocytes were fixed on day 7 of differentiation in 4% paraformaldehyde for

20 min at room temperature, washed (3 x 1X PBS), permeabilized in 0.1% Triton X-100 in

1X PBS for 10 min, and blocked (10% fetal bovine serum [FBS], 0.1% Tween 20 in 1X PBS) for 30 min. Anti-myosin heavy chain (Abcam) was applied overnight, followed by PBS washes (3 x 1X PBS), and incubation with goat anti-mouse Alexa 546 (Invitrogen) for 1 hr.

Cells were incubated in DAPI (200 ng/mL in ethanol) for 30 min and visualized by confocal microscopy on a Zeiss 710.

Proteomic analysis of Tbx20 affinity purifications

Protein preparations, conjugation of magnetic beads and immunoaffinity purification and mass spectrometry were conducted as previously reported (Waldron et al., 2016).

Briefly, immunoisolated proteins were resolved (~ 4 cm) by SDS-PAGE, and visualized by

Coomassie blue. Each lane was subjected to in-gel digestion with trypsin and analyzed by nanoliquid chromatography coupled to tandem mass spectrometry as previously reported

(Kaltenbrun et al., 2013). Tandem mass spectra were extracted by Proteome Discoverer

(ThermoFisher Scientific, ver 1.4), and searched with the SEQUEST algorithm against a theoretical tryptic peptide database generated from the forward or reverse entries of the mouse UniProt-SwissProt protein sequence database (2013/08) and common contaminants

(total of 43, 007 sequences). SEQUEST search results were analyzed by Scaffold (version

4.6.1, Proteome Software Inc) using the LFDR scoring scheme to calculate peptide and protein probabilities. Peptide and protein probabilities thresholds were selected to achieve ≤

1% FDR at the peptide level based on LFDR modeling and at the protein level, based on the number of proteins identified as hits to the reverse database. The spectral counts assigned to

30 proteins that satisfy these criteria and had a minimum of two unique peptides were exported to Excel for data processing.

Interaction bioinformatics analysis

Proteins identified by LC-tandem MS were filtered to exclude non-specific associations. Proteins were retained as specific interaction candidates if the proteins were assigned (1) at least ten spectral counts in the TBX20Avi;BirA condition; and (2) were uniquely identified or had at least a 4-fold spectral count enrichment in the TBX20Avi; BirA condition versus the control. Next, the subset of candidates assigned a nuclear or unknown

UniProt subcellular localization were retained for calculation of enrichment index values, as previously described (Joshi et al., 2013). Briefly, the relative protein abundance within the affinity purification was calculated using the NSAF approach (Zybailov et al., 2007), then normalized by each protein’s respective cellular abundance estimated in the PAX database

(Wang et al., 2015) (Mouse - whole organism, SC GPM 2014). Interaction candidates were ranked by their enrichment index and the top 50 proteins were analyzed by STRING

(Franceschini et al., 2013) for interaction network analysis. Interactions with a combined

STRING score of > 0.4 (medium confidence) were retained, exported, and visualized in

Cytoscape (ver. 3.3). Proteins within the network were assigned into broad protein functional classes based on annotations in the UniProtKB database.

Western Blot analysis

Western blots were probed with the following primary antibodies overnight at 4°C: mouse anti-V5 (Invitrogen) 1:5000; mouse anti-GFP (JL-8, Clontech) 1:10000; mouse anti-

HA-HRP (Cell Signaling #2999) 1:1000, mouse anti-GAPDH (Millipore) 1:1000; and chick anti-BirA (Abcam) 1:2000. After being rinsed, blots were rinsed in the following secondary antibodies for 1 hr at room temperature: anti-IgG2a-HRP (Jackson Immunoresearch)

31 1:10000. Antibody-antigen complexes were visualized using an ECL Western Blotting

Analysis System (Amersham).

Mice

Tbx20flox/+ mice were generously provided by Sylvia Evans (UCSD) (Cai et al., 2005).

The Casz1flox/+ mouse has been previously reported (Dorr et al., 2015). Histological sectioning and immunohistochemistry were done as reported except as noted (Dorr et al.,

2015). All mice are on a mixed B6/129/SvEv/CD-1 background and all mouse experiments were performed according to the Animal Care Committee at the University of North

Carolina, Chapel Hill.

Echocardiography

Cardiac function was assessed in conscious mice by thoracic echocardiography using

VisualSonics Vevo 770 ultrasound system (Visual Sonics, Inc.). All imaging was done by trained technicians blinded to the genotypes of the animals. Briefly, a topical hair removal agent was used on the chest and abdomen of mice. The mice were placed on a warmed table in the supine position for imaging. A 30 MHz pediatric probe was used to capture 2- dimensional guided M-mode views of the long and short axes at the level of the papillary muscle. VisualSonics Analytic software was used to determine mean ventricular wall and interventricular septum thickness, as well as the left ventricle diameter from at least 3 consecutive cardiac cycles. Means were used to calculate ejection fraction and fractional shortening.

Statistics

32 All statistical analysis was done using SAS JMP 10. Statistical significance between each pair of groups was calculated using Student’s T-test, while significance among more than 2 groups was calculated using ANOVA.

RESULTS

Defining the endogenous cardiac TBX20 interactome

To identify endogenous protein interactions that regulate TBX20 function, we introduced the Avitag, in-frame, to the carboxy terminus of Tbx20 through homologous recombination in mouse embryonic stem cells (ESCs) (Tbx20Avi)(Fig. S2.1A,B). Since the Avi- tag can be biotinylated through recognition of the Avi-tag sequence by the E. coli biotin ligase

BirA (Bayer and Wilchek, 1980; Maine et al., 2010), we generated a lentivirus expressing

BirA (Fig. 2.1A) and transduced it into mouse Tbx20Avi/+ ESCs. After hygromycin selection,

Tbx20Avi/+ ESCs that stably expressed BirA (Fig. S2.1C) were differentiated into induced cardiomyocytes (iCM) using a serum-free differentiation method that routinely generates cultures containing >60% cardiomyocytes (Fig. 2.1A, Movie S1) (Kattman et al., 2011).

Expression analysis at each day of differentiation confirmed the Tbx20Avi:BirA ESCs recapitulated the wild-type cardiomyocyte differentiation program (Fig. 2.1A-C). We further showed by Myosin Heavy Chain (MHC) expression and time lapse imaging that the

Tbx20Avi:BirA ESCs differentiated into beating neonatal cardiomyocytes (Fig. 2.1C, Movie

S1). Our analysis further verified that Tbx20Avi expression recapitulates endogenous Tbx20 expression with highest levels in immature cardiomyocytyes at day 4 and differentiated cardiomyocytes at day 7 (Fig. 2.1B).

Published data has demonstrated a requirement for TBX20 in adult mice, with loss of TBX20 leading to abrupt cardiac failure (Shen et al., 2011). Recently, mutations in TBX20 in humans were associated with DCM (Liu et al., 2008; Qian et al., 2008; Zhao et al., 2016).

To delineate the mechanisms of how TBX20 DCM-associated mutations circumvent the

33 essential requirements for TBX20 in cardiac development, we isolated and characterized the endogenous TBX20 cardiac interactome under physiological conditions from Tbx20Avi:BirA iCMs at day 7 of differentiation. As a control for non-specific interactions, identical affinity isolations were performed from BirA-negative iCMs (Fig. 2.1D,E). Proteins co-isolated from

TBX20Avi affinity purifications (APs) were analyzed by an SDS-PAGE tandem mass spectrometry-based proteomics approach, as in (Mathias et al., 2014). TBX20 was detected in the AP from BirA-expressing iCMs, with 19 unique tryptic peptides covering 54% of the

TBX20 sequence (out of a theoretical maximum coverage of ~75%) (Fig. 2.1E, Fig. S2.2A,B,

Table S1)

Identification of candidate high-confidence TBX20 interactions that have the potential to regulate cardiac functions was achieved using a multi-step bioinformatics approach based on the number of identified spectra per protein. First, interacting proteins identified by less than 10 spectra did not meet the identification requirement and were excluded from further analysis. Further, proteins identified in the BirA-expressing isolations were required to have at least a 4-fold increase in identified spectra over isolations from control iCMs. Due to the ascribed function of TBX20 as a critical cardiac transcription factor, we specifically focused on proteins with a nuclear or unknown subcellular localization. Finally, these interaction candidates were ranked by their AP enrichment (AP abundance versus whole cell abundance, Table S1), which we have previously used to highlight the most prominent associations suitable for functional validation (Joshi et al.,

2013; Tsai et al., 2012). Interestingly, the top 50 most enriched proteins in the TBX20 AP were predominately (32/50) annotated to Chromatin and Transcription gene ontologies

(Fig. 2.2A; Table S2.1). Functional annotation of these proteins in the STRING database

(Szklarczyk et al., 2015) revealed an interconnected network containing components of chromatin remodeling and RNA polymerase transcriptional complexes (Fig. 2.2A), including four components of the INO80 complex (Ino80, Actr5, Actr5, Nfrkb) and five components of

34 the RNA Pol II mediator complex (Med13, Med14, Med17, Med19, Med27). These data suggest that TBX20 predominantly acts to regulate transcription in neonatal cardiomyocytes through the INO80 and RNA Pol II mediator complex.

TBX20 complexes with CASZ1

In addition to identifying components of broadly expressed multiprotein chromatin machines, our analysis revealed the association of TBX20 with the essential cardiac transcription factor CASZ1 in the BirA-expressing iCMs (25 unique peptides and 21% sequence coverage) (Fig. 2.2B, Fig. S2.2C). Surprisingly, this was the only developmentally- regulated cardiac transcription factor we found to interact with TBX20 in Day 7 cardiomyocytes. The low estimated cellular abundance of CASZ1 and the relatively high AP enrichment ratio (22nd most enriched) (Table S1) highlighted CASZ1 as a potential in vivo

TBX20 interacting protein. We further confirmed the TBX20:CASZ1 interaction through reciprocal immuno-isolation of endogenous CASZ1 from adult cardiac nuclei (Fig. 2.2C) thus, verifying our ESC differentiation based approach can successfully identify bona fide

TBX20 interaction partners under physiological conditions. Since expression analysis and genetic fate mapping studies have shown CASZ1 is expressed only in cardiomyocytes and no other cardiac cell types (Dorr et al., 2015), and since we were unable to identify this interaction at Day 4 of iCM differentiation (data not shown), these studies imply the interaction between TBX20 and CASZ1 is temporally regulated and cardiomyocyte-specific.

Phylogenic analysis shows that TBX20 and CASZ1 are highly conserved across vertebrate orthologs (Charpentier et al., 2013a; Christine and Conlon, 2008), suggesting the

TBX20:CASZ1 interaction may also be evolutionarily conserved. To independently confirm the interaction and to determine whether it is conserved, we injected X. laevis embryos with the Xenopus orthologous mRNAs of TBX20 and CASZ1 and immunoaffinity purified Tbx20.

Immunoblotting of these isolates confirms the formation of a Tbx20:Casz1 interaction in X.

35 laevis embryos (Fig. S2.3). Taken together our findings are supportive of an evolutionarily conserved role for the formation of a TBX20:CASZ1 protein complex in differentiated cardiomyocytes.

Combined haploinsufficiency of Tbx20 and Casz1 results in dilated cardiomyopathy

To determine the biological relevance of the TBX20:CASZ1 interaction, we tested for genetic interaction between TBX20 and CASZ1 by generating mice with cardiac-specific heterozygous loss of Tbx20 and Casz1 (Tbx20flox/+; Casz1flox/+; Nkx2.5Cre)(Cai et al., 2005;

Dorr et al., 2015). Compound heterozygous mice, hereafter referred to as Tbx20flox/+;

Casz1flox/+, were born and appeared normal. However, beginning at 4 weeks of age we observed Tbx20flox/+; Casz1flox/+ mice exhibited decreased ambulation and, shortly thereafter, lethality with the percent of affected Tbx20flox/+; Casz1flox/+ mice increasing with age (Table 1).

We observed no overt phenotypes or lethality of single heterozygous Tbx20flox/+; Nkx2.5Cre or

Casz1flox/+; Nkx2.5Cre mice. Since we were able to demonstrate that loss of Casz1 does not affect Tbx20 expression in adult heart tissue and that loss of Tbx20 does not affect CASZ1 expression (Fig. 2.4S), these studies strongly support a genetic requirement for a functional interaction between TBX20 and CASZ1.

The Tbx20:Casz1 interaction is essential for normal cardiac function

Detailed physiological analysis of cardiac tissue in Tbx20flox/+; Casz1flox/+ mice at 4 weeks of age shows dilated ventricles (systolic left ventricular inner diameter of 1.6 mm compared to 1.4 mm for Casz1flox/+; Nkx2.5Cre and 1.2 mm for Tbx20flox/+; Nkx2.5Cre, ANOVA p

= 0.08) (Fig. 2.3A, Table 2.2; Movies S2.2-2.6: Movies show one example of each control and movies of two independent Tbx20flox/+; Casz1flox/+ mice) with an accompanying dramatic decrease in ventricular wall thickness (systolic interventricular septum thickness of 1.3 mm compared to 1.51 mm for Casz1flox/+; Nkx2.5Cre and 1.54 mm for Tbx20flox/+; Nkx2.5Cre, ANOVA

36 p = 0.06). (Fig. 2.3A). Thus, Tbx20flox/+; Casz1flox/+ mice display defining anatomical features of DCM prior to undergoing cardiac failure.

Since Tbx20flox/+; Casz1flox/+ mice display alterations in cardiac architecture associated with DCM prior to lethality, we sought to examine the cardiac function of Tbx20:Casz1 compound heterozygotes more closely. We conducted echocardiography and found

Tbx20:Casz1 compound heterozygotes exhibited significant decreases in ejection fraction

(58.8% in Tbx20flox/+; Casz1flox/+ compared to 82.2% in Nkx2.5Cre, 79.3% in Casz1flox/+;

Nkx2.5Cre, and 84.4% in Tbx20flox/+; Nkx2.5Cre, ANOVA p = 0.0010) and fractional shortening

(31.8% in Tbx20flox/+; Casz1flox/+ compared to 50.6% in Nkx2.5Cre , 47.4% in Casz1flox/+;

Nkx2.5Cre, and 53.5% in Tbx20flox/+; Nkx2.5Cre, ANOVA p = 0.0006) (Table 2, 3; Movies S2-6).

Moreover, these changes are associated with statistically significant increases in the left ventricular blood volume and the left ventricular diameter (Table 2, 3; Movies S2-6). These findings were observed in both female and male mice (Table S2, S3). Collectively, these findings suggest that the genetic interaction between Tbx20 and Casz1 is essential for normal cardiac homeostasis, and perturbation of this interaction leads to DCM.

Tbx20:Casz1 compound heterozygous hearts undergo cardiac remodeling

One of the defining clinical features of severe DCM is an accumulation of myocardial collagen leading to interstitial fibrosis, a contributing and compounding factor in cardiac dysfunction (Barison et al., 2015; Unverferth et al., 1986; Weber and Brilla, 1991). To determine if the severe cardiac dysfunction we observe in Tbx20:Casz1 compound heterozygotes is associated with advanced DCM, we examined collagen fibers and found robust collagen deposition in the interstitium of Tbx20flox/+; Casz1flox/+ hearts (Fig. 2.3B).

Despite the interstitial fibrosis and severely impaired systolic function, the fact that these mice survive to adulthood (Table 1) with some degree of cardiac function led us to hypothesize that Tbx20:Casz1 compound heterozygous cardiomyocytes undergo

37 compensatory pathological hypertrophy. To test this hypothesis, we measured cardiomyocyte cross-sectional areas and found that MF20-positive cells in compound heterozygotes were increased in size relative to controls (Fig. 2.3C). This data shows that disrupting the

TBX20:CASZ1 interaction leads to severe DCM accompanied by remodeling of myocardial architecture, all of which culminate in lethality.

A human DCM-associated TBX20 mutation disrupts the TBX20:CASZ1 interaction

Our data implies the TBX20:CASZ1 interaction is essential for normal cardiac homeostasis. To define the region of TBX20 that mediates interaction with CASZ1, we conducted immunoisolations with wild-type and deletion mutants in which either the T-Box or the C-terminus of TBX20 has been removed (Fig. 2.4A). Immunopurifications of CASZ1 in the presence of wild-type TBX20, TBX20ΔT-box, or TBX20ΔC, show that the T-box domain is required for interaction with CASZ1, but that the C-terminus is dispensable (Fig. 2.4A). In reciprocal studies, we find the four most amino-terminal zinc finger domains of CASZ1 are necessary for interaction with TBX20 (Fig. 2.4B). Both of these regions are shown to be highly conserved (Fig. 2.4C).

Recently, human TBX20 mutations have been associated with DCM however, only one of these mutations, TBX20F256I, co-segregates in a dominant manner with complete penetrance in a family with DCM (Zhao et al., 2016). Moreover, DCM was found in all affected family members reported as healthy during health assessments performed when they were juveniles. The functional relevance of the F256I mutation is further underscored by the finding that the amino acid disrupted by this mutation is 100% conserved across all

TBX20 orthologs and by the observation that no F256I mutations were identified in 600 control samples (Zhao et al., 2016). Interestingly, the F256I mutation associated with DCM lies within the TBX20 T-box domain, the region we found essential for interaction with

CASZ1 (Fig. 2.4A, 2.5A). To test if TBX20F256I perturbs the TBX20:CASZ1 interaction, we

38 performed immunopurifications of CASZ1 in the presence of wild-type TBX20 or TBX20F256I.

Results show that TBX20F256I markedly reduces interaction with CASZ1 (Fig. 2.5A,B). These data imply that the DCM mutation F256I may contribute to the development of cardiac disease by disrupting a critical physical interaction between TBX20 and CASZ1.

