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SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state.

ABSTRACT: Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood. A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) methyltransferases, particularly SetDB1, had the most profound effects on ES cells. Chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing (ChIP-Seq) and functional analysis revealed that SetDB1 and histone H3K9-methylated nucleosomes occupy and repress genes encoding developmental regulators. These SetDB1-occupied genes are a subset of the "bivalent" genes, which contain nucleosomes with H3K4me3 (H3K4 trimethylation) and H3K27me3 modifications catalyzed by Trithorax and Polycomb group proteins, respectively. These genes are subjected to repression by both Polycomb group proteins and SetDB1, and loss of either regulator can destabilize ES cell state.

SUBMITTER: Bilodeau S

PROVIDER: S-EPMC2779743 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state.

Bilodeau Steve S Kagey Michael H MH Frampton Garrett M GM Rahl Peter B PB Young Richard A RA

Genes & development 20091101 21

Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood. A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) methyltransferases, particularly SetDB1, had the most profound effects on ES cells. Chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing ...[more]

PMID: 19884255

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Project description:X chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Despite identification of the crucial long non-coding RNA Xist and involvement of specific chromatin modifiers in the establishment and maintenance of the heterochromatin of the inactive X chromosome (Xi), interference with known pathways only partially reactivates the Xi once silencing has been established. Here, we studied ATF7IP (MCAF1), a protein previously characterized to coordinate DNA methylation and histone H3K9 methylation through interactions with the methyl-DNA binding protein MBD1 and the histone H3K9 methyltransferase SETDB1, as a candidate maintenance factor of the Xi.We found that siRNA-mediated knockdown of Atf7ip in mouse embryonic fibroblasts (MEFs) induces the activation of silenced reporter genes on the Xi in a low number of cells. Additional inhibition of two pathways known to contribute to Xi maintenance, DNA methylation and Xist RNA coating of the X chromosome, strongly increased the number of cells expressing Xi-linked genes upon Atf7ip knockdown. Despite its functional importance in Xi maintenance, ATF7IP does not accumulate on the Xi in MEFs or differentiating mouse embryonic stem cells. However, we found that depletion of two known repressive biochemical interactors of ATF7IP, MBD1 and SETDB1, but not of other unrelated H3K9 methyltransferases, also induces the activation of an Xi-linked reporter in MEFs.Together, these data indicate that Atf7ip acts in a synergistic fashion with DNA methylation and Xist RNA to maintain the silent state of the Xi in somatic cells, and that Mbd1 and Setdb1, similar to Atf7ip, play a functional role in Xi silencing. We therefore propose that ATF7IP links DNA methylation on the Xi to SETDB1-mediated H3K9 trimethylation via its interaction with MBD1, and that this function is a crucial feature of the stable silencing of the Xi in female mammalian cells.

Project description:Seed germination and subsequent seedling growth define crucial steps for entry into the plant life cycle. For those events to take place properly, seed developmental genes need to be silenced whereas vegetative growth genes are activated. Chromatin structure is generally known to play crucial roles in gene transcription control. However, the transition between active and repressive chromatin states during seed germination is still poorly characterized and the underlying molecular mechanisms remain largely unknown. Here we identified the Arabidopsis PHD-domain H3K4me3-binding ALFIN1-like proteins (ALs) as novel interactors of the Polycomb Repressive Complex 1 (PRC1) core components AtBMI1b and AtRING1a. The interactions were confirmed by diverse in vitro and in vivo assays and were shown to require the AL6 N-terminus containing PAL domain conserved in the AL family proteins and the AtRING1a C-terminus containing RAWUL domain conserved in animal and plant PRC1 ring-finger proteins (including AtRNIG1a/b and AtBMI1a/b). By T-DNA insertion mutant analysis, we found that simultaneous loss of AL6 and AL7 as well as loss of AtBMI1a and AtBMI1b retards seed germination and causes transcriptional derepression and a delayed chromatin state switch from H3K4me3 to H3K27me3 enrichment of several seed developmental genes (e.g. ABI3, DOG1, CRU3, CHO1). We found that AL6 and the PRC1 H3K27me3-reader component LHP1 directly bind at ABI3 and DOG1 loci. In light of these data, we propose that AL PHD-PRC1 complexes, built around H3K4me3, lead to a switch from the H3K4me3-associated active to the H3K27me3-associated repressive transcription state of seed developmental genes during seed germination. Our finding of physical interactions between PHD-domain proteins and PRC1 is striking and has important implications for understanding the connection between the two functionally opposite chromatin marks: H3K4me3 in activation and H3K27me3 in repression of gene transcription.

Dataset Information

SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state.

Publications

SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state.

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