A pliable mediator acts as a functional, rather than an architectural bridge, between promoters and enhancers
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA497125 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8066680.fastq.gz | Fastqsanger.gz | |||
| SRR8066690.fastq.gz | Fastqsanger.gz | |||
| SRR8066700.fastq.gz | Fastqsanger.gz | |||
| SRR8066710.fastq.gz | Fastqsanger.gz | |||
| SRR8066720.fastq.gz | Fastqsanger.gz |
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Project description:Our mechanistic understanding of Mediator derives largely from studies of the 25-subunit yeast complex. Here we combine CRISPR-Cas9 genetic screens, degron assays, in situ Hi-C, and cryo-EM to dissect the function and structure of the 33-subunit mammalian Mediator (mMED). Deletion analyses in B, T and ES cells reveal that depletion of the entire complex blocks PolII recruitment genome-wide, while loss of non-essential subunits, including the Tail module, primarily affects promoters linked to multiple enhancers. Contrary to current models, we find that mMED is not required to tether regulatory DNA, a topological activity controlled predominantly by architectural proteins. Structurally, we show that alterations in the Tail module, particularly at the core-Tail interphase, effect crucial mMED-PolII contacts, providing a rationale for how TFs stabilize the mMED-PolII holoenzyme and promote gene expression. Our studies therefore reveal key insights into how Mediator functionally bridge promoters and enhancers to regulate transcription initiation in higher eukaryotes.
2019-08-01 | GSE121355 | GEO
Project description:While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
| S-EPMC7533040 | biostudies-literature
Project description:Understanding the topological configurations of chromatin can reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3-D interactions that undergo marked reorganization at the sub-Mb scale during differentiation. Distinct combinations of CTCF, Mediator, and cohesin show widespread enrichment in architecture at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant sub-domains. Conversely, Mediator/cohesin together with pioneer factors bridge short-range enhancer-promoter interactions within and between larger sub-domains. Knockdown of Smc1 or Med12 in ES cells results in disruption of spatial architecture and down-regulation of genes found in cohesin-mediated interactions. We conclude that cell type-specific chromatin organization occurs at the sub-Mb scale and that architectural proteins shape the genome in hierarchical length scales. Analysis of higher-order chromatin chromatin architecture in mouse ES cells and ES-derived NPCs. Analysis of CTCF and Smc1 occupied sites in ES-derived NPCs.
2013-05-24 | E-GEOD-36203 | biostudies-arrayexpress
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.
2016-10-31 | GSE82082 | GEO
Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter. We took a systematic approach to examine the genome-wide distribution (using ChIP-chip) of the various Mediator subunits. Mediator occupancy was also assayed in mutants for most of the CTD kinases and in strains where some CTD serines had been replaced by alanines. Mediator ChIPs were performed with Myc-tagged subunits, except in some preliminary experiments where polyclonal antibodies were used. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In some of the preliminary experiments, non immunoprecipitated DNA (input) was used as the control. In addition to Mediator ChIPs, the project includes TFIIB and RNAPII (Rpb3) ChIP-chip datasets. All ChIP-chip experiments were done in duplicates except for the preliminary experiments that were done in monoplicat for the most part. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
2014-03-24 | E-GEOD-55402 | biostudies-arrayexpress
Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter.
2014-03-24 | GSE55402 | GEO
Project description:Understanding the topological configurations of chromatin can reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3-D interactions that undergo marked reorganization at the sub-Mb scale during differentiation. Distinct combinations of CTCF, Mediator, and cohesin show widespread enrichment in architecture at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant sub-domains. Conversely, Mediator/cohesin together with pioneer factors bridge short-range enhancer-promoter interactions within and between larger sub-domains. Knockdown of Smc1 or Med12 in ES cells results in disruption of spatial architecture and down-regulation of genes found in cohesin-mediated interactions. We conclude that cell type-specific chromatin organization occurs at the sub-Mb scale and that architectural proteins shape the genome in hierarchical length scales.
2013-05-24 | GSE36203 | GEO
Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (ChIP-seq)
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Project description:The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We suggest that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.
2021-07-30 | GSE171981 | GEO
Project description:Regulatory elements called CpG islands (CGIs) are associated with the majority of mammalian gene promoters and have been proposed to play an important role in gene expression. The regulatory capacity of CGIs is thought to rely upon a family of proteins that recognise CGIs through their ZF-CxxC DNA binding domains to recruit chromatin-modifying activities to gene promoters. Through studying FBXL19, a poorly characterized ZF-CxxC domain-containing protein, we have discovered that it specifically interacts with the CDK8-Mediator complex in mouse embryonic stem cells (ESCs). Intriguingly, FBXl19 recruits CDK8-Mediator to a subset of CGI-associated promoters of repressed developmental genes in ESCs. We show that FBXL19-dependent recruitment of CDK8 in ESCs is required for the proper induction of the associated genes during ESC differentiation. This is consistent with early embryonic lethality of in FBXl19 deficient mice. Together, our observations highlight a novel role for FBXL19 in priming developmental gene expression, via recruitment of CDK8 to their CGI promoters, in order to support normal gene induction during differentiation and development.
2018-06-08 | GSE98755 | GEO