Determining the role of the histone variant H2AZ in mammalian development
Elaborate mechanisms have evolved to introduce meaningful variation into chromatin for the purposes of regulating gene expression and other DNA-mediated processes in response to environmental and developmental cues (Sha and Boyer, 2009; Surface et al., 2010). The replication-independent replacement of core histones with histone variants has recently emerged as a key mechanism for regulating chromatin states. However, we lack detailed knowledge of how most histone variants function. A main goal of the lab is to dissect the function of the H2A-type variant H2AZ because it has an essential, but uncharacterized role in metazoan development. In particular, H2AZ ablation causes embryonic lethality at gastrulation in mammals, but its function during this process has not been elucidated. While a link between H2AZ and gene regulation has been established in a range of organisms, these studies also have not revealed how H2AZ contributes to development. Recent work from my lab has shown that H2AZ occupies the promoters of large cohort of developmental genes in murine ESCs and that it is necessary for appropriate target gene regulation (Creyghton et al., 2008). We found that H2AZ is dispensable for maintenance of the ESC state, but that it is essential for proper execution of developmental gene expression programs during ESC differentiation. We further showed that H2AZ-deficient ESCs could not contribute to tissues in chimeric animals. Thus, our work established a critical role for H2AZ in regulating cell fate transitions that may explain its essential requirement during mammalian development.
Our current goal is to understand the mechanistic basis of H2AZ function. One approach that we are taking is to dissect the function of regions that have diverged between H2AZ and canonical H2A. For example, the H2AZ docking domain contains an extended acidic patch (AP) that is essential for viability in both Drosophila and Xenopus. Our data show that site-specific mutation in H2AZ-AP causes defects in chromatin incorporation and also in the regulation of gene expression during ESC differentiation. Moreover, using Fluorescence Recovery After Photobleaching (FRAP), we found that H2AZ-AP influences chromatin dynamics that is distinct from the action of H2A suggesting that H2AZ drives formation of specialized chromatin domains. Thus, our work establishes a functional role for the acidic patch domain in mediating chromatin dynamics that determine gene expression states and ultimately cell fate (Subramanian et al., in preparation). We are now pursuing the downstream effectors of this domain as structural predictions indicate that the acidic patch may form a novel interaction surface on the nucleosome. We have already identified putative interacting proteins by mass spec, and are currently dissecting how these contribute to H2AZ function during ESC differentiation.
In parallel with our ESC studies, we aim to establish the role of H2AZ at later stages of development. For example, what is the function of H2AZ after gastrulation? Does H2AZ mediate lineage commitment in progenitor populations and is it required for maintenance of cell state in later development? Notably, the histone H2A type variant H2AZ is encoded by two genes in the mouse, H2AFZ (H2AZ) and H2AFV, that differ by only 3 amino acids. These are present in most vertebrates, yet little is known about their independent functions. We determined that the two isoforms display distinct expression patterns and that the H2AZ homolog is significantly more abundant in ESCs whereas H2AFV is more highly expressed in specific differentiated cells and adult tissues. Thus, do the two different variants have distinct developmental roles? We have developed conditional H2AZ and H2AFV knock-out ESC lines that will allow us to establish how each variant functions to regulate chromatin states during lineage commitment and how they interface with downstream effectors to pattern gene expression programs in the developing embryo.
Dissecting the regulators of cell-type specific gene expression patterns during cardiomyocyte differentiation
A new focus in the lab is to elucidate the regulatory networks that govern cardiomyocyte differentiation because congenital heart defects (CHD) and cardiac disease is a leading cause of morbidity and mortality. Work from many different labs has provided considerable insight into transcription factors that control cardiac morphogenesis. However, we still do not have a precise understanding of how these events are coordinated to yield the various transitional stages of mammalian cardiac development. We employ an in vitro ESC-based cardiac differentiation system to elucidate how chromatin and transcriptional states transition during cardiac morphogenesis. We have performed high-throughput genomic methods and have developed novel computational tools to generate integrative maps of the regulatory architecture that controls the transition from pluripotency to a functional cardiomyocyte. This is particularly novel because most large-scale studies use a variety of different cell lines and tissues to infer transition states. Our data reveal key chromatin changes and the distinct sets of co-regulated coding and non-coding genes as well as genomic regulatory elements that drive transitions in cell state along a defined developmental pathway (Wamstad et al., Submitted). Dissecting the changing chromatin and transcriptional landscapes during cardiomyocyte differentiation provides a robust framework on which to better design stem cell based therapies for cardiac related diseases.
Two projects in particular have emerged out of this initial work:
Large-scale discovery and functional analysis of distal enhancer elements. An exciting and emerging area of biology in the post-genomics era has been the genome-wide identification of noncoding regulatory elements that are required for the correct patterning of gene expression critical for tissue-specific development in what was once thought to be “junk DNA”. Distal enhancers have emerged as key cis-regulatory elements that can affect gene transcription independent of their orientation or distance. Global identification of these regions as well as their contribution to target gene expression has been challenging because enhancers can often reside thousands of base pairs away from their target of regulation.