The TBX20F256I mutation acts to sterically inhibit the TBX20:CASZ1 interaction

To gain a structural understanding of how the TBX20F256I mutant is able to disrupt the

TBX20:CASZ1 interaction we conducted molecular modeling of the wild-type and TBX20F256I

T-box domain (Fig. 2.5B-C). The predicted structures were based on the range of fluctuations in the structure that occur over a period of 100ns (Movie S7). Three regions are highlighted which show conformational changes induced by the mutation (Fig. 2.5B). Our models find

F256 is not predicted to contact DNA but the conversion of phenylalanine to isoleucine at p.256 leads to steric clashes with the conserved T-box residues E258 and T259 (Fig. 2.4C).

The critical functional nature of this region of TBX20 is underscored by the complete conservation of amino acids at residues p.F256, p.E258, and p.T259 across 250 members of the T-box gene family (Fig. S2.4). Taken together, these findings imply TBX20F256I leads to a conformational change across the surface predicted to interact with CASZ1, and disruption of this interaction leads to alteration in DNA binding.

To determine the transcriptional consequences of TBX20F256I on the TBX20:CASZ1 interaction, we conducted transcriptional assays with TBX20, CASZ1 and TBX20F256I alone and in combination. Results demonstrate TBX20 synergistically acts with CASZ1 and that

TBX20F256I significantly diminishes transcriptional activation by TBX20:CASZ1. These data together with our structural studies provide a mechanistic basis for how F256I disrupts

TBX20:CASZ1 function.

Tbx20:Casz1 hearts dysregulate proteins associated with DCM

39 To identify the molecular pathways altered in DCM haploinsufficient mutant

(Tbx20flox/+; Casz1flox/+; Nkx2.5Cre) mouse hearts, we used quantitative multiplexed mass spectrometry to determine proteins with altered abundances relative to control hearts.

Proteins were extracted from nuclear-enriched mouse cardiac fractions of mutant and control

(Nkx2.5Cre) mice in duplicate and digested in-solution with trypsin. Peptides from each sample were labeled with different isobaric tandem mass tagging (TMT) reagents, pooled, fractionated, and analyzed by reverse phase nanoliquid chromatography coupled to a high resolution quadrupole Orbitrap tandem mass spectrometer. Using this strategy, 3,164 proteins were identified and quantified based on their respective sequenced peptides and

TMT reporter ions, respectively (Table S4).

To define the TMT ratio threshold for differential relative abundance, protein abundance values were compared between biological duplicates (Fig. 2.6-B, Fig. S2.6). For both the control and mutant replicates, the correlation of abundances was high (R2 = 0.99) and the majority of proteins had low dispersion from a 1:1 linear curve (Fig. 2.6A-B), indicating low biological and technical variation. Curve-fit analysis of TMT abundance ratio histograms for the control and mutant biological duplicates showed that, on average, 90% of the ratios varied less than ±30% (Fig. S2.6). Based on this result, a relative abundance ratio of at least ±1.3-fold between mutant and control mice in both replicates was used to identify a protein as differential. From the total number of quantified proteins, 175 met this criterion, of which 86 and 89 were up and down-regulated, respectively (Fig. 2.6C and Table S4).

To generate an initial picture of potentially dysregulated pathways, the known functional connectivity among differential proteins can be determined using databases of annotated pathways and protein-protein interaction. Towards this goal, known functional associations among the 175 differential proteins were scored based on the STRING bioinformatics database (Szklarczyk et al., 2017), and the relational networks visualized in

Cytoscape (Shannon et al., 2003) (Fig. 2.6D and Fig. S2.7). A high degree of interconnectivity

40 was observed among the differential proteins as 127 of the 175 annotated proteins had at least one other connection and each protein on average was connected to 4.6 neighbors. Network clustering was performed to identify subsets of highly connected proteins, which likely share similar functions. Overall, there are 10 functional clusters containing at least 3 proteins, indicated by the color-coding in Figure 2.6C. To identify the most significant biological processes and pathways that are perturbed in Tbx20:Casz1 DCM hearts, we performed comparative Gene Ontology over-representation analysis of the differentially regulated proteins using ClueGO (Bindea et al., 2009) (Fig. 2.6E). Consistent with studies demonstrating an association between DCM and inflammation (Muller-Werdan et al., 2016;

Ong et al., 2016), we found components of the pro-inflammatory response (i.e. complement activation) significantly up-regulated.

In addition to pro-inflammation, our data further found a dysregulation of mitochondrial proteins known to be associated with impaired cardiomyocyte contractile function in DCM (McNally et al., 2013). In line with these findings and the observation that reduced contractile force is linked to altered glycogen metabolism and cardiomyopathy (Ang et al., 2016; Chandramouli et al., 2015; Chavali et al., 2013), we found an over-representation of proteins associated with the glycogen metabolic pathway. We note that these were exclusively down-regulated proteins, represented by glycogen synthase (Gys1), glycogen phosphorylases (Pygm/Pygb), and phosphorylase kinase gamma 1 (Phkg1). Interestingly, proteins involved in glycogen regulation and in myosin-dependent muscle contractility were part of the same functional cluster (Fig. 2.6D, yellow nodes); however, their individual abundances were down- and up-regulated, respectively (Fig. 2.6D, circle vs. square nodes).

Taken together, these data confirm at the protein level the DCM pathology in TBX20:CASZ1

DCM mice.

TBX20:CASZ1-linked DCM is associated with dysregulation of cell-cell adhesion proteins

41 In addition to proteins previously reported to be associated with DCM, our analysis identified a distinct set of cell-cell adhesion proteins in Tbx20:Casz1 mutant hearts that were significantly overrepresented compared to the whole genome annotation (Fig. 2.6E, Fig. S2.7, yellow). These observations highlight the significant changes that are likely occurring in the extracellular and intracellular spaces and raise a key question. What are the signaling mediators that link these processes? To identify potential key mediators of TBX20:CASZ1

DCM, we constructed a gene-linked GO network for the Cellular Component ontology (Fig.

S2.8, S2.9). This network highlighted two interesting candidates, bone morphogenic protein

10 (Bmp10) and thrombospondin 1 (Thbs1), the former being a TGF-beta receptor ligand and the latter having roles in cell-cell adhesion as well as ER stress response (Lynch et al., 2012).

Overall, this systems-level proteome view of DCM provides potential downstream targets and pathways that may be influenced as a result of Tbx20 and Casz1 haploinsufficiency and suggests a role for cell-cell adhesion in mediating DCM.

DISCUSSION

One in five adults free of cardiovascular disease by the age of 40 are at risk of developing congestive heart failure over their lifetime (Lloyd-Jones et al., 2002; Roger et al.,

2012). Within this population DCM remains one of the leading causes of disease and death with nearly half of DCM cases genetically determined (Goerss et al., 1995; Grunig et al., 1998;

Keeling et al., 1995; Mestroni et al., 1999; Michels et al., 1992). To date, most DCM mutations have been identified in genes coding for components of the contractile apparatus or the cell cytoskeleton or factors involved in excitation-conduction coupling. Though these studies have provided insight into the pathology of DCM, the transcriptional regulation of DCM is poorly understood. Recently, studies have identified mutations in the T-box transcription factor TBX20 in DCM patients (Liu et al., 2008; Qian et al., 2008; Zhao et al., 2016).

However, these studies have not explained how DCM-associated mutations bypass early

42 essential requirements for TBX20. Here, we demonstrate TBX20 and CASZ1 physically and genetically interact in the adult heart and the TBX20:CASZ1 interaction is essential for cardiac homeostasis. We further find disruption of the TBX20:CASZ1 interaction in mice and humans leads to cardiomyopathy. Mice singly heterozygous for alleles of Tbx20 or Casz1 are asymptomatic, while Tbx20:Casz1 compound heterozygotes die post-natally, exhibiting systolic dysfunction at 4 weeks of age, as well as ventricular dilatation and interstitial fibrosis by 8 weeks of age. These cardiac defects, in the absence of coronary artery disease or substantially abnormal load, are defining features of DCM as seen in human patients

(Kimura, 2016; McMurray et al., 2012; Pinto et al., 2016).

CASZ1 and DCM

CASZ1 is a large para-zinc finger protein of unique structure and to date, there have been limited studies on the mechanisms of how CASZ1 regulates transcription (Charpentier et al., 2013b; Liu et al., 2006; Virden et al., 2012). These types of studies have been compromised by the lack of high-affinity high-specificity mammalian CASZ1 antibodies precluding approaches such as ChIP-seq. In addition, it remains unclear if CASZ1, as a para- zinc finger protein, directly binds DNA or is recruited via other transcription factors. Our structural studies favor a model by which the TBX20:CASZ1 interaction is required for DNA binding. This model predicts that the respective regions of TBX20 that binds CASZ1 is near to or contributes to the DNA binding interface and has the potential to impact CASZ1 binding.

This model is supported by the observation that TBX20F256I leads to a significant decrease in activation of the TBX20 target gene ANF/Nppa (Fig. 2.5D)(Zhao et al., 2016).

CASZ1 was first ascribed a role in vertebrate cardiovascular development in Xenopus

(Charpentier et al., 2013b; Christine and Conlon, 2008; Sojka et al., 2014). Subsequent genetic studies in mammals uncovered that like TBX20, CASZ1 functions in the embryonic heart to control cardiomyocyte proliferation with loss of CASZ1 leading to cardiac death by

43 E12.5 (Dorr et al., 2015; Liu et al., 2014). Our findings that Tbx20:Casz1 compound heterozygous mice die post-natally imply CASZ1 has a second and later role in cardiac homeostasis. This model is supported by the recent finding that mutations in CASZ1, like

TBX20, are associated with human DCM (Qiu et al., 2017).

TBX20:CASZ1 protein pathways and DCM

A previously published model of DCM, the phospholamban R9C transgenic mouse

(Schmitt et al., 2003), has also been studied by proteomic analysis (Gramolini et al., 2008;

Isserlin et al., 2010). This model exhibits impaired calcium regulation in cardiomyocytes, accompanied by decreased cardiac contractility and premature mortality (Schmitt et al.,

2003). The GO-associated proteome changes that we found in the haploinsufficient mice share similarities with the R9C mice. Specifically, both mouse models show up-regulation of actin-myosin cytoskeletal networks and down-regulation of mitochondria-associated proteins involved in fatty acid oxidation. Interestingly, proteomic analyses performed on ventricular tissues from human patients with inflammatory DCM also had similar findings (Hammer et al., 2011). Yet some functional protein classes in the haploinsufficient DCM mice were distinct, including an up-regulation of the complement system and greater coverage of down- regulated proteins in glycogen metabolic processes. While we found the haploinsufficient mice have evidence of differential regulation in calcium-binding proteins, not surprisingly, the R9C mice have more pervasive effects on calcium-dependent signaling, such as involving

ER stress responses. Though it is possible that these distinctions may also be due to differences in the progression of DCM.

One of the hallmarks of DCM is altered cardiomyocyte force transduction that is frequently associated with alteration in the composition or functions of intercalated discs; a cardiac specific structure at the contact site between cardiomyocytes (Li, 2014; Perriard et al.,

2003). Here, we observed a significant mis-regulation of proteins involved in cell-cell

44 adhesion in heart tissue from Tbx20:Casz1 mice. Moreover, these include three proteins which are encoded by genes that when mutated are causative to DCM; TTN, DES, and

PDLIM3. Thus, our findings imply that the TBX20:CASZ1 complex acts at least in part to control, directly or indirectly, integration of mechanical and possibly electrical integration or neighboring cardiomyocytes.

TBX20, CASZ1, and digenic inheritance of DCM

The observation that the TBX20F256I mutation leads to a decreased association with

CASZ1 and the finding that patients heterozygous for predicted TBX20 null mutations

(TBX20Q195X) (Kirk et al., 2007) also display DCM could suggest that the TBX20F256I mutation is acting in a haploinsufficient fashion. However, only 2 of the individuals within a single pedigree with the TBX20Q195X mutation display DCM while other individuals display a wide range of cardiac abnormalities (Kirk et al., 2007). Moreover, we screened the Exome

Aggregation Consortium (ExAc) reference set and have identified 4 variants in TBX20 in individuals that are asymptomatic. All variants lead to a premature stop codon in one of the

TBX20 alleles and all would be predicted to be functionally null (introduction of stop codons into exons 2, 4, 7 and 8)(Lek et al., 2016). Together, these findings would imply that the function of the TBX20:CASZ1 complex in DCM is not dose dependent but rather, individuals harboring the TBX20F256I mutation have a genetically sensitized background leading to the varying degree of penetrance potentially, carrying modifying genes in the CASZ1 pathway.

In cardiovascular disease, genetic mutations often result in varying degrees of penetrance, and in extreme examples the presence of a disease-causing mutation can be asymptomatic (Dahme et al., 2009; Faivre et al., 2007; Hobbs et al., 1989; Homsy et al.,

2015; Kathiresan and Srivastava, 2012; Zaidi et al., 2013). These phenomena have often been explained by the action of genetic modifiers in which one gene mutation is causative to CHD and a second mutation modifies the effect of the first. However, more recent studies suggest

45 an alternate or additional mechanism by which complete penetrance is achieved in human disease states by genetic variation at one or more loci (Cooper et al., 2013). In digenic inheritance, two genetic mutations are required for the clinical phenotype with either mutations alone being asymptomatic. Our findings provide an example of digenic inheritance in CHD and DCM and suggest mutations in TBX20 or CASZ1 could lead to susceptibility to

DCM but in many cases are not in themselves causative. We would envision these findings are not restricted to TBX20 and CASZ1 but rather are applicable to other genes and other forms of CHD, and genome sequencing of familial CHD will ultimately reveal a spectrum of additional CHD susceptibility alleles.

46 Figure 2.1 The TBX20Avi-BirA system for isolation of the TBX20 interactome. (A) Schematic diagram of Embryonic Stem Cell (ESC) transduction with Lenti-BirA and subsequent differentiation. (B) RT-PCR panel showing changes in gene expression ESCs undergo during the 8 days of differentiation into iCMs. (C) Immunohistochemistry of iCMs stained with the cardiomyocyte marker Myosin Heavy Chain (MHC) and counterstained with DAPI. (D)

Scheme of BirA-dependent biotinylation and streptavidin affinity isolation of TBX20 from iCMs. (E) Streptavidin affinity isolation of TBX20Avi confirmed in BirA-expressing iCMs by western blot analysis

47 Figure 2.2. Endogenous TBX20 interactome. (A) Functional interaction network of the top

50 most enriched TBX20 interaction candidates. Known functional relationships are based on the STRING mouse database. Nodes are labeled with the protein’s gene symbol and color-coded based on its primary UniProt annotated cellular role. (B) Proteomic detection of

TBX20 and CASZ1 in affinity purifitcations. The number of assigned spectra between control

(-BirA) and BirA-expressing (+BirA) iCMs, unique peptides, percent amino acid sequence coverage, and respective molecular weights are provided. (C) Co-immunoprecipitations from adult mouse hearts. CASZ1 immunoprecipitated using an antibody against CASZ1. TBX20 is detected as an interacting protein using Western blot analysis.

48 Figure 2.3 Double heterozygous hearts undergo pathological remodeling. (A-C) Hearts from mice aged 8-11 weeks. (A) Sections stained with hematoxylin and counter-stained with eosin.

(B) Sections stained with Picrosirius red and fast green, and visualized using bright field microscopy. (C) Polarizing light microscopy of Picrosirius red-stained sections. Thin collagen fibers stain green to yellow, while thicker collagen fibers stain orange to red. (D)

Sections were immunostained with tropomyosin (red, cardiomyocytes) and WGA (green, cell membranes). Region of left ventricular free wall shown. (E) Histogram of cardiomyocyte cross-sectional areas shown as mean ± sem of an average of 450+ cardiomyocytes per animal, 3 hearts per group. (F) Graph of left ventricular mass of control and mutant hearts.

ANOVA value was not significant when comparing the 4 groups. * p < 0.05, ** p < 0.0005.

Statistical significance between each pair of groups was calculated using Student’s T-test, while significance among more than 2 groups was calculated using ANOVA. A-C, scale bar is

300 um. D, scale bar is 20um.

49

F.

50 Figure 2.4. TBX20 and CASZ1 interact through their DNA binding domains. (A) (Top)

Schematic of full-length TBX20 and truncations. NLS, Nuclear Localization Signal, putative activation domain shown in green and putative repression domain in orange. (Bottom) Co- immunoisolations from X. laevis embryos expressing full-length CASZ1-V5 and either full- length HA-TBX20 or deletions shown in top panel. (B) (Top) Schematic of full-length CASZ1 and the truncations used in the co-immunoisolations. (Bottom) Co-immunoisolations from

X. laevis embryos expressing full-length HA-TBX20 alone, or in combination with either full-length CASZ1-V5 or truncations shown in the top panel. (C) (Top) Sequence alignment of TBX20 position 248 to 288 across 90 TBX20 orthologs. Height of letters is relative to conservation at that residue. (Bottom) Sequence alignment of CASZ1 at positions 601 to 650 across 90 CASZ1 orthologs.

51

52 Figure 2.5. TBX20F256I impairs the TBX20-CASZ1 interaction. (A) (Top) Full-length TBX20 showing the location of p.F256I. (Bottom) Co-immunoisolations of full-length wild-type

CASZ1 with wild-type TBX20 or TBX20F256I. (B) Ribbon models of the average structures of

TBX20 calculated from the 100 ns molecular dynamics simulations of the T-box domain.

Left panel: starting structure of wild-type TBX20 (cyan) including DNA for reference.

Right panel: an overlay of wild-type TBX20 (green) and TBX20F256I (magenta). p.F256 and p.I256 side chains are displayed in stick form. Regions designated 1, 2 and 3 in right panel undergo conformational changes induced by the mutation in the unbound form of the T-box domain. (C) Enlargement of the p.F256I mutation shown in panel B in a different side chain rotamer that also induces steric clashes. The p.F256I mutation is rendered in purple stick form, with small red discs indicating steric clashes between the side chain of p.I256 and T- box residues p.E258 and p.T259. (D) Transcriptional assay demonstrating the reduction in

ANF reporter activity due to the combined effects of TBX20F256I and CASZ1 as compared to the combined activity of wildtye TBX20 and CASZ1. Results represent average of 3 experiments ± SEM. * p < 0.05, **p < 0.005.

