Conserved Features of Chromatin Remodeling Enzymes: A Dissertation

Chromatin structure plays an essential role in the regulation of many nuclear processes such as transcription, replication, recombination, and repair. It is generally accepted that chromatin remodeling is a prerequisite step in gene activation. Over recent years, large multisubunit enzymes that regulate the accessibility of nucleosomal DNA have emerged as key regulators of eukaryotic transcription. It seems likely that similar enzymes contribute to the efficiency of DNA replication, recombination, and repair. These chromatin remodeling complexes can be classified into two broad groups: (1) the ATP-dependent enzymes, which utilize the energy of ATP hydrolysis to increase the accessibility of nucleosomal DNA; and (2) histone modifying enzymes that phosphorylate, acetylate, methylate, ubiquitinate, or ADP-ribosylate the nucleosomal histones (for review see Kingston and Narlikar, 1999; Muchardt and Yaniv, 1999; Brown et al., 2000; Vignali et al., 2000; Strahl and Allis, 2000). The mechanism by which these two groups of large, multi-subunit enzymes function to alter chromatin structure is enigmatic. Studies suggest that ATP-dependent and histone acetyltransferase chromatin remodeling enzymes have widespread roles in gene expression and perform both independent and overlapping functions. Interestingly, although both groups of enzymes appear to be distinct, several features of these enzymes have been conserved from yeast to man. Thus, understanding the role of these similar features will be essential in order to elucidate the function of remodeling enzymes, their functional interrelationships, and may uncover the fundamental principals of chromatin remodeling. In this study, we use a combination of yeast molecular genetics and biochemistry to dissect out the function of individual parts of these chromatin remodeling machines and to understand how these large macromolecular assemblies are put together. In addition, we also investigate the mechanism by which the ATP-dependent enzymes exert their regulatory effects on chromatin structure. Structure/function analysis of Saccharomyces cerevisiaeSwi3p (conserved in SWI/SNF complexes across all eukaryotic phyla) reveals a unique scaffolding role for this protein as it is essential for assembly of SWI/SNF subunits. We have also characterized a novel motif that has homology to the Myb DNA binding domain, the SANT domain, and that is shared among transcriptional regulatory proteins implicated in chromatin remodeling. Mutational analysis of this domain in yeast Swi3p (SWI/SNF), Rsc8/Swh3p (RSC), and Ada2p (GCN5 HATs) reveals an essential function for the SANT domain in chromatin remodeling. Moreover, our studies suggest that this novel motif may be directly involved in mediating a functional interaction with chromatin components (i.e. histone amino terminal domains). We have also directly compared the activities of several members of the ATP-dependent chromatin remodeling enzymes. Surprisingly, we find that these enzymes utilize similar amounts of ATP to increase nucleosomal DNA accessibility. In as much, we show that changes in histone octamer comformation or composition is not a requirement or consequence of chromatin remodeling by SWI/SNF. Taken together, these data suggest a similar mechanism for ATP-utilizing chromatin remodeling enzymes in which disruption of histone-DNA contacts occur without consequence to the structure of the histone octamer. These data have striking implications for how we view the mechanism of chromatin remodeling

Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10⁻⁹. We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10⁻⁴⁰) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10⁻⁴). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.National Heart, Lung, and Blood Institute (HHSN268201100005C)National Heart, Lung, and Blood Institute (HHSN268201100006C)National Heart, Lung, and Blood Institute (HHSN268201100007C)National Heart, Lung, and Blood Institute (HHSN268201100008C)National Heart, Lung, and Blood Institute (HHSN268201100009C)National Heart, Lung, and Blood Institute (HHSN268201100010C)National Heart, Lung, and Blood Institute (HHSN268201100011C)National Heart, Lung, and Blood Institute (HHSN268201100012C)National Heart, Lung, and Blood Institute (N01-HC-55015)National Heart, Lung, and Blood Institute (N01-HC-55016)National Heart, Lung, and Blood Institute (N01-HC-55018)National Heart, Lung, and Blood Institute (N01-HC-55019)National Heart, Lung, and Blood Institute (N01-HC-55020)National Heart, Lung, and Blood Institute (N01-HC-55021)National Heart, Lung, and Blood Institute (N01-HC-55022)National Heart, Lung, and Blood Institute (R01HL087641)National Heart, Lung, and Blood Institute (R01HL59367)National Heart, Lung, and Blood Institute (R01HL086694)National Human Genome Research Institute (U.S.) (U01HG004402)United States. National Institutes of Health (HHSN268200625226C)United States. National Institutes of Health (UL1RR025005)National Heart, Lung, and Blood Institute (HHSN268201200036C)National Heart, Lung, and Blood Institute (N01HC55222)National Heart, Lung, and Blood Institute (HHSN268200800007C)National Heart, Lung, and Blood Institute (N01HC85079)National Heart, Lung, and Blood Institute (N01HC85080)National Heart, Lung, and Blood Institute (N01HC85081)National Heart, Lung, and Blood Institute (N01HC85082)National Heart, Lung, and Blood Institute (N01HC85083)National Heart, Lung, and Blood Institute (N01HC85086)National Heart, Lung, and Blood Institute (U01HL080295)National Science Foundation (U.S.) (R01HL087652)National Heart, Lung, and Blood Institute (R01HL105756)National Heart, Lung, and Blood Institute (R01HL103612)National Heart, Lung, and Blood Institute (R01HL120393)National Institute on Aging (R01AG023629)National Center for Advancing Translational Sciences (U.S.) (UL1TR000124)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (DK063491)National Heart, Lung, and Blood Institute (N01-HC-25195)National Heart, Lung, and Blood Institute (2K24HL04334)National Heart, Lung, and Blood Institute (R01HL077477)National Heart, Lung, and Blood Institute (R01HL093328)National Heart, Lung, and Blood Institute (NIH R01HL105993)National Institute on Aging (N01AG62101)National Heart, Lung, and Blood Institute (N01AG62103)National Heart, Lung, and Blood Institute (N01AG62106)National Institute on Aging (1R01AG032098-01A1)United States. National Institutes of Health (HHSN268200782096C)National Cancer Institute (U.S.) (CA-34944)National Cancer Institute (U.S.) (CA-40360)National Cancer Institute (U.S.) (CA-097193)National Heart, Lung, and Blood Institute (HL-26490)National Heart, Lung, and Blood Institute (HL-34595

H2A.Z: a molecular rheostat for transcriptional control

The replacement of nucleosomal H2A with the histone variant H2A.Z is critical for regulating DNA-mediated processes across eukaryotes and for early development of multicellular organisms. How this variant performs these seemingly diverse roles has remained largely enigmatic. Here, we discuss recent mechanistic insights that have begun to reveal how H2A.Z functions as a molecular rheostat for gene control. We focus on specific examples in metazoans as a model for understanding how H2A.Z integrates information from histone post-translational modifications, other histone variants, and transcription factors (TFs) to regulate proper induction of gene expression programs in response to cellular cues. Finally, we propose a general model of how H2A.Z incorporation regulates chromatin states in diverse processes

Polycomb Repressive Complex 2 Regulates Lineage Fidelity during Embryonic Stem Cell Differentiation

Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, making it difficult to determine the precise roles of PRC2 during lineage commitment. Moreover, while studies suggest that PRC2 prevents DNA methylation, how these two epigenetic regulators coordinate to regulate lineage programs is poorly understood. Using several PRC2 mutant ESC lines that maintain varying levels of H3K27me3, we found that partial maintenance of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). In contrast, genes that function to specify other lineages failed to be repressed in these cells, suggesting that PRC2 is also necessary for lineage fidelity. We also found that loss of H3K27me3 leads to a modest gain in DNA methylation at PRC2 target regions in both ESCs and in SMNs. Our study demonstrates a critical role for PRC2 in safeguarding lineage decisions and in protecting genes against inappropriate DNA methylation.National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Training Grant T 32 GM007287)Smith Family Foundation (Contract LTR DATED 11/6/09

