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EsBAF is an essential component of the core pluripotency transcriptional network

ABSTRACT: Distinctive SWI/SNF-like ATP-dependent chromatin remodeling esBAF complexes are indispensable for the maintenance and pluripotency of mouse embryonic stem (ES) cells. To understand the mechanism underlying the roles of these complexes in ES cells, we performed high-resolution genome-wide mapping of the core ATPase subunit, Brg, using ChIP-Seq technology. We find that that esBAF, as represented by Brg, binds to genes encoding components of the core ES transcriptional circuitry, including Polycomb group proteins. esBAF colocalizes extensively with Oct4, Sox2 and Nanog genome-wide, and shows distinct functional interactions with Oct4 and Sox2 at its target genes. Surprisingly, no significant colocalization of esBAF with PRC2 complexes, represented by Suz12, is observed. Lastly, esBAF co-binds with Stat3 and Smad1 genome-wide, consistent with a direct and critical role in LIF and BMP signaling essential to maintain pluripotency. Taken together, our studies indicate that esBAF is both an essential component of the core pluripotency transcriptional network, and might also be a critical component of the LIF and BMP signaling pathways essential for maintenance of self-renewal and pluripotency.

ORGANISM(S): Mus musculus

PROVIDER: GSE14344 | GEO | 2009/01/20

SECONDARY ACCESSION(S): PRJNA111529

REPOSITORIES: GEO

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Project description:Signaling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs) by opposing MAP kinase signaling. This contrasts with most cell types where STAT3 signaling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent upon Brg, the ATPase subunit of a specialized chromatin remodeling complex (esBAF) found in ESCs. Brg is required to establish chromatin accessibility at STAT3 binding targets, in essence preparing these sites to respond to LIF signaling. Moreover, Brg deletion leads to rapid Polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg-activated targets genome-wide, including the target genes of the LIF signaling pathway. Hence, one crucial role of Brg in ESCs involves its ability to potentiate LIF signaling by opposing PcG. Contrary to expectations, Brg also facilitates PcG function at classical PcG target including all four Hox loci, reinforcing their repression in ESCs. These findings reveal that esBAF does not simply antagonize PcG, but rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency. Brg conditional embryonic stem cell (ESC) line was prepared from Brg(lox/lox);Actin-CreER mice that allow conditional deletion of Brg upon addition of 4-hydroxytamoxifen (4OHT, Sigma). Brg conditional ESCs were treated with 4-OHT for 72 hours to delete Brg. Brg protein levels completely abolished only at 48 hours (Brg-KO). RNA from Brg conditional ESCs (WT), Brg-KO ESCs, Brg conditional ESCs starved of LIF (Lif-WD) for 48 hours were extracted using Trizol reagent (Invitrogen), followed by RNeasy kit with DNaseI digestion to eliminate genomic DNA. Purified RNA was processed and hybridized onto Affymetrix Mouse MOE 4.0 3’ expression arrays according to the manufacturer's instructions. ChIP-Seq was used to detect and measure STAT3 occupancy and levels of H3K27me3 in the genome before and after the removal of the SWI/SNF ATPase Brg in mouse ESCs. Total of 4 samples (WT and KO STAT3; WT and KO H3K27me3). Input DNA was sequenced as control.

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EsBAF is an essential component of the core pluripotency transcriptional network

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