53

54 Figure 2.6. Proteomic analysis of mouse hearts with DCM reveals activation of complement cascades and decreased protein abundance involved in glycogen metabolic processes. (A-B)

Comparison of normalized protein abundances (black dots, N = 3164) quantified by TMT- based proteomics between biological replicates of (A) Nkx2.5Cre (Control, CTL) and (B)

Tbx20flox/+; Casz1flox/+; Nkx2.5Cre (Mutant) mouse hearts. (C) Comparison of average normalized TMT protein abundances between Mutant and CTL mouse hearts (N = 2).

Proteins classified as differentially abundant in the Mutant (TMT abundance ratios ≥ ±1.3- fold in both replicates) are indicated (red dots, N = 175). (D) Functional interaction network of differential proteins constructed using the STRING interaction database. Proteins with up and down-regulated abundances are represented by circle and square nodes, respectively, labeled with primary gene symbols. Proteins that did not have connectivity to at least one other protein were excluded. Nodes are color-coded by cluster connectivity, which was assigned by the ReactomeFI plug-in (see Methods). (see Supplementary Figure 2.7).

Network edges reflect known STRING database relationships (confidence score > 0.4). Edge thickness correlates with STRING confidence score (0.4 – 1). (E) Gene ontology (GO)-based network of selected over-represented Biological Processes represented by the differential proteins. Over-represented GO terms (p-value < 0.05), are colored according to the proportion of the total differential proteins annotated to that term that were up (yellow) or down (blue) regulated. Protein to GO terms assignments are indicated by network edges connecting the respective gene symbols (squares) with their assigned GO term. For the complete network see Supplementary Figure 2.8.

55

56 Table 2.1. Lethality in mutant and control mice at 4, 8 and 16 weeks. Lethality shown as percentage of the total shown in parenthesis.

57 Table 2.2. The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in young adult mice aged 4 - 7 weeks. Mean ± SEM. LVEF - Left Ventricular Ejection Fraction,

LVFS - Left Ventricular Fractional Shortening, LVVol;D - Left Ventricular Volume at End

Diastole, LVVol;S - Left Ventricular Volume at End Systole, LVID;D - Left Ventricular Inner

Diameter at End Diastole, LVID;S - Left Ventricular Inner Diameter at End Systole, IVS;D -

Interventricular Septum thickness at End Diastole, IVS;S - Interventricular Septum thickness at End Systole, LVPW;D - Left Ventricular Posterior Wall at End Diastole, LVPW;S

- Left Ventricular Posterior Wall at End Systole. * p < 0.05, ** p < 0.0005: indicated control group compared to compound heterozygotes. # p < 0.05, indicated group compared to

Tbx20flox/+; Nkx2.5Cre.

58 Table 2.3 The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in mice aged 8-11 weeks. Mean ± SEM. LVEF - Left Ventricular Ejection Fraction, LVFS - Left

Ventricular Fractional Shortening, LVVol;D - Left Ventricular Volume at End Diastole,

LVVol;S - Left Ventricular Volume at End Systole, LVID;D - Left Ventricular Inner Diameter at End Diastole, LVID;S - Left Ventricular Inner Diameter at End Systole, IVS;D -

Interventricular Septum thickness at End Diastole, IVS;S - Interventricular Septum thickness at End Systole, LVPW;D - Left Ventricular Posterior Wall at End Diastole, LVPW;S

- Left Ventricular Posterior Wall at End Systole. * p < 0.05, ** p < 0.005, *** p < 0.0005: indicated control group compared to compound heterozygotes.

59 SUPPLEMENTAL MATERIALS AND METHODS

DNA constructs

The pLenti-BirA plasmid was obtained from Addgene (Addgene plasmid 29649; principal investigator Eric Campeau) and modified to contain a polyadenylation signal for expression in mammalian cells. In collaboration with the UNC gene therapy core facility, the pLenti-BirA plasmid was packaged and purified into a concentrated lentivirus. The EGFP epitope tag was fused to Mus musculus Tbx20a cDNA and cloned into pSP64TxB as previously described (Kaltenbrun et al., 2013). The HA epitope tag was fused to full length

Xenopus laevis xTbx20a, and to truncations of xTbx20a lacking either the T-box (xTbx20ΔT- box) or the carboxy terminus downstream of the T-box (xTbx20ΔC) and cloned into pSP64TxB. The V5 epitope tag was fused to Xenopus laevis Casz1 cDNA and cloned into pSP64TxB (Christine and Conlon, 2008). The V5 epitope tag was also fused to truncations of xCasz1 retaining amino acids 1-830 (xCasz11-830-V5), amino acids 562-999 (xCasz1562-999-V5), or amino acids 786-1109 (xCasz1786-1109-V5). The mTbx20-EGFP plasmid was generated as previously described (Kaltenbrun et al., 2013). The mTbx20F256I-EGFP plasmid was generated following the manufacturer’s instructions for the Quick Change II Site-Directed

Mutagenesis Kit (Agilent #200521-5).

RNA extraction and RT-PCR

RNA was extracted using Trizol (Invitrogen) and purified on RNeasy columns

(Qiagen). cDNA synthesis was performed from 0.5-1 µg of RNA using random primers and

SuperScript II reverse transcriptase (Invitrogen). Expression levels were assessed using

GoTaq Green Master Mix (Promega) and Taq polymerase on a GeneAmp PCR System

(Applied Biosystems). PCR products were analyzed by 2.5% agarose gel electrophoresis.

Immunoprecipitations

60 Co-immunoprecipitations from adult hearts were done as previously described

(Waldron et al., 2016). Briefly, 5 adult hearts were ground to a powder using a chilled mortar and pestle. Powder was resuspended in homogenization buffer and homogenized using 20 strokes in a chilled glass vial using a Teflon dounce. Larger tissue clumps were separated from nuclei and excluded from further processing by passing the homogenate through a

10uM filter. The nuclei were collected by gentle centrifugation followed by sucrose-assisted concentration, then were rinsed 3X with homogenization buffer. Nuclei were pelleted and resuspended in 20 mM HEPES/1.2 % PVP/1X protease inhibitors, and frozen drop-wise in liquid nitrogen. Frozen pellets were solubilized in 5 mls lysis buffer, and homogenized using a hand-held polytron. Cell debris was pelleted by centrifugation, a fraction of the lysate was removed and reserved for input, and the remaining volume of cleared lysate was incubated with 5 mg magnetic beads conjugated with 50 ug rabbit anti-CASZ1 antibodies (Aviva Bio

Systems).

Preparation and injection of Xenopus laevis embryos was carried out as previously described (Wilson and Hemmati-Brivanlou, 1995). Embryos were staged according to

Nieuwkoop and Faber (Nieuwkoop and Faber, 1994). Plasmid DNA was linearized using the following enzymes: pSP64TxB-mTbx20a-EGFP with Xba1; pSP64TxB-HA-xTbx20, pSP64TxB-HA-xTbx20ΔT-box, and pSP64TxB-HA-xTbx20ΔC with BamH1; pSP64TxB-xCasz1-

V5, pSP64TxB-xCasz11-830-V5, pSP64TxB-xCasz1562-999-V5, pSP64TxB-xCasz1786-1109-V5 with

Sal1. Using the mMessage mMachine® SP6 Transcription Kit (Life Technologies) linear

DNA served as a template for synthesizing capped sense mRNA according to the manufacturer’s instructions.

X. laevis embryos were injected at the one cell stage with 500ng with mTbx20-EGFP and xCasz1-V5 alone and in combination. Immunoprecipitations were conducted as reported (Conlon et al., 2012; Greco et al., 2012). Briefly, embryos were deyolked, lysed, and homogenized, then cell debris was pelleted at 8000 rpm at 4°C, and protein concentration

61 was determined using a Pierce Coomassie Plus (Bradford) Assay Kit (Life Technologies

#23236). Five percent of the cell lysate was reserved for input and remaining fraction incubated with 2 µg/1 mg protein immunoprecipitation antibody. To isolate full-length xCasz1-V5 expressed with xTbx20 deletions, cell lysates were incubated with mouse anti-V5

(Invitrogen #R960CUS) overnight at 4°C while rotating. To isolate full-length HA-xTbx20 expressed with xCasz1 deletions, cell lysates were incubated with rabbit anti-HA antibodies

(Invitrogen #71-5500) overnight at 4°C while rotating. Protein G Agarose beads (Sigma

#P7700) were hydrated and stored at 4°C in a storage solution (20 mM NaH2PO4) as a 50% bead slurry until all other reagents were prepared for co-immunoprecipitation experiments.

Following overnight lysate-antibody incubation, 200 µL Protein G Agarose bead slurry was rinsed with 400 µL lysis buffer 3 times. After the final rinse, beads were pelleted at 3000 rpm for 30 sec, the supernatant was removed and 1 mL of the lysate-antibody mixture was used to transfer the beads to the remaining volume of lysate-antibody mixture. The lysate- antibody-bead mixture was incubated at 4°C with rotation for 2 hr. Immunoisolated protein complexes were separated from solubilized unbound factors by centrifugation at 3000 rpm for 30 sec at room temperature. Beads were rinsed with 400 µL lysis buffer 3 times. After the final rinse, proteins were eluted from beads in 60 µL sample buffer (50 mM dithiothreitol,

1X LDS sample buffer (Life Technologies #NP0007)) at 70°C for 15 min and resolved as previously described.

Cardiomyocyte cross sectional area

To determine cardiomyocyte cross-sectional area, paraffin sections were dewaxed and re-hydrated using standard protocols; and to determine cardiomyocyte mitotic index, frozen sections were rinsed with 1X PBS to remove OCT. Following these preparative steps, sections were treated with heat-induced antigen retrieval as previously described (Dorr et al., 2015). Briefly, sections were heated in sodium citrate buffer (10mM sodium citrate,

62 0.05% Tween 20, in water; pH 6.0) in a steamer for 10 min (for cardiomyocyte cross- sectional area experiments) or 20 min (for mitotic index experiments), cooled for 5-10 min, and rinsed in distilled water (3 x 5 min) and in 1X PBS for 5 min. Sections were blocked (10%

FBS, 1% TritonX-100 in 1X PBS) at room temperature for 20 min (for cardiomyocyte cross- sectional area experiments) or 1 hr (for mitotic index experiments), and probed with one of the following primary antibodies overnight at 4°C: mouse anti-tropomyosin (DHSB; cardiac, clone CH1) 1:50; anti-wheat germ agglutinin conjugated with Alexa fluor 647 (Life

Technologies) 1:1000; mouse anti-sarcomeric actin (DHSB) 1:50. Sections were then incubated with Alexa 488 goat anti-mouse IgG, H+L (Molecular Probes) 1:1000 for 30 min to 1 hr at room temperature, rinsed, incubated with 200 ng/mL DAPI (Sigma) for 10 min and mounted with Permafluor (Thermo Scientific).

Collagen Staining

For detection of myocardial collagen deposition, dewaxed and re-hydrated sections were incubated in 0.1 % Fast green (0.1% w/v Fast Green FCF (Fisher Scientific #BP123-10) in distilled water) for 10 min, tap water for 5 sec, 0.1 % Picrosirius red (0.1% w/v Direct red

80 [Sigma #365548] in saturated picric acid (Fisher Scientific #SP9200)) for 10 min, 95% ethanol for 5 min, 100 % ethanol (2 X 5 min), and xylene (2 X 5 min). Sections were mounted and images captured using the polarizing light feature on the BX61 microscope.

Colors were interpreted as previously described (Pereira et al., 2013; Tarantal et al., 2010).

Modeling and Molecular Dynamics Simulations of the TBX20 T-box Domain

The fold recognition server HHpred (Alva et al., 2016), along with BLAST pairwise sequence similarity searches (Altschul et al., 1990), identified the crystal structure of the T- box domain of human TBX1 (PDB ID 4a04) bound to DNA (El Omari et al., 2012) as a suitable template for structural modeling of the T-box domain of mouse TBX20. The T-box

63 domains of TBX1 and TBX20 are clear homologs, sharing 63% sequence identity. The model of the T-box domain of TBX20, including DNA and 8 buried water molecules, was built using MODELLER 9.16 (Webb and Sali, 2014).

Explicit solvent molecular dynamics (MD) simulations of the wild-type and F255I mutant apo-TBX20 T-box domains, with buried waters, were used to identify conformational changes that might elucidate the loss of CASZ1 binding in TBX20F256I. The mutant TBX20F256I model was generated in PyMOL (Schrodinger, 2015) using the

Mutagenesis wizard. Seven chloride ions were added using the LEaP module of Amber to neutralize the overall charge of the molecule, and then 14 Å of TIP3P water molecules surrounding the T-box domain were added to yield a rectangular box size of 78.7 Å by 81.7 Å by 98.4 Å containing a total of 16405 water molecules. The Amber ff12SB force field (Perez et al., 2007) was used for simulation runs using the pmemd.cuda module of the Amber 14 software suite applying periodic boundary conditions (Case et al., 2014). For equilibration,

10,000 steps of minimization were run, switching from steepest descent to conjugate gradient methods after 5000 steps. This was followed by 5 ps of NVT heating to 100 K, then

100 ps of NVT heating to 300 K, followed by 10 constant pressure (NTP at 1 atm) density equilibration runs of 500 ps each. The production run was 100 ns in the NTP ensemble at 1 atm. The particle mesh Ewald method for long-range electrostatic estimation was used with a 10 Å cutoff. Average structures and rmsd statistics over 0-100 ns of the production run were calculated using the cpptraj module of Amber.

Quantitative TMT-based mass spectrometry analysis of mice hearts

Protein abundance changes in the hearts of Tbx20flox/+; Casz1flox/+; Nkx2.5Cre

(mutant) mice were measured using a multiplexed, quantitative mass spectrometry proteomic approach. Hearts from mutant and Nkx2.5Cre (control) mice were perfused, dissected, flash frozen in liquid nitrogen, and stored at -80°C. In parallel, control and

64 mutant hearts (N = 2 each) were ground in a chilled mortar and pestle, and lysed in 4 mLs lysis buffer (150mM NaCl, 0.5% TritonX-100, 20mM K-HEPES, 110mM KOAc, 2mM MgCl2,

0.1% Tween20, 1uM ZnCl2, 1uM CaCl2, 1X protease inhibitors, 1X phosphatase inhibitors).

Once resuspended, lysates were homogenized using the Kinematica PT1200E tissue homogenizer and cleared of cellular debris via centrifugation at 3K rpm at 4°C for 10 min.

Cleared lysates were treated with 10U/mL RNAse A and DNAse for 30 min at room temperature. Following nuclease treatment, proteins were acetone-precipitated from tissue lysates and reconstituted in 7M urea. Heart protein lysates (75 ug) were simultaneously reduced and alkylated in 20 mM TCEP and 20 mM chloroacetamide, respectively, for 45 min at 37°C. Proteins were then digested to peptides in-solution using a Filter-aided Sample

Preparation (FASP) approach (Greco et al., 2016; Wisniewski et al., 2009) with modification. Briefly, Amicon-500 ultrafiltration devices were used to buffer exchange reduced and alkylated lysates by sequential washing (1 x 0.4 mL, 2 x 0.3 mL) in 0.1M

TrisHCl, pH 8.0, containing 8M urea, interleaved with centrifugation at 14,000 x g for 10 min at 20°C. After the final centrifugation, 20 uL of LC-MS grade water containing 1.5 ug of

LysC/trypsin (Promega) was added to the filter and incubated for 2 hrs at 37°C. Samples were diluted 1:4 with 20 mM TrisHCl, pH 8.0 and incubated for 4 hrs at 37°C. The filters were centrifuged as above, then washed 2 x 50 uL with water. The filtrate containing tryptic peptides was recovered, desalted over StageTips (Greco et al., 2016), concentrated to dryness, and dissolved in 0.1M HEPES, pH 8.2, containing 20% ACN. The peptide concentration was determined by the colorimetric peptide BCA assay kit (Thermo Scientific

Pierce).

For each sample, equivalent peptide amounts (30 ug) were labeled with 0.16 mg of

TMT6plex tagging reagent (Thermo Scientific Pierce #90061) (TMT label 128 and 129 for control duplicates and 130 and 131 for mutant duplicates) for 1 hr at RT. The labeling was quenched with hydroxylamine (0.33%) for 10 min at RT. The TMT-labeled peptides were

65 combined and separated by 2D StageTip fractionation. First, the peptides were separated into four fractions over C18-SCX StageTips with sequential elution (90 uL) in 0.05 M ammonium formate/20% acetonitrile (ACN), 0.05 M ammonium acetate/20% ACN, 0.05 M ammonium bicarbonate/20% ACN, and 0.1% ammonium hydroxide/20% ACN. Then, each fraction was diluted to 10% ACN, adjusted to 0.5% TFA, and further separated into three fractions over SDB-RPS StageTips with sequential elution (40 uL) in 0.2 M ammonium formate/0.5% formic acid (FA)/60% ACN, 0.2 M ammonium acetate/0.5% FA/60% ACN, and 5% ammonium hydroxide/80% ACN. The resulting 12 fractions were evaporated to near dryness and dissolved in ~5 uL of 1% FA/5% ACN.

Peptides (2 uL) were analyzed by nanoliquid chromatography-tandem mass spectrometry on a Dionex Ultimate 3000 UPLC coupled to an EASYSpray ion source and Q

Exactive HF quadrupole orbitrap mass spectrometer (Thermofisher Scientific). Briefly,

TMT-labeled peptides were separated over a 2.5 hr linear gradient (5 to 40% solvent B; solvent A: 0.1% FA, solvent B: 0.1% FA/97% ACN) by reverse phase chromatography

(EASYSpray C18 column, 75 um x 50 cm). As peptides eluted from the column, a data- dependent MS acquisition method was employed, which consisted of an analytical MS scan, measuring intact peptide ions, followed by up to 15 tandem MS fragmentation events

(resolution: 30,000 at m/z = 400) on the most intense ions.

Informatics analysis of quantitative MS data

Tandem MS spectra collected across the 12 LC-MS/MS runs were analyzed by

Proteome Discoverer (Thermofisher Scientific, ver 2.1 SP1). In summary, a Processing workflow containing the SEQUEST algorithm was used to search tandem MS spectra against a UniProt-SwissProt mouse database and common contaminants (2016-07, 16,810 sequences), generating peptide spectrum matches. Percolator was used to calculate spectrum q-values based on reverse sequence database searches. For TMT-based

66 quantitative analysis, the reporter ion quantifier module extracted TMT signal-to-noise values from MS/MS spectra. A Consensus workflow was employed which defined peptide and protein filters to limit the false discovery rate to ≤ 1%, then assembled peptides into protein groups with strict parsimony, and retained quantitative values from spectra that (1) were unique to the protein group, (2) had a co-isolation threshold of < 25%, and (3) had an average S/N of > 10. TMT S/N values were normalized between TMT reporter channels based on the total sum of the S/N values within each channel over all identified peptides.

Protein abundances were calculated as the sum of normalized spectrum S/N values for each channel within each protein group. Protein groups and associated TMT abundances for each sample were exported to Excel and only those containing a minimum of 2 quantified unique peptides were considered for further analysis. The TMT abundance ratio for the experimental comparison (mutant versus control) was calculated as the abundance of each mutant replicate abundance versus the average abundance in the control (N = 2). The fold- change threshold for differential protein abundance was selected empirically based on 5-

95% confidence interval limits of the TMT ratio distribution for biological replicates (e.g. mutant 1 vs. mutant 2), which provides an estimate of the biological and technical variability. Protein groups meeting this threshold (±1.3-fold, see Supplemental Figure 2.4) for both mutant replicate conditions were considered significant and analyzed by GO and pathway over-representation analysis.

Network and GO analysis of quantitative MS data

The corresponding primary gene symbols for differentially up and down-regulated protein group were analyzed by Cytoscape (v3.4.0) (Shannon et al., 2003) using the ClueGO plug-in (v2.3.2) (Bindea et al., 2009). Gene Ontology-based networks were assembled using the Cluster comparison function. GO networks contained terms that were (1) over- represented in the subset of differential genes versus the reference mouse genome

67 (Bonferroni corrected p-value < 0.05), and (2) had at least 4 annotated genes or ≥ 66% of the differential genes were either up or down-regulated. STRING protein networks were constructed within Cytoscape platform at a confidence score cut-off of 0.4, using only the differential genes to establish network connectivity. STRING networks were assigned functional clusters using the ReactomeFI PlugIn (Wu et al., 2010).

68 Figure S2.1 Construction of the Tbx20Avi allele. (A) Schematic representation of the strategy for targeting the Avi-tag by homologous recombination in-frame downstream of exon 8 (E8) of the Tbx20 genomic sequence. Neo - neomycin resistance gene. PGK - phosphoglycerate kinase-1 promoter. Exons colored black are coding exons, white represent untranslated exons (3’ UTR). Schematic of the endogenous locus (Top), the Avi-tag targeting construct

(Middle), schematic of the Tbx20 recombined locus (Bottom)(Tbx20Avi). (B) Southern blot of HindIII fragments of the Tbx20 locus, showing successful integration of the Avi-tag into one allele of the Tbx20 locus. (C) Immunoblot showing stable levels of BirA protein after

BirA transduction and 4 days of directed differentiation.

69 Figure S2.2 MS/MS identification of TBX20 and CASZ1. (A) Sequence coverage (yellow) of

TBX20 detected by MS/MS. (B) Representative MS/MS spectrum of TBX20 peptide identified in TBX20 (+BirA) isolation from Day 7 iCMs. (C) Representative MS/MS spectrum of CASZ1 peptide identified in TBX20 (+BirA) isolation from Day 7 iCMs.

70 Figure S2.3 The TBX20-CASZ1 interaction occurs with both the Xenopus laevis and Mus musculus protein isoforms. (A) Co-immunoprecipitations of murine isoforms of TBX20-

EGFP and Flag-CASZ1 were from HEK293 cells. (B) Co-immunoprecipitations from

Xenopus leavis embryos ectopically expressing TBX- 20-EGFP alone, or in combination with

CASZ1-V5.

71 Figure S2.4 TBX20 is highly conserved at p.F256, p.E258, and p.T259. (A) Sequence conservation bracket of TBX20 (highlighted) and 250 human T-box transcription factors and their various isoforms. (B) Sequence logo showing conservation at amino acid positions 251 to 271 of the TBX20 protein sequence. * indicates p.F256, and # indicates p.E258 and p.T259, the two highly conserved amino acids that interact with p.F256.

72

73

74 Figure S2.5. TBX20 and CASZ1 expression in the left ventricles of control and mutant hearts.

(A) In situ hybridization showing the expression of Tbx20 transcripts in adult cardiac tissue.

(B) Immunohistochemical analysis of CASZ1 in adult cardiac tissue. Boxed regions are enlarged in the images on the bottom row. Green – cardiomyocytes, blue – nuclei, red –

CASZ1. Scale bar is 10 uM.

75 Figure S2.6 Distribution of TMT ratios for biological duplicates. Distribution of TMT abundance ratios (log2) for the biological duplicates of Nkx2.5Cre (Control, CTL) (top) and

Tbx20flox/+; Casz1flox/+; Nkx2.5Cre (Mutant) (bottom) samples. The table summarizes the mean, standard deviation (SD), and 5-95% interval for each distribution, determined from curve-fit analysis (Mathwave EasyFit software).

76 Figure S2.7 STRING network of differential proteins coded by Mut/CTL TMT abundance ratio. Functional interaction network of differential proteins constructed using the STRING interaction database. The layout of the main interconnected network (left) is identical to

Figure 6D. Proteins with up and down-regulated abundances are represented by circle and square nodes, respectively, labeled with primary gene symbols. The 42 proteins that did not have connectivity to at least one other protein are shown on the right. Nodes are color-coded by log2 TMT ratio. Network edges reflect known STRING database relationships (confidence score > 0.4). Edge thickness correlates with STRING confidence score (0.4 – 1).

77 Figure S2.8 Over-represented GO Biological Process Network for Differential Proteins. Over- represented GO terms (p-value < 0.05) are color-coded according to the proportion of the total differential proteins annotated to that term that were up (yellow) or down (blue) regulated. Protein to GO terms assignments are indicated by network edges connecting the respective gene symbols (squares) with their assigned GO term. A subset of this network is illustrated in Figure 2.6E.

78 Figure S2.9 Over-represented GO Cellular Component Network for Differential Proteins.

Gene ontology (GO)-based network of over-represented Cellular Components represented by the differential proteins. Over-represented GO terms (p-value < 0.05) are color-coded according to the proportion of the total differential proteins annotated to that term that were up (yellow) or down (blue) regulated. Protein to GO terms assignments are indicated by network edges connecting the respective gene symbols (squares) with their assigned GO term.

79 Table S2.1. Proteins that interact in the endogenous TBX20 transcriptional network isolated from Day7 iCMS. This table can be found online as a part of the data supplement for the manuscript.

80 Table S2.2 Cardiovascular physiology in young adult female and male mice. Mean ± SEM.

LVEF - Left Ventricular Ejection Fraction, LVFS - Left Ventricular Fractional Shortening,

LVVol;D - Left Ventricular Volume at End Diastole, LVVol;S - Left Ventricular Volume at

End Systole, LVID;D - Left Ventricular Inner Diameter at End Diastole, LVID;S - Left

Ventricular Inner Diameter at End Systole, IVS;D - Interventricular Septum thickness at

End Diastole, IVS;S - Interventricular Septum thickness at End Systole, LVPW;D - Left

Ventricular Posterior Wall at End Diastole, LVPW;S - Left Ventricular Posterior Wall at End

Systole. * p < 0.05, ** p < 0.005: comparisons between the indicated controls and the sex- matched compound heterozygotes.

81 Table S2.3 Cardiovascular physiology in mature adult female and male mice. Mean ± SEM.

LVEF - Left Ventricular Ejection Fraction, LVFS - Left Ventricular Fractional

Shortening, LVVol;D - Left Ventricular Volume at End Diastole, LVVol;S - Left Ventricular

Volume at End Systole, LVID;D - Left Ventricular Inner Diameter at End Diastole, LVID;S -

Left Ventricular Inner Diameter at End Systole, IVS;D - Interventricular Septum thickness at

End Diastole, IVS;S - Interventricular Septum thickness at End Systole, LVPW;D - Left

Ventricular Posterior Wall at End Diastole, LVPW;S - Left Ventricular Posterior Wall at End

Systole. * p < 0.05: comparisons between the indicated controls and the sex-matched compound heterozygotes.

82 Table S2.4. Proteins showing significant changes in TBX20-CASZ1 mutant mouse hearts.

This table can be found online as a part of the data supplement for the manuscript.

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94

CHAPTER 3: DISCUSSION

Developmental Requirements for Protein-Protein Interactions

In this dissertation proposal, I test the hypothesis that the cardiac transcription factors TBX20 and CASZ1 biochemically interact, and that this transcriptional complex performs critical functions in the cardiomyocyte. This structural-functional interaction is especially intriguing considering the global scale of the cellular environment. Such a scale introduces inter-protein proximity, inter-protein binding affinities, protein levels, and protein turnover rates as factors that influence the capacity for proteins to form networks (Li et al., 2008; Subbotin and Chait, 2014). Countless molecules and organelles being packed into a compact environment necessitates considering protein functions in the context of the networks in which they exist. These factors must also be considered when designing and applying molecular therapeutics for disease treatment.

In the context of the cardiomyocyte, previous in vivo and in vitro work has established a requirement for protein-protein interactions in development (Brown et al.,

2005; Stennard et al., 2005; Vincentz et al., 2008). In vitro assays have also supported combinatorial transcriptional activity, with TBX20 showing enhanced transcriptional activity on Anf, Mef2c, or Gja5 promoters when combined with either NKX2-5, GATA4,

GATA5, SMAD1, SMAD5, or ISL1, alone or in various combinations (Brown et al., 2005;

Debenedittis et al., 2011; Kaltenbrun et al., 2013; Singh et al., 2009; Stennard et al., 2003;

Takeuchi et al., 2005). Together, these data indicate TBX20 can function in transcriptional networks to regulate expression of its target genes.

Generating Pluripotent Material for Isolating Endogenous TBX20 Complexes

95 As part of this thesis dissertation, I collaborated on a project to isolate an endogenous cardiac TBX20 transcriptional complex from cardiac tissue. Although isolating TBX20 from embryonic or adult hearts would best represent an endogenous purification, suboptimal antibody specificity and affinity precluded this option as a feasible method for immunoisolating TBX20. Additionally, designing a cell culture system using pluripotent cells produces material with high plasticity and potential for differentiation into various tissue types. To isolate endogenous TBX20 co-factors we collaborated to first design a vector to target the AviTag epitope in-frame to the C-terminus of the TBX20 coding sequence using homologous recombination. The AviTag is a small synthetic peptide that has been used extensively in affinity purifications (Bayer and Wilchek, 1974; Bayer and Wilchek, 1980;

McCormick, 1965). Previous work from our research group has shown that introduction of the AviTag in vivo has no effect on the function of the tagged protein or on animal health or viability (Waldron et al., 2016). Stem cells have become a great tool for generating several tissue types. Once differentiated, these tissues are a feasible material for studying the morphological, physiological, and molecular changes that can occur as a result of drug treatments and mechanical or genetic disruptions.

Generating the Tbx20Avi allele was done in mouse embryonic stem cells due to the high level of conservation of the mouse TBX20 sequence to the human TBX20 sequence

(97.5% identity), and due to the well-established protocols for differentiating mouse embryonic stem cells (ESCs) into cardiomyocytes (Kattman et al., 2011). These in vitro differentiation protocols induce molecular and physiological changes that mimic the endogenous cardiomyocyte differentiation program. Endogenously, a subset of cells within the cardiac mesoderm undergo specification, determination, patterning, and differentiation in a sequential and overlapping manner to become the cardiomyocytes of the linear heart tube. This tube will then elongate, loop, and septate to form the 4-chambered heart. To induce differentiated cardiomyocytes (iCMs), Tbx20Avi ESCs virally transduced to express

96 BirA were treated with optimized levels of VEGF, Activin A, and BMP4 over a period of 7 days according to a modified version of the Kattman et al. protocol (Kattman et al., 2011).

Over this period, cells undergo a series of changes, and at day 3 (D3) of the differentiation, cells dramatically, but transiently upregulate Mesp1, a marker of cardiac mesoderm (Saga et al., 2000; Saga et al., 1999). Upon Mesp1 downregulation the specified cells then become determined to adopt a cardiac fate, marked by upregulation of Isl1, Tbx5, Mef2c, and Nkx2.5.

Finally, terminal differentiation is marked by expression of cardiac troponin (cTNT).

An intermediate step of cardiomyocyte induction is cardiac mesoderm induction.

This can be assessed using KDR (Flk-1) and Pdgfr-alpha expression. Flk-1, which encodes

VEGFR2, is expressed at the induction of cardiac mesoderm (Ema et al., 2006), but also marks non-cardiac mesodermal populations such as endothelial and vascular smooth muscle cell progenitors (Kattman et al., 2006). Pdgfr-alpha, however, is expressed in cardiac precursors in the mesoderm (Kataoka et al., 1997) and is downregulated once these precursors become determined (Prall et al., 2007). Therefore, when assayed on the third day

(D3) of induction, the mesodermal population that will give rise to cardiomyocytes is both

Flk1+ and PDGFRa+, and this population has been shown to comprise more than 60% of the cell population (Kattman et al., 2011). Additional populations resulting from this induction protocol are Flk1+/PDGFR- cells, Flk1-/PDGFR+ cells, Flk1-/PDGFR- cells. Flk1+/PDGFR- cells, which are ~15% of the D3 population, are non-cardiac mesodermal cells that will give rise to vascular cells and hematopoietic lineages (Ema et al., 2006; Kattman et al., 2006).

Flk1-/PDGFR+ cells, which are also ~15% of the D3 population may give rise to extraembryonic mesodermal cell types (Takakura et al., 1997). Flk1-/PDGFR- cells, which are less than 10% of the D3 population, may consist mostly of uninduced stem cells. These results demonstrate the existence of a heterogeneous cell population within the iCM culture system, possibly including vascular endothelial cells and hemangioblasts. Although both

TBX20 and CASZ1 were expressed throughout the iCM induction, TBX20Avi protein

97 networks isolated prior to iCM terminal differentiation lacked CASZ1 demonstrating a cardiac-specific interaction between TBX20 and CASZ1 that is required post-cardiac progenitor specification.

CASZ1 is a Novel TBX20 Interaction Partner

Using a mass spectrometry approach, we identified CASZ1 as a new TBX20 interaction partner. Although this is the first publication identifying an endogenous cardiac interaction network for TBX20, several groups have directly or indirectly shown TBX20 to function synergistically with other transcription factors in the heart. In Xenopus laevis, morpholino-mediated TBX20/TBX5 depletion led to cardiac hypoplasia and cardiac looping defects, with heart development failing to progress beyond the looping stage (Brown et al.,

2005), and in adult mice, TBX20-NKX2-5 compound heterozygosity led to mild ventricular dilation accompanied by systolic dysfunction (Stennard et al., 2005). These works provided the first evidence of the critical functions that endogenous TBX20 interaction networks play in establishing and maintaining homeostasis in the cardiac environment.

Complementing these in vivo haploinsufficiency studies, TBX20 has also been shown to interact with the Groucho corepressors TLE1 and TLE3 in embryonic day 10.5 (E10.5) mouse embryos (Kaltenbrun et al., 2013), and with several additional transcriptional cofactors in cell culture assays. Despite these previously identified interaction partners of

TBX20, we only identified the cardiac transcription factor CASZ1, which we validated by co- immunoprecipitating full-length TBX20 and CASZ1 from independent assays, mapping the interaction domains of TBX20 and CASZ1, and performing reciprocal co- immunoprecipitations from adult hearts.

Failing to identify published TBX20 interaction partners such as TBX5 in our affinity purifications from iCMs may have occurred for one or more of the following reasons. (1)

Interactions identified developmentally could be transient and therefore only identifiable in

98 a temporally and spatially narrow developmental window. (2) A subset of the interactions identified in vitro may represent false-positives, because they are identified under conditions and in systems that do not mimic the endogenous setting. (3) Low relative abundance of some transcription factors prevents the possibility of identifying these proteins in TBX20 transcriptional networks. (4) Protein solubilization conditions are determined empirically for the bait protein and therefore are not broadly optimal for all proteins in the cardiac tissue. Therefore, insoluble proteins will be discarded in the sample preparation steps preceding mass spectrometry. (5) Stringent cut-offs are used for the mass spectrometry peptide identification and subsequent protein assignment. As a result, proteins identified with low confidence are excluded from the final list of identified proteins. A reasonable, but costly, solution for the technical hurdles would be to solubilize proteins under 2-3 stringencies, compare and contrast the abundances of the bait and prey proteins, and select the best buffer based on optimal isolation of both bait and prey. Future investigations employing manipulation of one or more of these factors, may prove useful in identifying additional protein interactions.

Early Detection as a DCM Intervention

The best offense in familial DCM is early detection. DCM has been identified in infants as young as a few months old and can be severe enough to affect quality of life, even in young children. DCM is the most common cardiomyopathy in children, with most cases having an unknown cause (Wilkinson et al., 2010). Although most cases are detected based on severe cardiac dysfunction that affects quality of life, methods to identify the potential for cardiac dysfunction should be employed prior to the onset of severe dysfunction. Using this strategy, individuals with a family history of DCM, heart failure, or premature death that could be attributable to cardiovascular defects, should be screened for mutations in DCM-

99 related genes. This early detection method allows for earlier medical intervention and treatment.

In keeping with identifying DCM mutations earlier, in this thesis I am identifying

CASZ1 as a new gene associated with DCM pathogenesis. During the preparation of this thesis, two articles were published linking CASZ1 to cardiovascular disease. In one, CASZ1 was linked to DCM after screening 138 unrelated individuals with idiopathic DCM. The

K351X mutation was identified in a patient with a family history of DCM (Qiu et al., 2017).

This mutation produced a stop codon that terminated CASZ1 between the second nuclear localization signal and the first zinc finger, and produced a truncated CASZ1 protein that was transcriptionally inactive in in vitro luciferase assays. In an independent study screening 172 unrelated individuals with CHD, Huang et al. identified the heterozygous CASZ1 mutation

(Huang et al., 2016) L38P in a patient with double outlet right ventricle and VSD. This mutation truncated CASZ1 just downstream of the first and essential nuclear localization signal (Virden et al., 2012). Analysis of the patient’s family revealed a history of VSD.

CASZ1 is the newest addition to the list of cardiac transcription factors that have been shown to be essential for cardiac development while also harboring mutations found to be causal in human DCM. This list includes TBX20, NKX2-5, ISLET1, GATA4/6, TBX5, and

HAND1. Isl1 is expressed in the second of the two groups of cells that comprise the heart.

This group of cells is termed the secondary heart field. Isl1 ablation in mice leads to proliferation and migration defects, as well as cell death in cardiac progenitor cells derived from the secondary heart field. In Isl1 mutant mice, disrupted contribution of secondary heart field cells to the linear heart tube results in failed elongation of the linear heart tube.

Failure of the heart to undergo looping also occurs, and is closely followed by lethality (Cai et al., 2003). These defects are similar to those seen in TBX20 null mice, which exhibit arrested cardiac development just prior to looping stage, accompanied by failed linear heart tube elongation, and cardiac hypoplasia due to proliferation defects. Tbx5 null mouse hearts

100 initiated very early stages of looping morphogenesis, but failed to complete it, and ventricular myocardium was remarkably hypoplastic (Bruneau et al., 2001). These defects culminated in lethality at the same stage in embryonic development as the Isl1 and Tbx20 null mice.

Similar to Isl1, Tbx20, and Tbx5 null mice, NKX2-5 is also required for looping morphogenesis (Biben et al., 2000; Tanaka et al., 1999). Although Nkx2-5 mutants showed overall growth retardation, these animals did not demonstrate ventricular proliferation defects at the developmental stage just prior to looping of the heart. In contrast to the mutants listed above, Hand1 mutants showed gestational lethality later in development once the heart completed looping and chamber formation. Mutant hearts displayed VSD with varying degrees of severity, and these defects lead to lethality in late embryonic development, in the neonate, or in the adult mouse (McFadden et al., 2005). Gata4 is essential for cardiomyocyte cell cycle regulation and proliferation, and mice homozygous null at the Gata4 locus die during gestation directly following the looping stage, displaying proliferation and septation defects within the ventricle (Rojas et al., 2008). Finally, Gata6 mutant mice survive until the looping stage of the heart, and display defective looping and cardiac hypoplasia (Zhao et al., 2005).

Altogether, TBX20/5, NKX2-5, ISLET1, GATA4/6, and HAND1 are required for cardiomyocyte proliferation and for elongation and/or looping of the linear heart tube.

Because these factors are required for some overlapping cellular functions and morphological processes in the heart, they may function in the same pathway and may therefore lead to very similar phenotypes when mutated in humans. The essential cardiac transcription factors, with the addition of Casz1, broaden our understanding of how the mammalian heart develops. Complementing this, identifying the factors that are criticial in the developing myocardium, also populates a list of potential targets that should be suequenced in CHD and in cardiomyopathies such as DCM. Of the list of approximately 13

101 transcription factors required for mammalian heart development using genetic manipulation tools in mice, 7 have been linked to DCM, and that total increases when considering which of those factors have also been linked to CHD.

Tbx20-Casz1 Compound Heterozygosity Leads to DCM

In addition to identifying a novel cardiac transcriptional network, another major finding in this dissertation is the fundamental need for the TBX20-CASZ1 interaction in cardiac function. Compound Tbx20-Casz1 heterozygosity leads to DCM, interstitial fibrosis, mild hypertrophic cardiomyopathy, and lethality in both female and male mice. We included female mice in our analyses because they have cardio-protective mechanisms that mitigate the effects of cardiac dysfunction and prolong the progression from molecular perturbation to heart failure (Bush et al., 1987; Dent et al., 2010a, b; Stampfer et al., 1985). This cardio- protection also allowed us to focus specifically on the effects of Tbx20-Casz1 heterozygosity, rather than just characterizing a rapid progression to heart failure and death.

The Tbx20-Casz1 mutants at the focal point of our study were heterozygous floxed at both the Tbx20 and Casz1 loci (Casz1flox/+; Tbx20flox/+), with cardiac-specific recombination events that lead to compound heterozygosity being driven by Nkx2.5Cre. Here, we observed lethality in our mutants, but not in our Tbx20flox/+; Nkx2.5Cre mice or in our Casz1flox/+;

Nkx2.5Cre mice. Mouse embryos with cardiac Casz1 heterozygosity were viable and displayed no overt phenotypes (Dorr et al., 2015). Tbx20 heterozygosity has been reported to cause very mild DCM in adult mice (Stennard et al., 2005). The first 3 exons of Tbx20 were replaced with lacZ in the germline of these mice, but not specifically in cardiac tissue.

Therefore, early expression of Tbx20 in cardiogenic tissue prior to specification may indicate an early requirement for Tbx20 in those tissues, that when lost, produces a more severe phenotype than bringing Tbx20 into heteroyzgosity beginning in specified cardiogenic tissue.

102 Additionally, Nkx2.5Cre has been predicted to contribute to cardiac phenotype due to a combination of off-target effects and Nkx2.5 heterozygosity, since the wildtype Nkx2.5 coding sequence has been replaced with Cre recombinase (Moses et al., 2001). Because we were unable to detect any qualitative or quantitative defect in the single heterozygous

Tbx20flox/+; Nkx2.5Cre and Casz1flox/+; Nkx2.5Cre mice, our data fail to support a genetic interaction between Tbx20 and Nkx2.5 or between Casz1 and Nkx2.5 postnatally.

Proteomic Changes in Tbx20-Casz1 Mutant Mice

To identify proteomic changes in mutant hearts, we processed mutant and control adult hearts for tandem mass spectrometry, and used Uniprot Swiss Prot databases to assign peptides to proteins with high confidence. When the identified proteins were grouped according to GO term, compound mutant hearts exhibited upregulation of factors associated with humoral immune response, cell-matrix adhesion, repression of wound healing, and regulation of homeostasis.

Among the factors mis-regulated in TBX20/CASZ1 mutants was the essential calcium regulators cardiac Ryanodine receptor, which is encoded by Ryr2, and

Sarcoplasmic/endoplasmic ATPase (SERCA), which is encoded by Atp2a3. Interestingly,

Ryr2 and Atp2a3 are absent from several published lists showing genes that are significantly misregulated in DCM patients. However, Ryr2 is significantly down-regulated in TBX20 mutant mice (Shen et al., 2011), and another ryanodine receptor encoded by Ryr3 is down- regulated in the hearts of CASZ1 mutant mice (unpublished data). Therefore, I propose a mechanism whereby TBX20 and CASZ1 interact to regulate expression of these important regulators of calcium transients within the cell, and when calcium transients are outside homeostatic conditions, it becomes disruptive to cardiomyocyte contraction and detrimental to cardiomyocyte survival.

103 One of the factors found to be upregulated in the Tbx20-Casz1 mutants was Desmin, an essential cytoskeletal protein upregulated in Tbx20 knockout mice (Shen et al., 2011).

Mutations in Desmin cause human idiopathic DCM (Japp et al., 2016; Kimura, 2016;

Raghow, 2016; van Spaendonck-Zwarts et al., 2011; Viegas-Pequignot et al., 1989), and it is upregulated in at least one case of murine DCM (Nishigori et al., 2016). Ablating Desmin in mice led to DCM, exercise-induced hypertrophy, and lethality (Milner et al., 1999). These results support roles for Desmin both upstream and downstream of DCM onset, in human

DCM pathophysiology and in mouse models of DCM

Compensatory and Decompensatory Cardiac Remodeling in Response to Tbx20-Casz1 Heterozygosity

Cardiac remodeling encompasses changes to the structure or function of the heart in response to physiological, pathological, or mechanical insult (Cohn et al., 2000). Some of these remodeling events include cardiomyocyte hypertrophy (concentric augmentation), cardiomyocyte lengthening (eccentric augmentation), ventricular dilation, collagen accumulation, and scar formation. These changes allow the heart to maintain volumetric load despite focal or global injury that reduces the heart’s functional capacity. Concentric hypertrophy is generally one of the initial changes that occurs in the myocardium to compensate for dysfunction on the cell or tissue level. Concentric hypertrophy is an increase in the cross-sectional area of the cardiomyocyte that, based on the extent of myocardial involvement, can lead to increased ventricular wall thickness. In our DCM model, the traditional hallmarks of DCM were accompanied by increased cardiomyocyte cross-sectional area, termed hypertrophy, which commonly results in increased ventricular wall thickness and constitutes a distinct cardiac phenotype not commonly presenting with DCM. Because

Tbx20/Nkx2-5 mutant mice exhibited quantitative thinning of the ventricular wall, we posit hypertrophy to be a secondary phenotype affiliated with compensatory remodeling. At no

104 point during our assessment of Tbx20/Nkx2-5 mutants aged 4 to 32 weeks do we detect increased ventricular wall thickness as an indicator for a primary hypertrophic cardiomyopathy phenotype.

Overall, this thesis project supports the need for characterizing transcription factor networks, and considering the combined functions of these networks in the cardiac environment, while presenting a new model for DCM pathogenesis. Although no clinically identifiable feature delineates the transition from primary cardiac insult to compensatory remodeling and progression to heart failure, characterizing molecular changes that define these phases is invaluable to understanding the pathogenesis and progression of heart disease. The TBX20/CASZ1 DCM presented here is an excellent model for studying DCM pathogenesis in the absence of specific mutations within contractile components. This model shows a slow progression to heart failure which allows for ample to time closely investigate

DCM pathogenesis. Due to variable penetrances and the fact that some mutant animals survive as long as wildtype controls, our model represents an excellent one for studying and optimizing DCM therapies such as stem cell injection.

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110

111 APPENDIX: A GRO/TLE-NURD COREPRESSOR COMPLEX FACILITATES TBX20-DEPENDENT TRANSCRIPTIONAL REPRESSION2

PREFACE

The appendix is composed of evidence identifying factors that interact in complex with the T-box transcription factor TBX20. These factors include members of the

Nucleosome remodeling and deacetylase complex as well as the Groucho corepressors. This was the first publication identifying TBX20 transcriptional networks, and therefore provided valuable insight into TBX20 function. This work was completed in collaboration in Erin

Kaltenbrun, Todd Greco, Christopher Slagle, Li Tuo, and Ileana Cristea and was published in the Journal of Proteomic Research. My contribution to the work was validating the interaction between TBX28 and TBX20 by performing co-immunoprecipitation experiments from HEK293 cells transiently expressing tagged versions of both proteins.

INTRODUCTION

The development and maturation of a functional heart is a complex process that involves distinct but overlapping phases of specification, proliferation, migration, differentiation, and morphogenesis. Disturbances in any of these processes can lead to a number of congenital heart defects. Currently, congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death (Reller et al., 2008; van der Linde et al.,

2011). Recent studies have demonstrated that human patients with dilated cardiomyopathy, atrial septal defects, or mitral valve disease carry mutations in the transcription factor TBX20 , while upregulation of TBX20 gene expression has been reported in patients with tetralogy of Fallot (Hammer et al., 2008; Kirk et al., 2007; Liu et al., 2008; Qian et al., 2008). Tbx20 is a member of the T-box family of transcription factors, all of which share a well-conserved DNA binding domain known as the T-box and have

2 Kaltenbrun E. Greco T.M., Slagle C.E., Kennedy L.M., Tuo L., Cristea I.M., Conlon F.L. (2013) A Gro/TLE- NuRD Corepressor Complex Facilitates Tbx20-Dependent Transcriptional Repression. J Prot Res. 12: 5395- 5409.

112 diverse roles in embryonic development. Tbx20 has been identified in many organisms, including Drosophila , zebrafish, Xenopus , and mouse, and in all species examined Tbx20 transcripts are strongly expressed throughout the developing heart (Ahn et al., 2000; Brown et al., 2003; Conlon and Yutzey, 2010; Griffin et al., 2000; Iio et al., 2001; Kaltenbrun et al.,

2013; Meins et al., 2000). Results from genetic analysis and protein depletion studies are consistent with a role for Tbx20 during the early stages of vertebrate heart development; hearts lacking Tbx20 show a progressive loss of cardiomyocytes, a failure of the heart to undergo looping and chamber formation, and defects in cardiomyocyte maturation (Brown et al., 2005; Cai et al., 2005; Mandel et al., 2010; Singh et al., 2005; Stennard et al., 2005;

Takeuchi et al., 2005). Collectively, these studies suggest that the sequence, expression, and function of Tbx20 are evolutionarily conserved from flies to human. Similar to other T-box factors, Tbx20 is localized to the nucleus, binds DNA in a sequence-specific manner, and modulates transcription of downstream target genes (Brown et al., 2005; Cai et al.,

2005; Sakabe et al., 2012; Shen et al., 2011; Singh et al., 2005; Stennard et al., 2003;

Stennard et al., 2005; Takeuchi et al., 2005). Results from a number of studies have shown that Tbx20 can act to both promote and repress target gene expression in the heart; however, it is unclear how Tbx20 initiates a transcriptional repressive program within the same cells in which it also acts as a potent transcriptional activator. It has been proposed that protein cofactors may act to specify Tbx20 transcriptional activity (Sakabe et al., 2012). A model in which protein cofactors act as determinants of Tbx20 activity has several unresolved issues because few in vivo Tbx20 cofactors have been identified.

Additionally, there is uncertainty about the precise mechanism by which binding of Tbx20 to

DNA results in either activation or repression of a target gene. In vitro assays have been used to demonstrate interactions between Tbx20 and a suite of cardiac transcription factors that include Tbx5, Nkx2.5, Gata4, Gata5, and Islet1 (Brown et al., 2005; Stennard et al., 2003), although none of these interactions have been shown to occur in vivo in the embryonic

113 heart. Indeed, the presence of DNA-binding motifs for Nkx2.5, Gata4, and Tbx5 in the promoter regions of Tbx20 target genes, in combination with evidence that these transcription factors act combinatorially to promote target gene expression, suggests that cardiac transcription factors are important cofactors for Tbx20 to activate gene expression in the developing heart (Sakabe et al., 2012; Stennard et al., 2003; Takeuchi et al., 2005).

However, it is not well understood how Tbx20 functions as a transcriptional repressor, as cofactors that may act as functional corepressors have not been identified. Therefore, the precise mechanisms by which Tbx20 regulates distinct gene programs in the heart remain unclear. To begin to address these questions, we have undertaken, to our knowledge, the first proteomic study aimed at identifying Tbx20 protein interactions. Using affinity purification mass spectrometry (AP-MS) (Miteva et al., 2013), we have systematically characterized Tbx20-containing transcriptional complexes. With this approach, we have identified a unique Tbx20 chromatin remodeling network that includes the Groucho-related proteins Transducin-like Enhancer of Split 1 and 3 (TLE1/3), Metastasis-associated Protein 1

(MTA1), the histone-binding proteins RBBP4 and RBBP7, RUVB-like 1 and 2,

Nucleolin, Nucleophosmin, Histone Deacetylase 2 (HDAC2), and the Tbox repressor Tbx18.

We provide evidence that Tbx20 recruits TLE1/3 through an evolutionarily conserved N- terminal engrailed homology 1 (eh1) binding motif, and we demonstrate that recruitment of

NuRD complex components requires binding of TLE3 to Tbx20. We find that TLE family members are expressed in mouse embryonic heart tissue and that Tbx20 interacts with both

TLE1 and TLE3 in vivo during heart development, representing the first endogenous

Tbx20 interactions identified in embryonic heart tissue to date. Finally, we observe that the

Tbx20− TLE interaction is essential for Tbx20 transcriptional activity in the embryo, and we define a unique set of genes that are repressed by the Tbx20-TLE transcriptional complex.

We propose a model in which Tbx20 binds to TLE factors to assemble a chromatin

114 remodeling and deacetylase complex on target gene loci to repress distinct genetic programs in the forming heart.

METHODS

DNA Constructs

Mouse Tbx20a cDNA was fused to EGFP and cloned into the pMONO-neo-mcs plasmid (Invitrogen) for expression in HEK293 cells. The Tbx20eh1mut-EGFP construct was generated by site-directed mutagenesis (Stratagene) of phenylalanine 18 (F18L) and serine 19 (S19I) using the primers 5′-

CTCTCGAGCCAATGCCTTAATCATCGCCGCGCTTATGTC- 3′ and 5′-

GACATAAGCGCGGCGATGATTAAGGCATTGGCTCGAGAG- 3′ according to the manufacturer’s instructions. To generate the Tbx20-HA construct, mouse Tbx20a cDNA was fused to an HA epitope and cloned into pMONO-neo-mcs. The pCMV2-TLE1-Flag construct was generously provided by Dr. Stefano Stifani (Buscarlet et al., 2009). The pCMX-

TLE3 plasmid was kindly provided by Dr. Peter Tontonoz (Villanueva et al., 2011). Tbx18-

Flag was generously provided by Dr. Chen-Leng Cai (Nie et al., 2010). Xenopus Injections and Animal Cap Isolation Xenopus laevis embryos were staged according to Nieuwkoop and

Faber (Nieuwkoop and Faber, 1994) and injected with 1 ng of Tbx20 or Tbx20-EGFP mRNA at the one-cell stage using established protocols (Goetz et al., 2006; Smith and Slack,

1983). Animal caps were excised at stages 8−9 and cultured in 1X modified Barth’s saline

(MBS) until sibling embryos reached stage 13. Activin-treated caps were cultured in 8 units/mL Activin in 1X MBS.

