Project description:NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB (NFYB) and NF-YC (NFYC)) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5,000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (non-modified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively co-localizes with FOS in all genomic contexts, and at promoters and enhancers this often occurs in the absence of JUN and the AP-1 motif. NF-Y also co-associates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve co-association with FOS. Scrambled control (shSCM) and NF-YA pLKO.1-shRNAs were designed by Sigma-Aldrich. The puromycin resistance cassette was replaced with an EGFP cassette. Viral production and transduction were carried out as previously described (Benatti et al. 2011). HeLaS3 cells were transduced with shSCM or shNF-YA viral supernatants, in triplicate, and cells collected after 48 hr of incubation. Total RNA was prepared by Trizol extraction and Qiagen RNeasy kit purification, converted to biotinylated aRNA and hybridized to U133 Plus 2.0 GeneChip expression arrays using the 3’ IVT Express Kit (Affymetrix, USA) following the manufacturer’s protocol. Arrays were RMA normalized (Irizarry et al. 2003), gene expression levels calculated, differential expression determined using the following R packages from the Bioconductor project: affy (Gautier et al. 2004), limma (Smyth 2004). HelaS3 cells were transduced with shSCM or shNF-YA in triplicate for 48hrs.

Project description:NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB (NFYB) and NF-YC (NFYC)) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5,000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (non-modified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively co-localizes with FOS in all genomic contexts, and at promoters and enhancers this often occurs in the absence of JUN and the AP-1 motif. NF-Y also co-associates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve co-association with FOS. Scrambled control (shSCM) and NF-YA pLKO.1-shRNAs were designed by Sigma-Aldrich. The puromycin resistance cassette was replaced with an EGFP cassette. Viral production and transduction were carried out as previously described (Benatti et al. 2011). HeLaS3 cells were transduced with shSCM or shNF-YA viral supernatants, in triplicate, and cells collected after 48 hr of incubation. Total RNA was prepared by Trizol extraction and Qiagen RNeasy kit purification, converted to biotinylated aRNA and hybridized to U133 Plus 2.0 GeneChip expression arrays using the 3’ IVT Express Kit (Affymetrix, USA) following the manufacturer’s protocol. Arrays were RMA normalized (Irizarry et al. 2003), gene expression levels calculated, differential expression determined using the following R packages from the Bioconductor project: affy (Gautier et al. 2004), limma (Smyth 2004).

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.

Dataset Information

Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors [ChIP-seq]

Publications

Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors.

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