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EXPRES Report Summary

Project ID: 281781
Funded under: FP7-IDEAS-ERC
Country: Israel

Final Report Summary - EXPRES (Chromatin and transcription in ES cells: from single cells to genome wide views)

How embryonic stem cells (ESCs) maintain their dual capacity to self-renew and to differentiate into all cells of the organism is still one of the fundamental questions in modern biology. Derived from the inner cell mass (ICM) of the developing blastocyst, ESCs are pluripotent and self-renewing, while maintaining a normal karyotype. As such, they hold great promise for basic research, developmental studies and cell-based therapy. ESCs must be plastic enough to accommodate substantial changes in cell fate and lineage commitment. Work from several labs, including our own, over the past several years, highlighted a key role for chromatin plasticity and epigenetic regulation, providing ESCs their necessary flexibility. In this project (ExprES), I aimed to reveal chromatin plasticity and global transcription regulation, including non-polyadenylated transcription, in undifferentiated ESCs and during differentiation. I aimed to delineate the mechanisms that support chromatin plasticity in ESCs and to reveal the transcriptional output of ESCs (focusing here on non-polyadenylated transcription), and decipher the chromatin context, regulation and function of these transcripts. Specifically, I aimed to:

a. Identify and characterize chromatin proteins that are important for the stem cell state;
b. Dissect the mechanisms underlying chromatin plasticity in ESCs;
c. Provide genome-wide views of transcription in ESC differentiation, and decipher their chromatin context, regulation and function;

To accomplish the first aim and to identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation. D-CAP allowed us to identify several additional chromatin proteins with roles in ESCs including HP1, SF3B1 and SET.

In order to identify what supports chromatin plasticity in ESCs, we investigated several potential mechanisms that regulate chromatin in ESCs. Using epigenetic drugs and mutant ESCs lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome's epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells. We further developed tools to study, for the first time, chromatin protein dynamics in vivo in developing mouse embryos and found an exaggerated dynamic behavior at the 2-cell stage. Together, these data suggest that chromatin protein dynamics is a surrogate for pluripotency.

Finally, we focused on the transcriptional output in ESCs. The transcriptional landscape in ESCs and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. We also show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs. We further used the bioinformatic tools we developed to generate a webtool which is freely available to all researchers enabling unbiased epigenomic analysis in mouse and human ESCs.

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