Isolation of Tbx20-EGFP Protein Complexes

pMONO-Tbx20-EGFP or Tbx20eh1mut-EGFP plasmids were transfected into HEK293 cells using FuGENE (Roche Applied Science). Tbx20-EGFP complexes and GFP complexes

115 were immunoaffinity purified from cells using in-house developed rabbit polyclonal anti-

GFP antibodies conjugated to magnetic beads, as previously described (Cristea et al., 2005).

Briefly, HEK293 cells expressing Tbx20-EGFP or GFP alone were washed with cold PBS, harvested from the plate by scraping with a plastic spatula, and pelleted at 1500 rpm for 10 min at 4 °C. The cell pellet was resuspended in 100 µL/1 g 20 mM HEPES, pH

7.4, containing 1.2% polyvinylpyrrolidone and protease inhibitors and snap frozen in liquid nitrogen. Cells were lysed by cryogenic grinding using a Retsch MM 301 Mixer Mill

(10 cycles Å~ 2.5 min at 30 Hz) (Retsch, Newtown, PA), and the frozen cell powder was resuspended in optimized lysis buffer (5 mL/1 g cells) (20 mM K-HEPES pH 7.4, 0.1 M

KOAc, 2 mM MgCl2, 0.1% Tween-20, 1 µM ZnCl2, 1 µM CaCl2, 150 mM NaCl, 0.5% Triton

X-100 containing protease and phosphatase inhibitors). Cell lysates were homogenized using a Polytron (Kinematica) step (2 Å~ 15 s) and pelleted at 8000 rpm at 4 °C. Cleared lysates were rotated with 7 mg of magnetic beads (M270 Epoxy Dynabeads, Invitrogen) coupled to anti-GFP antibodies for 1 h at 4 °C. The magnetic beads were then washed in lysis buffer (6 Å~ 1 mL) (without protease and phosphatase inhibitors) and eluted from the beads in 40 µL 1x LDS Sample Buffer (Invitrogen) at 70 °C for 15 min. Eluted proteins were alkylated with 100 mM iodoacetamide for 1 h at room temperature and subjected to mass spectrometry analysis.

Mass Spectrometry Analysis of Tbx20-EGFP Protein Complexes

Immunoisolates were analyzed by mass spectrometry as previously described (Tsai et al., 2012) with minor differences. Briefly, reduced and alkylated eluates were partially resolved by SDS-PAGE on 4−12% Bis-Tris NuPAGE gels (Invitrogen) and stained using SimplyBlue Coomassie stain (Invitrogen). Each lane was divided into 1 mm slices and binned into 8 wells of a 96-well plate. Gel slices were destained in 50 mM ammonium bicarbonate (ABC) containing 50% acetonitrile (ACN). Proteins were digested

116 in-gel with 20 µL of 12.5 ng/µL trypsin in 50 mM ABC for 5 h at 37 °C. Tryptic peptides were extracted in 0.5% formic acid for 4 h at room temperature, followed by 0.5% formic acid/50% ACN for 2 h at room temperature. The extracted peptides were concentrated by vacuum centrifugation to 10 µL and desalted either online (trap column, Magic C18 AQ,

100 µm Å~ 2.5 cm) or offline using StageTips. Desalted peptides (4 µL) were separated online by reverse phase C18 (Acclaim PepMap RSLC, 1.8 µm, 75 µm Å~ 25 cm) over 90 min at 250 nL/min using a Dionex Ultimate 300 nanoRSLC and detected by an LTQ Orbitrap

Velos or XL mass spectrometer (Thermofisher Scientific, San Jose, CA). The mass spectrometer was operated in data-dependent acquisition mode with dynamic exclusion enabled. A single acquisition cycle comprised a single full-scan mass spectrum (m /z = 350−

1700) in the Orbitrap (r = 30,000 at m /z = 400), followed by collision-induced dissociation (CID) fragmentation in the linear ion trap of the top 10 (XL) or 20 (Velos) most intense precursor ions. The FT full scan target value was 1E6 with a maximum injection time of 300 ms. IT tandem MS target values were 5E3 (XL) or 1E4 (Velos) with a maximum injection time of 100 ms. CID fragmentation was performed at an isolation width of 2.0 Th, normalized collision energy of 30, and activation time of 30 (XL) or 10 ms (Velos).

Data Processing and Functional Protein Analyses

MS/MS spectra were extracted from Thermo RAW files and searched by Proteome

Discoverer/SEQUEST (version 1.3, Thermo Fisher Scientific) against the UniProt

SwissProt protein sequence database (release 2010− 11) containing forward and reverse entries from human and the mouse Tbx20a sequence plus common contaminants (20,324 forward sequences). SEQUEST search parameters were as follows: full enzyme specificity with 2 missed cleavages, precursor and fragment tolerances, 10 ppm and 0.5 Da, fixed modification, carbamidomethylation of cysteine, and variable modifications, oxidized methionine and phosphorylation of STY. SEQUEST peptide spectrum matches (MSF files)

117 were loaded into Scaffold software (ver. 3.5.1, Proteome Software, Inc.), subjected to an X!

Tandem refinement search, and then analyzed by PeptideProphet and ProteinProphet algorithms to determine peptide and protein probabilities. The high mass accuracy option for probability scoring was enabled. The following peptide modifications were included in the X! Tandem refinement search: deamidation of NQ and acetylation of K and the amino- terminus. Protein groups were assembled by Scaffold and filtered by a minimum of two unique peptides. Probability thresholds were empirically defined to achieve <1% peptide and protein FDR, as assessed by matches to the reverse database. Protein descriptions, accession numbers, and their respective unique peptides and unweighted spectrum counts were exported to Excel for further analysis. Specific Tbx20 protein interactions were identified by spectral counting enrichment analysis13 comparing the Tbx20-EGFP versus EGFP alone conditions. The following criteria were applied to each individual replicate (N = 3): (1) only proteins with ≥ 5 spectral counts were retained; (2) only those proteins that had a spectral count enrichment of ≥ 2.5- fold versus GFP alone were retained; and (3) using gene ontology

(GO) annotations, proteins assigned a “ nuclear” localization ontology term were retained

(Supporting Information Table A1; see Supporting Information Table A2 for proteins excluded for not being associated with a nuclear localization ontology term). The proteins that fulfilled these criteria in all three replicates were imported into Cytoscape

(Smoot et al., 2011) for classification into functional subgroups according to biological processes using the plugin ClueGO (Supporting Information Table A3) (Bindea et al., 2009). Proteins within GO term clusters were analyzed in STRING using protein accessions as input (Szklarczyk et al., 2011). Comparison of interaction protein abundance versus estimated average proteome abundance, defined here as an enrichment index, was used to identify prominent interaction candidates from the GO-classified nuclear proteins, as described in ref 13. To calculate this enrichment index, first, a protein’ s spectral counts was normalized within each biological replicate (N = 3) by the Tbx20 spectral count ratio of

118 each individual replicate/average. Then, normalized spectral counts were converted to NSAF

(normalized spectral abundance factor) values (Zybailov et al., 2007) and further normalized by estimated proteome abundance from the human subset of the PAX

(protein abundance across organisms) database (Wang et al., 2012) (Supporting

Information Table A4). Tbx20 spectral counts were excluded from this analysis. We have previously shown that calculating this enrichment index aids in identifying high confidence interactions that prominently associate with the bait (Tsai et al., 2012).

Analysis of Tbx20-EGFP and Tbx20eh1mut-EGFP Protein Complexes Using Mass Spectrometry-Based Label-Free Analysis

Tbx20-EGFP and Tbx20eh1mut -EGFP complexes were immunoisolated and analyzed by LC-MS/MS as described above. Peptide and protein identifications were filtered using the criteria described above, including the requirement of at least five spectra per protein identified for either wild-type or mutant isolations and the requirement that all proteins identified in the wild-type isolation be at least 2.5-fold enriched over isolations from EGFP- expressing cells. To determine differences in interacting proteins between wild-type and mutant Tbx20 isolations, a combination of spectral counting and MS1 label-free approaches was employed. For spectral counting analysis, the spectral counts for putative protein interactions in the Tbx20eh1mut isolation were normalized by the Tbx20 spectral count ratio of eh1mut/wild-type isolation. Then, the fold difference in spectral counts of putative interactions (Tbx20eh1mut isolation relative to wild-type Tbx20) was used to identify differential interacting proteins. For selected Tbx20 interactions, label-free quantification was performed by MS1 peak area measurements using the MaxQuant software (ver 1.4.0.8)

(Cox and Mann, 2008). Relative quantification was performed using the MaxQuant “ intensity” measurements and the “ Match between runs” feature enabled, which quantified nonsequenced peptide features based on accurate mass, retention time, and

119 sequence information collected from other data-dependent LC-MS/MS runs. Only unmodified peptides designated as unique at the protein group level were used to calculate summed protein intensities. The intensity ratio of Tbx20 in the eh1mut/wildtype isolation was used to normalize protein interaction ratios. The normalized fold-differences of putative interactions (Tbx20eh1mut isolation relative to the wild-type Tbx20) were used as a measure of differential interaction. Interacting proteins with fold-differences ≤ 0.5 were considered to be significantly reduced in mutant isolations (Supporting Information Tables A5 and

A6).

Construction of a HEK293 HDAC2-EGFP Stable Cell Line

The HDAC2 ORF was amplified from an HDAC2 plasmid (gift from E. Seto, Moffitt

Cancer Center) and inserted into the pLXSN-C-EGFP-FLAG vector to create the HDAC2-

EGFP-flag fusion, as in ref 20. The Phoenix retrovirus expression system (Orbigen, San

Diego, CA) was used to transduce HEK293 cells to express the HDAC2-EGFP-FLAG fusion according to the manufacturer’ s instructions. The transduced cells were selected in 300 mg/L G418 (EMD, Gibbstown, NJ) and sorted by FACS (Vantage SE with TurboSort II,

Becton Dickinson, Franklin Lakes, NJ) to obtain a stable cell line. The nuclear localization and deacetylation activity of the GFP-tagged HDAC2 were confirmed.

Isolation of Endogenous Tbx20 from Mouse Embryonic Hearts

Pregnant CD1 females were sacrificed on embryonic day 10.5 (E10.5) and the embryos removed. Embryonic hearts (n = 25) were dissected from the embryos in cold PBS and snap-frozen in liquid nitrogen. Embryonic hearts were cryogenically lysed, and endogenous Tbx20 protein complexes were immunoaffinity purified as described using 5 mg magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to anti-Tbx20 antibodies

(Santa Cruz Biotechnology). The isolated proteins were analyzed by Western blotting.

120

Immunofluorescence and Immunoblotting

For immunofluorescence of HEK293 cells, cells were cultured in 8-well chamber slides pretreated with poly-D -lysine. For live imaging of EGFP fluorescence, cells were transfected with pMONO-Tbx20-EGFP. Forty-eight hours later, the cells were rinsed with

1X PBX and DAPI was added (200 ng/mL in 1X PBS) for 30 min. Cells were imaged by confocal microscopy on a Zeiss 710. Antibodies used for immunoblotting include mouse anti- GFP (JL8) (Clontech Living Colors Monoclonal), mouse anti- Flag (M2) (Sigma), goat anti-TLE1 (N-18) (Santa Cruz Biotechnology), rabbit anti-TLE3 (M-201) (Santa

Cruz Biotechnology), mouse anti-GAPDH (Millipore), and goat anti-Tbx20 (Santa Cruz

Biotechnology).

RNA Extraction and RT-PCR

RNA was extracted using Trizol (Invitrogen) and purified on RNeasy columns

(Qiagen). cDNA synthesis was performed from 0.5 to 1 µ g of RNA using random primers and SuperScript II reverse transcriptase (Invitrogen). Expression levels were assessed using

GoTaq Green Master Mix (Promega) and Taq polymerase on a GeneAmp PCR

System (Applied Biosystems). PCR products were analyzed by 2.5% agarose gel electrophoresis.

In Situ Hybridization

E10.5 embryos were fixed whole in 4% paraformaldehyde, and 16 µ m tissue sections were cut for in situ hybridization. Fragments corresponding to the 5′ UTR of Tbx20 , TLE1 , and TLE3 transcripts were cloned into a pStrataclone cloning vector (Strataclone Blunt PCR

Cloning Kit, Agilent Technologies), and then digoxigenin-labeled antisense riboprobes

121 were generated and hybridized to the tissue sections using standard techniques. Sections were imaged on an Olympus BX61 Upright Wide Field microscope.

RNA-seq and Differential Expression Analyses

RNA was extracted from stage 13 Xenopus laevis embryos injected with Tbx20-EGFP or Tbx20eh1mut-EGFP mRNA. As a control, RNA was also extracted from uninjected stage

13 siblings. The Illumina Tru-seq RNA sample prep kit was used for cDNA library preparation. Libraries were sequenced using an Illumina HiSeq 2500 system (Vanderbilt

Technologies for Advanced Genomics, Vanderbilt University). Each sample (two replicates per condition) generated between 32 million and 38.5 million single-end, 50-bp reads. These were mapped to a set of 8879 Xenopus laevis transcripts using the default options of

Bowtie2 (version 2.1.0) (Langmead and Salzberg, 2012) as described previously (Amin et al.,

2014; Tandon et al., 2013), with a unique-mapping rate of 45− 47%. Bowtie2 output fi les were converted to binary forms and sorted using Samtools (version 0.1.19) (Li et al., 2009a).

Transcript assemblies and expression level quantification were performed on each sample using the default settings of Cufflinks (version 2.0.2) (Trapnell et al., 2010). The transcript lists were subsequently merged using the default settings of the Cuff merge script in

Cufflinks to produce a comprehensive list of transcripts represented in all our Cufflinks assemblies (Supporting Information Tables A7 and A8). Differential expression analysis was performed on all samples using the default settings of the Cuff-diff program in Cufflinks, with the bias detection and correction option (-b) and the multimapping correction option (- u) (Supporting Information Tables A7 and A8). Gene ontology analysis was performed using

Gorilla (Eden et al., 2009) (Supporting Information Table A9 and A10). Heat maps were generated using the heatmap.2 function of the g-plots package (R package version

2.11.3, R Foundation for Statistical Computing, Vienna, Austria) in R (version 3.0.1, R

Foundation for Statistical Computing, Vienna, Austria).

122

RESULTS

Tbx20-EGFP Is Localized to the Nucleus and Transcriptionally Active

Identification of critical Tbx20 protein cofactors in a high throughput manner has been hampered by a lack of antibodies against Tbx20 that are suitable for directed proteomics analyses. Additionally, there are no cell lines that recapitulate endogenous

Tbx20 expression and thus could provide sufficient material for large-scale proteomics studies of Tbx20 protein complexes. Since the main goal of this set of studies was to determine the general transcriptional mechanisms by which Tbx20 functions, we generated human embryonic kidney (HEK293) cells expressing Tbx20 tagged at the C- terminus with EGFP (Figure A1A). HEK293 cells have been used as a cell culture model for studies on the transcriptional activity of Tbx20, indicating that this cell line contains the necessary cohort of transcriptional cofactors required for Tbx20-mediated transcriptional regulation. In agreement with its known role in transcription, Tbx20-EGFP localizes to the nucleus when expressed in HEK293 cells, as shown by live GFP fluorescence microscopy

(Figure A1B). To confirm that the EGFP-tagged Tbx20 is transcriptionally active, we made use of a Xenopus animal cap assay. The animal cap is a region of the Xenopus blastula and early gastrula stage embryo (stages 8/9) that consists of naive pluripotent cells. Animal caps normally contribute to skin and nervous tissue; however, a recent study demonstrated that animal caps excised from embryos injected with Tbx20 mRNA express the early mesoderm marker Xbra but not the skeletal muscle marker Myf5, indicating that Tbx20 can induce cell fate changes in the early embryo (Stennard et al., 2003). To assess whether EGFP-tagged

Tbx20 has the same ability to induce gene expression changes as untagged Tbx20, we injected Tbx20 and Tbx20-EGFP mRNA into one-cell stage Xenopus laevis embryos. By stage 9, Tbx20-EGFP protein is localized throughout the animal pole of the embryo, as shown by live GFP fluorescence microscopy (Figure A1C). At this stage, animal caps were

123 excised from Tbx20- and Tbx20- EGFP-injected embryos and cultured in isolation until uninjected sibling whole embryos reached stage 13. After extraction of RNA, we assessed the expression of the early mesodermal markers chordin and Xbra , and as a negative control, Myf5 , by RT-PCR. The levels of induction were compared to expression of these genes within whole embryos, untreated caps, and, as a positive control, caps treated with the mesoderm-inducing factor Activin. Both EGFP-tagged Tbx20 and untagged Tbx20 induce chordin expression to the same degree as Activin (Figure A1D). Further, Tbx20 and

Tbx20- EGFP induce moderate levels of Xbra compared to Activin induced caps; however, there was no induction of the muscle marker Myf5 by either version of Tbx20 or by

Activin treatment. Untreated caps did not express any of the tissuespecific markers tested.

These data indicate that EGFP-tagged Tbx20 is transcriptionally active and retains the ability to modulate downstream gene expression.

Proteomic Analysis of Tbx20-EGFP Interactions Reveals Association with a Unique Chromatin Remodeling Network

To systematically identify Tbx20-associated proteins, we performed immunoaffinity purifications of Tbx20-EGFP complexes from HEK293 cells using a high affinity in- house developed antibody against GFP (Cristea et al., 2005). In parallel, as controls, we performed immunoaffinity purifications from cells expressing EGFP alone (Supporting

Information (SI) Tables A1 and A2). Immunopurified proteins were partially resolved by

SDSPAGE, digested in-gel with trypsin, and analyzed by nLC tandem MS (MS/MS) on an

LTQ Orbitrap XL or an LTQ Orbitrap Velos. Three independent biological replicates were performed (SI Tables A1 and A2); two of these immunopurifications were performed in the presence of DNase to eliminate interactions mediated by binding of factors on adjacent

DNA sequences. Raw MS/MS spectra from each experiment were analyzed by SEQUEST database searches (Proteome Discoverer) and loaded into Scaffold for further analysis.

124 Protein identifi cations from all three replicates were filtered using stringent confidence parameters (see Materials and Methods). A spectral counting approach was employed to assess enrichment of protein interactions with Tbx20-EGFP relative to EGFP alone. First, proteins were required to be reproducibly present, detected by at least five spectral counts. Second, proteins showing less than 2.5-fold spectral count enrichment over proteins coisolated with EGFP alone were deemed nonspecific and were excluded. Further, given the nuclear localization of Tbx20-EGFP, proteins lacking a nuclear gene ontology term were excluded as likely nonspecific associations occurring during whole-cell lysis (SI Table A2). In total, 114 proteins with annotated nuclear localization passed our spectral count and fold enrichment criteria, 97% of which occur in all three biological replicates. There were no proteins identified as unique to a single Tbx20-EGFP isolation; however, three proteins, while detected in all three isolations, failed to pass our stringent criteria in one of the three experiments (SI Table A1). To assess the Tbx20 nuclear interaction network, we classified the proteins passing our spectral count and fold enrichment criteria into functional subgroups by Cytoscape, using the ClueGO plugin (Bindea et al., 2009; Smoot et al., 2011). Specifically, proteins were assigned into GO term clusters according to biological function ontologies. When we examined Tbx20 nuclear interactions, the most prominent biological function category contained 53 proteins related to RNA processing (Figure A2A and SI Table A3). This is expected, as it is well established that the DNA template that is being actively transcribed is often closely associated with the RNA processing machinery and suggests that a portion of Tbx20 binding is closely associated with active transcription.

Nuclear interactions also included 13 proteins involved in nucleosome assembly (Figure A2A and SI Table A3). Nucleosome assembly functions were represented by such GO terms as chromatin remodeling, DNA conformation change, and nucleosome organization, indicating a crucial role for Tbx20 in modification of chromatin architecture. Sixteen proteins were assigned to DNA repair/ synthesis, and 10 proteins were assigned to nuclear

125 transport (Figure A2A and SI Table A3). Collectively, these putative Tbx20 interactions represent different complexes and functions of Tbx20 throughout the nucleus. Given the lack of knowledge regarding the molecular mechanisms of Tbx20-mediated transcriptional regulation, we reasoned that the specific protein functions represented within the

“nucleosome assembly” category could provide new insight on the potential roles of Tbx20 in gene regulation. Moreover, since chromatin remodeling and transcription repression complexes are often large multiprotein complexes, we speculated that the specific proteins represented within the “ nucleosome assembly” category were likely to be interconnected. To test this hypothesis, we analyzed the proteins within this functional cluster (Table A1 and SI Table A3) using STRING, a knowledge database of known and predicted protein− protein interactions (Szklarczyk et al., 2011), with the aim of generating a predictive Tbx20 interaction network. Eight of these proteins form a highly interconnected network containing chromatin remodeling and deacetylase functions and include the nucleolar proteins Nucleophosmin (NPM1) and Nucleolin (NCL), as well as core components of the Nucleosome Remodeling and Deacetylase (NuRD) complex (MTA1, RBBP4, RBBP7, and HDAC2)Ű a major ATP-dependent chromatin remodeling complex with important roles in transcription and chromatin assembly (reviewed in (Bowen et al., 2004)) (Table A1 and

Figure A2B, gray lines). NCL and MTA1 were integrated into this network based upon their known or predicted functional association with components of the chromatin remodeling network (Ikura et al., 2000; Li et al., 1996; Sardiu et al., 2008; Xue et al., 1998).

Additionally, this network includes two members of the INO80 chromatin remodeling complex, the ATPases RUVBL1 and RUVBL2. The INO80 complex is a highly conserved, multisubunit, ATP-dependent chromatin remodeling complex that contributes to both transcriptional activation and repression (Jin et al., 2005; Shen et al., 2000). As this is the first demonstration that Tbx20 associates with chromatin remodeling proteins, it was unclear how Tbx20 might be functionally linked to this chromatin modification network.

126 There is evidence that MTA family proteins interact directly with transcription factors at target gene loci (Roche et al., 2008); however, different transcription factors have been shown to bind to different regions of individual subunits of the NuRD complex (Fujita et al.,

2004; Li et al., 2009b). To attempt to identify the functional link between Tbx20 and the chromatin remodeling network, we first examined the list of nuclear-enriched proteins for additional components of a chromatin modification network that may have been excluded in our original analysis due to incomplete GO or functional annotation. Surprisingly, this search uncovered the transcriptional corepressors Transducin-like Enhancer of split 1 and

3 (TLE1 and TLE3) and the T-box transcriptional repressor Tbx18. TLE family members are orthologs of the Drosophila Groucho protein and have been previously demonstrated to bind directly to T-box factors, including Tbx18 and Tbx15, through an engrailed homology 1 (eh1) binding motif and achieve transcriptional repression by recruiting histone deacetylases

(Chen et al., 1999). Based upon the reported functional relationships between TLE proteins,

T-box factors, and HDACs, we incorporated TLE1/3 and Tbx18 into the STRING network (Table A1 and Figure A2B, black lines). To examine if Gro/TLE factors could serve as proximal interacting partners, linking Tbx20 to chromatin remodeling complexes, we estimated the relative enrichment of proteins within the interaction network. As previously described (Miteva et al., 2013; Tsai et al., 2012), the relative protein enrichment within the immunoisolates was estimated by normalizing their relative protein abundances (NSAF values)(Zybailov et al., 2007) to their proteome abundances from the PAX database (pax- db.org) (SI Table A4). This relative enrichment analysis was performed for the 114 proteins enriched in Tbx20 immunoisolations and in the ″ nuclear″ GO subcellular localization (SI

Table A3). Respective enrichment index values were then illustrated in the Gro/TLE- chromatin remodeling network (Figure A2B). We hypothesized that proteins within the interaction network having greater enrichment indices would represent more proximal and perhaps essential Tbx20 interactions. Indeed, TLE1 and TLE3 comprised two of

127 the most highly enriched components of the interaction network. This supports a critical role for these proteins in regulating Tbx20 function, suggesting they may be directly linked to Tbx20 (Figure A2B, black dashed lines).

Tbx20 Forms Protein Complexes with TLE1/3, Tbx18, and HDAC2

While our AP-MS analysis of Tbx20 complexes does not establish which proteins are direct interactions (Figure A2B, black dashed lines), other T-box proteins, Tbx15 and

Tbx18, have been reported to bind directly to TLE3 via N-terminal eh1 binding motifs (Farin et al., 2007), indicating that the eh1 motif may represent a common motif used by T-box transcription factors to bind Gro/TLE family members. Therefore, to determine whether Tbx20 directly recruits Groucho corepressors via an eh1 binding motif, we next investigated the interaction between Tbx20 and TLE1/3. Tbx20 contains an N-terminal eh1 binding motif that is fully conserved in all vertebrate orthologs of Tbx20 (Figure A3A). To confirm an interaction between Tbx20 and TLE1/3 and determine whether these interactions require the eh1 motif, we generated a Tbx20eh1mut-EGFP expression construct in which the eh1 motif has been ablated by site-directed mutation of phenylalanine 18 and serine 19 to an isoleucine and a leucine, respectively (Tbx20F18I; S19L-EGFP ). Reciprocal immunoisolations of TLE1 and TLE3 complexes were performed in the presence of wild-type

Tbx20-EGFP or Tbx20eh1mut -EGFP. Mutation of the eh1 motif significantly reduced the ability of Tbx20-EGFP to be coisolated with either TLE1 or TLE3, suggesting that Tbx20 binds TLE1/3 directly through this motif (Figure A3B and C). The finding that Tbx20 (Farin et al., 2007) interacts with both a Gro/TLE complex and the Groucho dependent repressor

Tbx18 suggests that Tbx20 and Tbx18 may heterodimerize to regulate a common set of targets in a Groucho-dependent manner. To further investigate the interaction between

Tbx20 and Tbx18, we transfected HEK293 cells with Tbx18-Flag alone or in the presence of

Tbx20-EGFP . Immunopurification with an anti- Flag antibody and Western blot analysis

128 revealed efficient coisolation of Tbx20 (Figure A3D). Collectively, these data imply a role for a Tbx20-Tbx18 repressor complex during vertebrate development. To investigate the link between the Tbx20-TLE1/3 complex and HDAC2, we generated an HEK293 cell line stably expressing HDAC2 tagged at the C-terminus with EGFP. To confirm an interaction between Tbx20 and HDAC2, we performed reciprocal isolations of HDAC2-EGFP in the presence or absence of Tbx20-HA. Tbx20 was successfully coisolated with HDAC2-EGFP

(Figure A3E). As a number of studies indicate that binding to Groucho cofactors results in recruitment of deacetylase machinery (Chen et al., 1999; Choi et al., 1999; Yochum and

Ayer, 2001), we also performed isolations of HDAC2-EGFP in the presence of overexpressed TLE3 and Tbx20. Interestingly, excess TLE3 results in a substantial increase in the amount of Tbx20 associated with HDAC2, suggesting that TLE3 bridges an interaction between Tbx20 and HDAC2 (Figure A3E). However, less TLE3 associates with HDAC2 when

Tbx20 is overexpressed; therefore, an alternate explanation is that Tbx20 competes with

TLE3 for binding to HDAC2.

Label-free Mass Spectrometry Analysis Reveals That Tbx20-TLE Binding Triggers Recruitment of NuRD Components

To distinguish between these possibilities and to assess precisely which components of the chromatin modification network (Figure A2B) are dependent on the Tbx20−

TLE3 interaction, we used label-free mass spectrometry to assess the diff erences between

Tbx20-EGFP and Tbx20eh1mut -EGFP protein complexes. To do this, we expressed Tbx20-

EGFP and Tbx20eh1mut -EGFP in HEK293 cells and performed parallel isolations of EGFP- tagged Tbx20 complexes, as described above. Changes in the relative abundance of interacting chromatin remodeling factors between wild-type and mutant Tbx20 complexes were assessed using two label-free quantification approaches: (1) spectral counting and (2)

MS1 peak area quantification (see Materials and Methods). To correct for the total amount

129 of isolated Tbx20 complexes between conditions, we normalized the quantitative values for associated proteins identified in mutant Tbx20 complexes by the ratio of wild- type/eh1mut Tbx20-EGFP. Also, Tbx18 was excluded from label-free quantification due to the presence of only one tryptic peptide that was exclusive to Tbx18 (the other peptide was isobaric with a tryptic peptide from Tbx20). Consistent with our previous Western blot analysis (Figure A3B and C), we no longer identified TLE1 or TLE3 in the Tbx20eh1mut mutant isolation (Figure A4A and SI Table A5). We also observed an eh1mut-dependent spectral count fold decrease that was specific to components of the NuRD complex, though with variation in the magnitude of the decrease between proteins (Figure A4A). To address this variation, we also employed MS1 -based quantification, which allows more precise measurement of relative abundance, particularly for proteins with few spectral counts (e.g., in the mutant isolation, SI Table A5) and/or with shared tryptic sequences (e.g., RBBP4 and

RBBP7). MS1 -based analysis confirmed the spectral counting result for TLE1/3, suggesting a relative decrease of at least 10-fold in the eh1mut vs wild-type Tbx20 (SI Table A6).

Further, we observed that association with HDAC2, MTA1, and the histone binding proteins

RBBP4/ RBBP7 was significantly reduced and to a similar extent in the eh1mut compared to wild-type Tbx20, indicating that the interaction of Tbx20 with NuRD complex components is partially Groucho-dependent (Figure A4A and B and SI Table A5 and A6). These data suggest that TLE corepressors play a central role in the formation of a Tbx20 transcriptional repressive complex by recruiting core components of the NuRD complex including HDAC2.

Overall, these results demonstrate that this AP-MS approach can efficiently isolate and identify specific Tbx20 interactions, which may help to define the mechanisms involved in

Tbx20-mediated gene regulation.

Endogenous Tbx20 Interacts with TLE Factors in Mouse Embryonic Hearts

130 Our proteomic analysis suggests that Tbx20 is linked to a transcriptional repressive complex via direct interaction with a TLE family member. To assess the endogenous

Tbx20 association with TLE family members in vivo at the time in embryogenesis at which

Tbx20 has been shown to function, we first examined the expression of all of the TLE family members in mouse embryonic day 10.5 (E10.5) heart tissue by RT-PCR. At this stage of development, Tbx20 is uniformly expressed throughout the four-chambered embryonic heart (Kraus et al., 2001), where it is required for proper transcriptional regulation of cardiac chamber-specific genes (Cai et al., 2005; Singh et al., 2005; Stennard et al., 2005;

Takeuchi et al., 2005). All of the TLE family members are highly expressed at this time, with the exception of TLE2 , which is expressed at relatively low levels (Figure A5A). To further confirm that Tbx20 colocalizes with TLE1/3 within the same cells of the embryonic heart, we performed in situ hybridization for Tbx20 , TLE1 , and TLE3 transcripts on serial tissue sections from an E10.5 heart. This analysis revealed that TLE1 and TLE3 are expressed broadly with Tbx20 throughout the myocardium (Figure A5B− G). Importantly, we were able to detect an interaction between endogenous Tbx20 and both TLE1 and TLE3 in the embryonic heart, indicating that Tbx20 binds both TLE factors in vivo during heart development (Figure A5H). This finding represents the first demonstration of endogenous

Tbx20 protein− protein interactions from an embryonic heart. These data further validate our proteomic approach and demonstrate that Tbx20 assembles a TLE repressor complex in the embryonic heart at the time at which Tbx20 functions in cardiac development.

Ablation of Tbx20-TLE Binding Disrupts Tbx20-Mediated Transcriptional Repression in Xenopus Embryos

Having established that Tbx20 assembles a TLE corepressor complex in the forming heart, we next sought to establish the consequences of Tbx20-TLE binding on Tbx20 biological activity. To address this issue, we examined whether Tbx20eh1mut mutant protein

131 could promote morphogenetic changes in the embryo when ectopically expressed in developing Xenopus embryos. Xenopus embryos were injected at the one-cell stage with

Tbx20-EGFP or Tbx20eh1mut-EGFP mRNA and allowed to develop until control uninjected embryos reached tailbud stages (stage 23/33). Ectopic expression of Tbx20-

EGFP drastically disturbs cell movements during gastrulation, leading to a marked shortening of the anterior/posterior axis, consistent with a previous report5a (Figure A6A and B). Strikingly, these defects are partially rescued by ablation of the eh1 binding motif, indicating that recruitment of TLE factors is critical for the ability of Tbx20 to promote changes in morphogenesis (Figure A6C). This finding is supported by the observation that

Tbx20-EGFP and Tbx20eh1mut -EGFP are expressed at comparable levels from injected mRNAs, as shown by western blotting for GFP (Figure A6D). Our findings are consistent with an essential role for Tbx20- TLE repressor complexes in embryonic heart formation.

To gain insights into what molecular processes and genes are regulated by Tbx20-TLE complexes, we used high throughput sequence analysis to examine the transcriptomes in

Tbx20- EGFP and Tbx20eh1mut-EGFP -injected embryos. Specifically, we sought to identify genes downregulated by Tbx20-TLE repressor complexes. We chose stage 12.5 (early neurulastage) embryos for analysis since misexpressed Tbx20 leads to gene expression changes at this stage (Figure A1D), and to eliminate the detection of gene expression changes induced by endogenous Tbx20 transcripts, which can be detected as early as stage 16 (Brown et al., 2003). A total of 201 genes were significantly downregulated at least 2-fold by ectopic

Tbx20 when compared to control uninjected embryos (SI Figure A1). Of these 201 genes, 173 were not repressed by ectopic expression of Tbx20eh1mut protein (86% of genes downregulated by wild wildtype Tbx20; SI Figure A1 and Table A9), indicating a role for Tbx20-TLE complexes in negative regulation of this group of genes. Gene ontology enrichment analysis revealed a trend whereby the 173 genes not repressed by Tbx20eh1mut were significantly enriched for functions involving anterior/posterior pattern specification,

132 regulation of cell differentiation and transcription, regulation of the Wnt signaling pathway, and tissue patterning (Figure A6E and SI Table A10), cellular processes known to be associated with Gro/TLE repressive function in the embryo (Buscarlet and Stifani, 2007).