Using a high-throughput genomic approach, we have discovered numerous distal enhancer elements in pluripotent ESCs as well as several adult cell types. We showed that specific histone modification patterns could distinguish enhancers as either active or poised, or inactive, and showed that genes connected to poised enhancers could predict the future developmental potential of the cell (Creyghton et al., 2010). Our cardiomyocyte differentiation system provides a unique opportunity to study enhancer state transitions during lineage commitment. To this end, we have defined a large set of both poised and active enhancers based on chromatin modification patterns during cardiomyocyte differentiation (Wamstad et al., Submitted). Remarkably, our work shows that enhancer utilization is highly cell type specific. We also find that enhancer state transitions are dynamic and non-random and seem to occur during narrow windows of developmental time. These findings provide new details about how tissue specific expression patterns are established early in development. Additional efforts aim to dissect the mechanistic principles by which enhancers exert their regulatory functions.
Long non-coding RNA as regulators of developmental gene expression patterns. Long intergenic noncoding RNAs (lncRNAs) have emerged as an exciting new cast of players with roles in modulating gene expression. LncRNA expression appears to be developmentally regulated making these non-coding RNAs good candidates for modulating developmental gene expression programs. However, a role for lncRNAs in development has not yet been clearly demonstrated. We have identified a large set of lncRNAs that are differentially expressed during cardiomyoctye differentiation and that may function to regulate gene expression during this process. We initially focused on one particular candidate, Braveheart, because it showed high expression in both ESCs and the heart, but low expression in most other tissues. We find that shRNA-mediated depletion of Braveheart leads to a failure to activate a gene network important for specification of cardiomyocytes from early pre-cardiac mesoderm (Klattenhoff et al., In preparation). We are actively pursuing many different avenues to elucidate its mechanisms of action.
Our ultimate goal is to dissect how lncRNAs function within the transcriptional regulatory landscape that defines cardiomyocte differentiation and heart development. We have begun to analyze additional lncRNA candidates by performing a high-throughput shRNA screen using a variety of functional readouts. We will also analyze the function of candidate lncRNAs in heart development in vivo. We expect that this work will reveal new regulatory elements important for modulating gene expression programs during lineage commitment, and will provide key insights that significantly improve our ability to use stem cells for cardiac regenerative therapies.
Wamstad JA, Alexander JM, Truti R, Shrikumar A, Li F, Ding H, Capra JA, Erwin G, Kattman SJ, Keller GM, Srivastava D, Levine SS, Holloway AK, Pollard KS, Boyer LA#, Bruneau BG#. (2012) Coordinated epigenetic regulation of developmental transitions in cardiac differentiation. #co-corresponding authors.
Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, Hanna J, Lodato MA, Frampton GM, Sharp PA, Boyer LA, Young RA, Jaenisch R. (2010) Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci U S A. 107, 21931-21936.
Surface L, Thornton SR, Boyer LA (2010) Polycomb group proteins set the stage for early lineage commitment. Cell Stem Cell 7, 288-298.
Lu R, Markowetz F, Unwin RD, Leek J, Airoldi EM, Boyer LA, Troyanskaya OG, Whetton AD, Lemischka IR (2009) Systems-level dynamic analyses of fate change in murine embryonic stem cells. Nature 462, 358-362.
Sha K, Boyer LA (2009) The chromatin signature of pluripotent cells. StemBook [Internet] Havard Stem Cell Institute 2008-2009 May 31.
Creyghton MP*, Markoulaki SM*, Levine SS, Hanna J, Lodato MA, Sha K, Young RA, Jaenisch R, Boyer LA (2008) H2AZ co-occupies target genes with Polycomb group proteins in embryonic stem cells and is required for proper execution of developmental programs. Cell 135, 649-661. *These authors contributed equally.
Boyer LA*, Plath K*, Zeitlinger J, Brambrink T, Medeiros L, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK, Bell GW, Otte AP, Vidal M, Gifford DK, Young RA, Jaenisch R (2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349-353. Epub 2006 April 19. *These authors contributed equally. Accompanied by Preview in Cell 125, 233-236; Science 312, 349.
Lee TI*, Jenner RG*, Boyer LA*, Guenther MG*, Levine SS*, Chevalier B, Murray HL, Johnstone SE, Kumar RM, Zucker JP, Yuan B, Bell GW, Herbolsheimer E, Hannett NM, Cole MF, Sun K, Guenther MG, Odom DT, Volkert TL, Bartel DP, Gifford DK, Melton DA, Jaenisch R, Young RA (2006) Control of developmental regulators in human embryonic stem cells. Cell 125, 310-313. *These authors contributed equally. Featured Article and Accompanied by Preview in Cell 125, 233-236; Science 312, 349.
Boyer LA*, Lee TI*, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA (2005) Core transcriptional regulatory network in human embryonic stem cells, Cell 122, 947-956. *These authors contributed equally. Featured Article and Accompanied by Preview in Cell 122, 828-830