Project description:Aim: To determine the state of methylation of DNA molecules that are physically engages with either poised or active enhancers. Methods: We used ChIP-bisulfite-sequencing with H3K4me1 antibody to map the methylation of DNA bound to enhancer. We used ChIP-sequencing with RNApolII-PS5 antibody to map actively transcribing transcription start sites. We used reduced representation bisulfite sequencing (RRBS) to map total DNA methylation. Results - conclusion: DNA molecules that are physically found in H3K4me1 chromatin are hypomethylated while DNA found in enhancers that are associated with active transcription is further demethylated.
Project description:The TATA binding protein (TBP) is a transcription factor that binds specifically to a DNA sequence called the TATA box upstream of the transcription start site. To gain insight into the genes occupied by TBP, chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) was performed to determine the genome-wide binding targets. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. ChIP was performed using an antibody against TBP.
Project description:The TATA binding protein (TBP) is a transcription factor that binds specifically to a DNA sequence called the TATA box upstream of the transcription start site. To gain insight into the genes occupied by TBP, chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) was performed to determine the genome-wide binding targets.
Project description:We performed a comprehensive ChIP-on-chip analysis for 28 transcription factors on a large set of known and novel TBP binding sites experimentally identified via ChIP-cloning. A large fraction of identified TBP binding sites is located in introns or lacks a gene/mRNA annotation and found to direct transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG- and non-CpG islands suggesting distinct transcription initiation pathways. Keywords: TBP, Promoter, ChIP-on-chip, transcription factor profiling
Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating genome-wide promoter methylation profiles in order to identify methylation changes likely to affect gene expression levels. MCF-10A cells were infected with MBD2 lentivirus in order to increase MBD2 expression. DNA was extracted from both infected and non-infected cells and enriched for methylated DNA through the method of methylated DNA immunoprecipitation (MeDIP). Enriched and total DNA was hybridized in different channels to 244K custom microarrays (Agilent Technologies) that contained probes covering at 100bp-spacing all transcription start sites from -800bp to 200bp (human, Ensembl v44) and all microRNAs in miRBase from 250bp before to 250bp after the microRNA. Three technical replicates were hybridized for infected and non-infected cells.
Project description:The Swi2/Snf2-family ATPase Mot1 displaces TBP from DNA in vitro, but the global relationship between Mot1 and TBP in vivo has been unclear. We therefore mapped the distribution of Mot1 and TBP on native chromatin at base-pair resolution. Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding. Using midpoint-versus-length mapping to assess the spatial relationship of Mot1 and TBP on chromatin, we find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action. Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association. Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA:dT) tracts that may contribute to nucleosome exclusion. We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites.
Project description:The Swi2/Snf2-family ATPase Mot1 displaces TBP from DNA in vitro, but the global relationship between Mot1 and TBP in vivo has been unclear. We therefore mapped the distribution of Mot1 and TBP on native chromatin at base-pair resolution. Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding. Using midpoint-versus-length mapping to assess the spatial relationship of Mot1 and TBP on chromatin, we find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action. Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association. Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA:dT) tracts that may contribute to nucleosome exclusion. We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites. We have analyzed the genomic distributions of yeast TBP and Mot1 using Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin and sequencing (ORGANIC-seq).
Project description:In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Herein, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBPT114A CRISPR/Cas9 edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprograming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.