Indeed, upon ranking the 173 genes uniquely repressed by Tbx20 overexpression, the 40 genes most differentially regulated between Tbx20- and Tbx20eh1mut expressing embryos included hox genes involved in anterior/ posterior patterning (hoxa7 , hoxc10 , hoxd4 , hoxc9 , hoxa2 ) (Gaunt et al., 1986), transcription factors important for control of cellular differentiation (irx3 , tbx3 , gata3 , and nkx6.2 ), and a number of Wnt signaling pathway components ( f rzb , dkk1 , frzb2 ) (Figure A6F). Notably, Irx3 has recently been identified as a direct ChIP-seq genomic target of Tbx20 in the adult heart, and a recent study fi nds that

Tbx20 directly regulates Wnt pathway genes in the endocardial cushions, suggesting that our RNA-seq analysis identified primary mediators of Tbx20 cardiac function (Cai et al.,

2013; Shen et al., 2011). Additionally, early, ectopic expression of Tbx20 leads to downregulation of a number of genes with important roles in cardiac muscle tissue morphogenesis, including the cardiac transcription factors nkx2.5 , isl1 , pitx2 , and foxc1

(Figure A6E and F and SI Table A10). Interestingly, these cardiac genes are not downregulated by Tbx20eh1mut protein (Figure A6E and F and SI Table A10), implying that, in the absence of other cues (i.e. transcriptional coactivators), Tbx20 may act in combination with TLE repressor complexes to repress cardiac promoters. Collectively, these results provide additional evidence that Tbx20-mediated repression is facilitated by

Gro/ TLE complexes.

CONCLUSIONS

Despite the critical role of Tbx20 in cardiac development, the precise mechanisms by which Tbx20 regulates distinct gene programs in the heart are not understood. Studies in mouse knockout models of Tbx20 indicate that Tbx20 is required for proper patterning and

133 morphogenesis of working myocardium (Cai et al., 2005; Singh et al., 2005; Stennard et al.,

2005; Takeuchi et al., 2005). Thus, activating and/or repressive activity of Tbx20 on target genes underlies the primary cardiomyocyte lineage split into specialized chamber and nonchamber myocardium. To identify and characterize the determinants of

Tbx20 transcriptional activity in the heart, it is essential to identify Tbx20 interacting proteins. Our study constitutes the first unbiased analysis of Tbx20 protein interactions.

Using a proteomics/bioinformatics approach, we identified a unique transcriptional repression network that includes Groucho corepressors, components of the NuRD complex, components of the INO80 chromatin remodeling complex (RUVBL1 and RUVBL2), and the

T-box repressor Tbx18.

A Tbx20-TLE-NuRD Repressor Complex

The Tbx20 homologue Midline was recently demonstrated to bind Groucho directly in an eh1-dependent manner in Drosophila whole embryos, and this interaction was required for proper transcriptional repression of the wingless gene during segmentation of the ectoderm (Formaz-Preston et al., 2012). Our studies confirm and expand upon this work by demonstrating that (1) vertebrate Tbx20 interacts with Groucho homologues, (2) vertebrate Tbx20 interacts with at least two members of the Groucho-related TLE family,

TLE1 and TLE3, in human cells and in the mouse embryonic heart through the eh1 motif, and (3) Tbx20− TLE interactions directly result in the recruitment of components of the chromatin remodeling NuRD complex including HDAC2. These data suggest that recruitment of Gro/ TLE corepressor complexes and subsequent deacetylation of target loci represent an evolutionarily conserved mechanism by which Tbx20 functions and, thus, one mode by which Tbx20 promotes inactive chromatin states during development. A thorough expression analysis of TLE factors in the developing heart has not been published, although it has been reported that TLE1 and TLE3 transcripts were not detectable in the

134 mouse embryonic heart by in situ hybridization (Santisteban et al., 2010). Our data, however, suggests that most members of the mouse TLE family are expressed in the heart at stage E10.5 of embryogenesis. Further, we fi nd that TLE1 and TLE3 transcripts are broadly expressed throughout the E10.5 myocardium by in situ hybridization. Finally, TLE1 and

TLE3 proteins coisolate with endogenous Tbx20 at E10.5. The availability of other TLE factors in the heart, and our results showing that Tbx20 interacts with both TLE1 and TLE3, suggest that Tbx20 may interact with multiple members of this family to cooperatively regulate genes in the heart. As such, it will be interesting to determine whether other TLE family members have distinct temporal and spatial expression patterns in the forming heart or whether they can act redundantly on Tbx20 target genes. Our proteomic and biochemical analyses of Tbx20 complexes indicate that binding of TLE factors by Tbx20 results in the recruitment of core components of the NuRD complex (Joshi et al., 2013), specifically, the histone binding proteins RBBP4 and RBBP7, MTA1, and HDAC2. Historically, requirements for the NuRD complex in early cell fate decisions in the embryo (Hendrich et al., 2001;

Marino and Nusse, 2007) have prevented the identification of a role for NuRD specifi cally in the heart; however, HDAC2 is ubiquitously expressed in developing myocardium (Hang et al., 2010), and mice that are mutant for both HDAC1 and -2 die neonatally of cardiac arrhythmias and dilated cardiomyopathy (Montgomery et al., 2007). Taken together, these studies implicate an important role for a Tbx20-NuRD association in developing cardiomyocytes. Interestingly, interactions with the remainder of the chromatin remodeling network were unaffected by the eh1 mutation, suggesting that they are recruited independently of the eh1 binding motif and TLE recruitment. Of note, RUVBL2 (also called

Reptin) was demonstrated to co-occupy the Hesx1 promoter with TLEs and HDAC1 to silence Hesx1 expression during mouse pituitary development (Olson et al., 2006), indicating that this protein, although still present within Tbx20eh1mut mutant complexes, may be part of an intact TLE corepressor complex via recruitment by a as yet unidentified

135 binding motif within Tbx20. The finding that Tbx20 represses cardiac genes when ectopically expressed in early Xenopus embryos indicates that Tbx20 activating or repressive activity is likely context dependent. Previous studies have reported that Tbx20, in combination with other cardiac transcription factors, primarily acts to promote cardiac gene expression while repressing genetic programs of other tissues (Mandel et al., 2010;

Sakabe et al., 2012; Stennard et al., 2003). In accordance with this, we identified genes essential for vasculogenesis, eye development, neural crest development, and motor neuron development downregulated by Tbx20 misexpression. Given that Tbx20 misexpression also downregulates cardiac transcription factors, one possibility is that additional tissue-specific factors or cues are needed to antagonize default repression achieved by Tbx20. Indeed, we observed that Tbx20eh1mut protein fails to repress these genetic programs, implying that displacement of the TLE corepressor complex is sufficient to abolish the Tbx20 repressive activity on these genes. As such, it would be interesting to determine whether TLE-mediated repression can be out-competed by cardiac coactivators in the context of a cardiac progenitor or myocyte. This principle has been illustrated previously by the role Gro/TLE plays in cells that do not receive Wg/Wnt signaling. Binding of Gro/TLE to Tcf/ Lef repressors on

Wg/Wnt target genes blocks recruitment of β -catenin, an obligatory Tcf/Lef coactivator.

Effective Wg/Wnt signaling leads to accumulation of β -catenin, which subsequently can out-compete Gro/TLE for binding to Tcf/ Lef (Cinnamon and Paroush, 2008). Thus, the level of Wg/Wnt signaling via β -catenin accumulation dictates which cofactor binds Tcf/Lef, leading to transcription activation or repression. Our data therefore highlight a unique mechanism by which TLE-mediated repression may function as a context-dependent regulatory switch required for Tbx20 to alternate between activator and repressor.

A Tbx20-INO80 Complex

136 The INO80 chromatin remodeling complex is a very large protein complex with 11 to

16 members, including the ATPdependent helicases Ino80 and SRCAP, the DNA helicases Pontin (Ruvbl1, Tip49, Tip49a), and Reptin (Ruvbl2, Tip48, Tip49b), actin, and various actin-related proteins (Arp4, Arp5, Arp8, β -actin, Arp7, Arp9) (Jin et al., 2005;

Shen et al., 2000). The INO80 complex alters chromatin accessibility, resulting in activation or repression of target genes (Cai et al., 2007; Ford et al., 2007; Klopf et al., 2009). Two members of the complex, Pontin and Reptin, have opposing acitivities within the INO80 complex, and this antagonistic relationship has been shown to play a role in cardiac growth in zebrafi sh (Rottbauer et al., 2002). An ENU-induced mutation in Reptin leads to cardiac hyperplasia and embryonic lethality. This mutation is an activating mutation in Reptin , increasing the ATPase activity of Reptin complexes, thus overriding Pontin inhibition of

Reptin activity, and increasing the transcriptional repressor activity of the complex. Mutant

Reptin was subsequently shown to have a stronger repressive effect on β - catenin/TCF- mediated transactivation, leading to the hypothesis that Pontin/Reptin complexes regulate β

-catenin-mediated activation of cell cycle genes such as cyclin D and c-Myc to control the balance between proliferation and differentiation in the developing zebrafish heart. Our data suggests that Tbx20 interacts with a RUVBL1-RUVBL2 protein complex independently of its association with a TLE repressor complex, suggesting that Tbx20 may also recruit the INO80 complex for gene regulation. Tbx20 has a well-documented role in cardiomyocyte proliferation (Cai et al., 2005) and seems to exert opposite effects on cell proliferation in embryonic versus fetal cardiomyocytes (Chakraborty and Yutzey, 2012).

Therefore, it is tempting to speculate that association of Tbx20 with the INO80 complex in fetal cardiomyocytes underlies this switch in the activity of Tbx20 transcriptional complexes.

A Tbx20-Tbx18 Complex

137 An unexpected finding of these studies is that Tbx20 interacts with the tissue-specific transcription factor Tbx18. In the developing heart, Tbx18 expression overlaps with that of

Tbx20 in a subset of cardiomyocytes within the interventricular septum and a portion of the left ventricle at a stage when the heart is undergoing chamber specialization and expansion, processes that are both dependent on proper Tbx20 function (Christoffels et al.,

2009; Conlon et al., 2012; Kaltenbrun et al., 2011; Zeng et al., 2011). Thus, interaction with

Tbx18 in this subset of cardiomyocytes provides one potential mechanism through which

Tbx20 may function to regulate regionally distinct gene programs in the heart. Tbx18 is also expressed in the epicardium, an epithelial monolayer that covers the myocardium and is a critical source of signals and cells for the underlying myocardium (Kraus et al., 2001).

Recently, a microarray analysis of isolated epicardial cells revealed Tbx20 as an epicardium enriched transcription factor (Huang et al., 2012), opening the possibility for a Tbx18-Tbx20 transcriptional complex within the epicardium. Tbx18 also plays a prominent role in formation of the myocardial sinus horns that make up the venous pole of the heart; however, it is not clear whether Tbx20 is coexpressed with Tbx18 within this tissue (Christoffels et al.,

2006). Additionally, our lab has previously reported that Tbx20 interacts physically with

Tbx5 (Brown et al., 2005), suggesting that heterodimerization with other T-box factors may represent an important mechanism by which Tbx20 regulates gene expression in the embryo. Interestingly, Tbx18 has also been shown to interact with TLE3 via an eh1 binding motif within the N-terminus of the protein (Farin et al., 2007). In this study, transcriptional assays demonstrated that Tbx18 can repress activation of the Nppa /ANF promoter by the cardiac transcription factors Gata4, Nkx2.5, and Tbx5. This finding was interpreted as Tbx18 abrogation of Nppa / ANF expression through competition with Tbx5, a second

Tbox protein, for T-box binding sites (TBEs) within the Nppa/ ANF promoter. Similarly,

Tbx18 is predicted to repress Tbx6- mediated activation of the Notch ligand Delta-like 1

(Dll1) in anterior somites through competition with Tbx6 (Farin et al., 2007).

138 Collectively, these data imply a model in which Tbx18 competes with other T-box activators for occupancy of TBEs and subsequently achieves repression by recruiting a TLE corepressor complex to the target gene. Here, we have demonstrated that Tbx20 and Tbx18 physically interact, and Tbx20 recruits a TLE repressor complex similar to that reported for

Tbx18. Therefore, a second model predicts that Tbx18 and Tbx20 may be acting cooperatively as repressors on a common set of target genes. Further studies are needed to distinguish between these possibilities. In summary, the goal of our study was to expand upon the current knowledge of the Tbx20 transcriptional network. By combining immunoaffinity purification with proteomic and functional network analysis, we have identified a Tbx20 transcription repression network with chromatin remodeling and deacetylase functions. In particular, our results reveal a crucial role for Gro/TLE-NuRD corepressor complexes in facilitating Tbx20-mediated transcriptional repression. We also identified Tbx18 as a Tbx20 interaction, raising the question of whether Tbx20 transcriptional repression relies on cooperative activity of Tbx20 and other cardiac transcription factors, similar to what has been shown for Tbx20 transcriptional activation in the presence of the activators Gata4, Nkx2.5, and Tbx5 (Stennard et al., 2003; Takeuchi et al., 2005). Future studies will aim to delineate the biological role of these repressive interactions, particularly as they relate to regulation of the cardiogenic program.

139 Figure A1. Tbx20-EGFP is nuclear-localized and transcriptionally active. (A) Schematic of

EGFP-tagged (green) Tbx20 expression construct, showing the N-terminal (white), T-box

(black), transactivation (blue), and repression (brown) domains. Numbers denote amino acid residues. (B) Tbx20- EGFP is localized to the nucleus in HEK293 cells, as confirmed by live GFP fluorescence and colocalization with DAPI. (C) Tbx20-EGFP mRNA was injected at the 1-cell stage into Xenopus laevis embryos. Expression of Tbx20-EGFP in the animal pole of stage 9 Xenopus embryos was confirmed by live GFP fluorescence. (D) RT-PCR analysis of the mesodermal genes chordin and Xbra and the skeletal muscle gene Myf5 in stage 13 whole embryos, stage-matched untreated animal caps, Activin-treated animal caps, Tbx20- injected animal caps, and Tbx20-EGFP-injected animal caps. The housekeeping gene Gapdh was used as a loading control for all RT-PCR reactions.

140 Figure A2. Shotgun proteomics of Tbx20-EGFP protein complexes reveals association with a chromatin remodeling and Groucho transcriptional protein network. (A) GO enrichment analysis of Tbx20 interactions using ClueGO clustering according to biological function ontologies. (B) The Cytoscape network was assembled from automated retrieval and manual curation of protein functional associations using STRING analysis (gray lines) and literature curation (solid black lines), respectively. Potential interactions/functional associations with

Tbx20 are indicated by black dashed lines. Nodes are labeled with respective gene symbols, and enrichment index values (see Materials and Methods) are represented by node size and blue color intensity. Functional protein groupings are indicated in closed circles.

141 Figure A3. Tbx20 interacts with TLE1/3, HDAC2, and Tbx18. (A) Protein sequence alignment of an N-terminal eh1 binding motif in Tbx20 demonstrating complete conservation of the eh1 motif across all vertebrate homologues of Tbx20. h, human; m, mouse; x, Xenopus; z, zebrafish. (B) Reciprocal immunoisolations of TLE1-Flag complexes from HEK293 cells expressing either Tbx20-EGFP or Tbx20eh1mut-EGFP. (C)

Reciprocal immunoisolations of TLE3 complexes from HEK293 cells expressing either

Tbx20-EGFP orTbx20eh1mut-EGFP. (D) Reciprocal immunoisolations of Tbx18-Flag complexes from HEK293 cells expressing Tbx18-Flag in the presence or absence of Tbx20-

EGFP. (E) Reciprocal immunoisolations of HDAC2-GFP from HEK293 cells expressing

Tbx20-HA and/or TLE3. IP, immunoprecipitation; WB, Western blot.

142 Figure A4. Tbx20 assembles a Gro/TLE-NuRD repression complex via the eh1 binding motif. Wild-type Tbx20-EGFP and Tbx20eh1mut-EGFP were immunoaffinity purified from

HEK293 cells with associated proteins and analyzed by mass spectrometry. (A) Fold differences for each interaction illustrated in part B are shown for the isolated Tbx20eh1mut mutant versus wild-type Tbx20. (B) The relative size of the circles indicates increased or decreased relative abundance of each interaction as determined by the MS1-based fold- change for mutant versus wild-type Tbx20 isolations.

143 Figure A5. Endogenous Tbx20 interacts with TLE1/3 in mouse embryonic hearts. (A) RT-

PCR analysis of TLE family members and cardiac-specific markers Nkx2.5 and Tbx20 in

E10.5 heart tissue. All samples derived from embryonic hearts dissected at E10.5. (B, C)

Section in situ hybridization analysis for Tbx20 expression on a cryosection of an E10.5 heart. Neighboring sections from the same embryonic heart were used to assess TLE1 (D, E) and TLE3 (F, G) expression. (H) 25 hearts were dissected, and endogenous Tbx20 complexes were isolated with an antibody against Tbx20, analyzed by SDS-PAGE, and immunoblotted with antibodies against TLE1 and TLE3. In parallel and as a control, a mock immunoprecipitation was performed in the absence of Tbx20 antibody: ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle.

144 Figure A6. RNA-sequencing reveals disrupted transcriptional output in Tbx20eh1mut - expressing embryos. Xenopus embryos were injected at the one-cell stage with Tbx20 (B) or

Tbx20eh1mut (C) mRNA and allowed to develop until sibling uninjected embryos (A) reached tadpole stages. (D) Total protein equivalent to one uninjected, Tbx20-EGFP, or Tbx20eh1mut embryo (stage 12.5) was analyzed by SDS-PAGE and immunoblotted with an antibody against GFP. Western blot shows comparable protein levels from injected mRNAs. (E) Gene ontology grouping (GOrilla) of the 173 genes downregulated at least 2-fold by wild-type

Tbx20, but not by Tbx20eh1mut. GO terms enriched in this gene set were plotted by the log of their FDRadjusted p-values. (F) Heat map depicting differential regulation of genes by wild- type Tbx20 and Tbx20eh1mut. The 173 genes downregulated at least 2-fold only by wild- type Tbx20 were ranked by the fold change ratio between the mutant and wild-type. The 40 genes with the greatest disparity between the mutant and wild-type are shown.

145 Table A1. Tbx20-Associated Proteins Identified by LC-MS/MS. Numbers represent an average across three experimental replicates.

146 Figure SA1. Venn diagram summarizing overlap of regulatory capabilities between wild-type

eh1mut Tbx20 and Tbx20 .

147 Supplemental Information Tables 1-10 are available at pubs.acs.org.

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