Getting to the heart of the matter: long non-coding RNAs in cardiac development and disease

Cardiogenesis in mammals requires exquisite control of gene expression and faulty regulation of transcriptional programs underpins congenital heart disease (CHD), the most common defect among live births. Similarly, many adult cardiac diseases involve transcriptional changes and sometimes have a developmental basis. Long non‐coding RNAs (lncRNAs) are a novel class of transcripts that regulate cellular processes by controlling gene expression; however, detailed insights into their biological and mechanistic functions are only beginning to emerge. Here, we discuss recent findings suggesting that lncRNAs are important factors in regulation of mammalian cardiogenesis and in the pathogenesis of CHD as well as adult cardiac disease. We also outline potential methodological and conceptual considerations for future studies of lncRNAs in the heart and other contexts.National Heart, Lung, and Blood Institute (Bench to Bassinet Program U01HL098179)National Heart, Lung, and Blood Institute (Bench to Bassinet Program U01HL098188

0578: Valvular atrial fibrillation and the risk of stroke and deaths: additional prognostic value of the CHA2DS2-VASc score

PurposeThe CHA2DS2-VASc score has been validated and is widely used to stratify the risk of thromboembolism in patients with non-valvular atrial fibrillation (AF). We sought to investigate whether this score could also be useful to predict the risk of stroke and death in patients with valvular AF.MethodsBetween 1998 and 2011, 1,592 consecutive patients, hospitalised for AF, 300 with valvular AF (mitral and/or aortic valve disease) and 1,292 with non-valvular AF were enrolled in the cohort. All patients were followed-up at least 6 months and cardiovascular events recorded. The end-point was defined as the first occurrence of stroke or death. The Cox analysis was adjusted on warfarin, antiplatelet and antiarrhythmic treatments at discharge.ResultsMean age was 73±14 years in valvular AF and 68±15 in non-valvular AF (p=0.0001). At baseline, in the valvular AF group CHA2DS2-VASc score were = 0 for 14 (5%) patients, = 1 for 28 (9%), ? 2 for 258 (86%). Non-valvular AF CHA2DS2-VASc scores were = 0 for 158 (12%), = 1 for 189 (15), ?2 for 945 (73%). The difference was statistically significant (p<0.0001). During a mean follow-up of 4.6±3.5 years, the patients with valvular AF experienced 154 (51%) and the patients with non-valvular AF experienced 409 (32%) strokes or deaths. The Kaplan-Meier curves (figure) show that patients with a CHA2DS2-VASc score ?2 were at higher risk of stroke or death. The adjusted Cox model, showed that valvular AF (HR, 1.57, 95%CI 1.30-1.89, p<0.0001) and a CHA2DS2-VASc score ?2 (HR, 5.30, 95%CI 3.77-7.45, p<0.0001) were predictors of risk of stroke or death.ConclusionThese results suggest that a CHA2DS2-VASc score ?2 is associated with a higher risk of stroke and deaths, at mid-term follow-up, in patients with valvular AF (figure next page).Abstract 0578 - Figure: Kaplan-Meier survival curve

Cell size is a determinant of stem cell potential during aging

Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.Peer reviewe

Braveheart, a Long Noncoding RNA Required for Cardiovascular Lineage Commitment

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.Damon Runyon Cancer Research Foundation (DRG 2032-09)Damon Runyon Cancer Research Foundation (DFS 04-12)European Molecular Biology Organization (Long-term Fellowship)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098179)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098188)Smith Family FoundationPew Charitable Trusts. Program in the Biomedical Science

Emergent mechanical control of vascular morphogenesis

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD