The RNA Polymerase II factor RPAP1 is critical for Mediator-driven transcription and cell identity
ABSTRACT: The RNA Polymerase II Associated Protein 1 (RPAP1) is conserved across metazoa and critical for stem cell differentiation in plants, however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of lineage specifying factors and for viability. Accordingly, inhibition of RPAP1 triggers somatic cell de-differentiation and facilitates reprogramming into pluripotent stem cells. Conversely, interfering with RPAP1 in ES cells severely impairs their differentiation capacity. Mechanistically, we show that RPAP1 is essential for the interaction between Pol II and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific Pol II factor POLR2M/Gdown1 and the CTD phosphatase RPAP2. In agreement, depletion of RPAP1 disturbs the loading of Pol II and Pol II Ser5 phosphorylation levels and impairs expression of super-enhancer-driven genes. We conclude that Mediator-RPAP1-Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.
Project description:In this study, we generate genomic maps of Mediator, Pol II, TBP and TFIIH, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type (WT) strains and med17-ts mutants from Saccharomyces cerevisiae. Some of the data, concerning WT strains are also deposited at ArrayExpress under accession number E-MTAB-1595 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1595). There are 2 series of experiment: 1- WT (see E-MTAB-1595) and mutants med17-98, med17-444, and med17-670 (this submission) 2- WT and mutant med17-444 (this submission).
Project description:In this study, we generate genomic maps of Mediator, Pol II, TBP, TFIIH, TFIIA, TFIIB, TFIIE, TFIIF, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type strains from Saccharomyces cerevisiae and in a mutant for the Mediator essential subunit Med10
Project description:Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites and recruit the Mediator complex which contacts the pre-initiating RNA Polymerase II (Pol II) complex assembled at the start site of the gene. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. Here we use quantitative live cell super-resolution and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 (± 0.9) s, and 12.1 (± 1.4) s respectively, Mediator and Pol II also form large and stable clusters in stem cells (~15 stable clusters per cell). The large and stable Mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Mediator and Pol II colocalize in the large clusters. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both Mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable Mediator clusters. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We conclude that Mediator and Pol II associate in diffraction-sized condensates with a defined lifetime dependent on active transcription in living stem cells. Overall design: H3K27ac, RPB1, and Dendra2 ChIP-seq in WT and Dendra2-RPB1, Halo-MED19 tagged (DRHM) R1 mouse ES cells in the ES state and the EpiLC state.
Project description:In this study, we generate genomic maps of Mediator, Rad2, Pol II, TBP and TFIIH, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type strains from Saccharomyces cerevisiae. A related study involving ChIP-chip analysis of Rad2 occupany is also deposited at ArrayExpress under accession number E-MEXP-3875 ( http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-3875 ).
Project description:Pol II(G) is a novel form of RNA polymerase II that contains a tightly associated Gdown1 polypeptide. Whereas Pol II suffices for robust activator-dependent transcription in the absence of Mediator, Pol II(G) is highly dependent upon Mediator in a biochemically defined in vitro system. However, the mechanism(s) whereby Gdown1 alters the coactivator-dependence of Pol II is unknown. Here we show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II in vitro, resulting in inhibition of a critical function of TFIIF in facilitating PIC assembly. The apparent inability of Pol II(G) to associate with TFIIF suggests that Gdown1 must be removed from Pol II for transcription initiation. However, Mediator can actually help Pol II(G) bind to the promoter prior to subsequent Mediator functions. Complementary cell-based analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where Pol II(G) appears to modulate transcription. Our findings indicate that differential regulation of subsets of genes by Pol II and Pol II(G) occurs through the regulation of TFIIF and consequent alterations in Mediator requirements. Genome-wide localization of PolII and Gdown1 in human IMR90 fibroblast cells using ChIP-seq
Project description:Temporal mapping of C/EBPα and –β binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries. Timeseries ChIP-seq of transcription factors C/EBPα and –β, and Pol II in time points 0, 3, 8, 16, 24, 36, 48, 168 hours after partial hepatectomy. Additionally, an IgG mock to 0h was sequenced, as well as the transcription factor Egr1.
Project description:Mediator, a transcriptional co-activator complex, is recruited to enhancers by DNA-binding activators via its tail module, and it interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC) via its head module. Although Mediator has been described as a general transcription factor, it is unclear if it is essential for Pol II transcription and/or is a required component of the PIC in vivo. We comprehensively analyze this issue by measuring Pol II transcription upon rapid depletion of individual or multiple Mediator subunits from the nucleus. Depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in Pol II transcription. Transcriptional effects are stronger on SAGA-dependent genes as opposed to TFIID-dependent genes. Thus, Mediator modules that associate either with the enhancer or with the core promoter confer partial transcriptional activity. Furthermore, Pol II transcription can occur when Mediator is not detected at core promoters. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in Pol II transcription that is roughly comparable to the effect observed upon anchor-away-mediated depletion of Pol II or TBP. These results provide strong evidence that Mediator is essential for Pol II transcription in vivo, but that it is not a required component of the PIC. Overall design: Chip-seq performed with 8WG14 antibodies before and after the anchor-away of 7 Mediator subunits, as well as in a WT strain, in a TBP anchor-away strain, an Rpb-1 anchor-away strain, and in the "Triple" mutant, where the Mediator tail is deleted and Med17 is anchored away.
Project description:ChIP-chip was performed to identify the genomic binding locations for the termination factors Nrd1, and Rtt103, and for RNA polymerase (Pol) II phosphorylated at the tyrosine 1 and threonine 4 position of its C-terminal domain (CTD). In different phases of the transcription cycle, Pol II recruits different factors via its CTD, which consists of heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Here we show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, and that this impairs recruitment of termination factors. Tyr1 phosphorylation levels rise downstream of the transcription start site (TSS), and decrease before the polyadenylation (pA) site. Tyr1-phosphorylated gene bodies are depleted of CTD-binding termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation blocks CTD binding by these termination factors, but stimulates binding of elongation factor Spt6. These results show that CTD modifications can not only stimulate but also block factor recruitment, and lead to an extended CTD code for transcription cycle coordination.
Project description:TNF alpha is one of the inflammatory mediator and induce genes mainly by transcriptional factor, p65, in endothelial cells. This time, we performed a time course study to detect the change of localization of p65 and Pol II. To identify p65 and Pol II binding sites, we used chromatin immunoprecipitation with deep sequencing (ChIP-seq) of HUVECs treated with or without TNF alpha for 30 mins. Cells were starved before stimulation longer than 16 hours. HUVECs were used within the first 6 passages. For crosslinking, 10 mM of EGS in 50% glacial acetic acid was used for 45 min, followed by 20 min of 1% paraformaldehyde treatmet was used.
Project description:Co-transcriptional splicing of introns is a defining feature of eukaryotic gene expression. We show that the mammalian spliceosome specifically associates with the S5P CTD isoform of RNA polymerase II (Pol II) as it elongates across spliced exons of protein coding genes, both in human Hela and murine lymphoid cell lines. Immuno-precipitation of MNase digested chromatin with phospho CTD specific antibodies reveals that components of the active spliceosome (both snRNA and proteins) form a specific complex with S5P CTD Pol II. Furthermore a dominant splicing intermediate formed by cleavage at intron 5’ss results in the tethering of upstream exons to this complex at all spliced exons. These are invariably connected to upstream spliced constitutive and less frequently to alternative exons. Finally S5P CTD Pol II accumulates over spliced exons but not adjacent introns. We propose that mammalian splicing employs a rapid, co-transcriptional splicing mechanism based on CTD phosphorylation transitions.