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Kin28 regulates the transient association of Mediator with core promoters.

ABSTRACT: Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-terminal domain is mutated to replace Ser5 with alanine, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is released quickly from promoters after phosphorylation of Ser5 by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

SUBMITTER: Jeronimo C

PROVIDER: S-EPMC3997488 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Kin28 regulates the transient association of Mediator with core promoters.

Jeronimo Célia C Robert François F

Nature structural & molecular biology 20140406 5

Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-ter ...[more]

PMID: 24704787

Similar Datasets

Kin28 Regulates the Transient Association of Mediator with Core Promoters

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

Kin28 Regulates the Transient Association of Mediator with Core Promoters

2014-03-24 | GSE55402 | GEO

Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae.

Project description:Transcription of eukaryotic mRNA-encoding genes by RNA polymerase II (Pol II) begins with assembly of the pre-initiation complex (PIC), comprising Pol II and the general transcription factors. Although the pathway of PIC assembly is well established, the mechanism of assembly and the dynamics of PIC components are not fully understood. For example, only recently has it been shown that in yeast, the Mediator complex normally occupies promoters only transiently, but shows increased association when Pol II promoter escape is inhibited. Here we show that two subunits of TFIID, Taf1 and Taf4, similarly show increased occupancy as measured by ChIP upon depletion or inactivation of Kin28. In contrast, TBP occupancy is unaffected by depletion of Kin28, thus revealing an uncoupling of Taf and TBP occupancy during the transcription cycle. Increased Taf1 occupancy upon Kin28 depletion is suppressed by depletion of TBP, while depletion of TBP in the presence of Kin28 has little effect on Taf1 occupancy. The increase in Taf occupancy upon depletion of Kin28 is more pronounced at TFIID-dominated promoters compared to SAGA-dominated promoters. Our results support the suggestion, based on recent structural studies, that TFIID may not remain bound to gene promoters through the transcription initiation cycle.

| S-EPMC7192586 | biostudies-literature

Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters.

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 propose 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.

| S-EPMC8384189 | biostudies-literature

Transient intracellular acidification regulates the core transcriptional heat shock response.

Project description:Heat shock induces a conserved transcriptional program regulated by heat shock factor 1 (Hsf1) in eukaryotic cells. Activation of this heat shock response is triggered by heat-induced misfolding of newly synthesized polypeptides, and so has been thought to depend on ongoing protein synthesis. Here, using the budding yeast Saccharomyces cerevisiae, we report the discovery that Hsf1 can be robustly activated when protein synthesis is inhibited, so long as cells undergo cytosolic acidification. Heat shock has long been known to cause transient intracellular acidification which, for reasons which have remained unclear, is associated with increased stress resistance in eukaryotes. We demonstrate that acidification is required for heat shock response induction in translationally inhibited cells, and specifically affects Hsf1 activation. Physiological heat-triggered acidification also increases population fitness and promotes cell cycle reentry following heat shock. Our results uncover a previously unknown adaptive dimension of the well-studied eukaryotic heat shock response.

| S-EPMC7449696 | biostudies-literature

A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction.

Project description:The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.

| S-EPMC3648612 | biostudies-literature

Phosphorylation of the Pol II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters.

Project description:Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is cotranscriptionally recruited to the 5' end of ARG1 in a manner stimulated by interaction of the BUR1 C terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5 and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.

| S-EPMC2683426 | biostudies-literature

Engineered Promoters for Potent Transient Overexpression.

Project description:The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods.

| S-EPMC4752495 | biostudies-literature

The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

Project description:The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

| S-EPMC5834202 | biostudies-literature

Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex.

Project description:The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli are poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

| S-EPMC8203292 | biostudies-literature

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