Project description:Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter, but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.
Project description:The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator – histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis, that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization.
Project description:The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator – histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis, that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. Med8-TAP strain ChIPed with IgG beads vs. Input in Saccharomyces cerevisiae
Project description:Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT and loss of these factors abolishes enhancer-promoter contact. This work not only provides a new model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia, but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.
Project description:The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses ChIP Seq to examine global localization of Scc2 in the presence or absence of MED14.
Project description:The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses RNA-Seq analysis to study the effect of these mutations on gene expression.
Project description:The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses RNA-Seq analysis to study the effect of these mutations on gene expression.
Project description:The Mediator co-activator complex directs gene specific expression by binding distal enhancer-bound transcription factors through its Med1 subunit while bridging to RNA Polymerase-II (Pol-II) at gene promoters. In addition, Mediator scaffolds epigenetic modifying enzymes that determine local DNA accessibility. We previously found that deletion of Med1 in cardiomyocytes deregulates more than 5000 genes and promotes acute heart failure and hypothesize Med1 deficiency disrupts enhancer-promoter coupling. Using ChIP-seq, we find Pol-II pausing index is increased in Med1 knockout versus floxed control hearts primarily from decreased Pol-II occupancy at the majority of transcriptional start sites. Med1-dependent gene expression correlates strongly with histone H3 K27 acetylation while H3 K27 tri-methylated levels are increased and inversely correlate with absolute expression levels. Furthermore, Med1 deletion leads to dynamic changes in acetyl-K27 associated super-enhancer regions and their enriched transcription factor binding motifs that are consistent with altered gene expression. Our findings suggest that Med1 is important in establishing enhancer-promoter coupling in the heart by facilitating the recruitment of Pol-II to gene promoters, determining chromatin accessibility within genes and enhancer regions and altering transcription factor binding motifs that are likely important in directing gene-specific expression.