Anti-Pol II pS5 co-immunoprecipitation in PHF3 WT, KO and ΔSPOC HEK293T cells
Ontology highlight
ABSTRACT: The analysis of differential interactome of Pol II pS5 in PHF3 WT, KO and ΔSPOC HEK293T cells using Pol II pS5 (4H8) antibody coupled to Protein G beads.
Project description:The analysis of differential interactome of Pol II pS5 in DIDO WT, KO and ΔSPOC HEK293T cells using Pol II pS5 (3E8) antibody coupled to Protein G beads (no crosslinking).
Project description:The wheat Pol II enzyme was purified, and a transcription initiation complex was assembled on the potato spindle tuber viroid (PSTVd) RNA template. The transcription initiation complex was characterized using LC-MSMS.
Project description:Transcription-coupled DNA repair (TCR) removes transcription-blocking DNA lesions through the coordinated assembly of repair factors on arrested RNA polymerase II (Pol II). This process requires the site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase. Pol II ubiquitylation is facilitated by ELOF1, a recently identified TCR factor. Using cryogenic electron microscopy, biochemical assays, and cell biology, we reveal that ELOF1 functions as an adaptor to correctly dock CRL4CSA onto Pol II and facilitate the recruitment of UVSSA. The ELOF1-CSA-UVSSA interaction positions CRL4CSA on arrested Pol II, leading to its ubiquitylation upon ligase activation by neddylation. ELOF1-mediated repositioning enables a TFIIS-like element in UVSSA to reach the Pol II active site and the pore region to prevent Pol II re-activation. These findings provide the structural and molecular basis underlying the transition of Pol II from a transcriptionally active to an arrested state, primed for DNA repair.
Project description:Bmi-1, Ring1B, H3K27me3, Ser2 Pol II, Ser 5 Pol II binding pattern in WT and Psip1 KO MEFs Menin occupancy is studied over Hox genes and several non-hox genes Bmi-1, Ring1B, H3K27me3, Ser2 Pol II, Ser 5 Pol II ChIPs from WT and Psip1 KO MEFs
Project description:Under current models for signal-dependent transcription in eukaryotes, DNA-binding activator proteins regulate the recruitment of RNA polymerase II (Pol II) to a set of target promoters. Yet, recent studies, as well as our results herein, show that Pol II is widely distributed (i.e., "preloaded") at the promoters of many genes prior to specific signaling events. How Pol II recruitment and Pol II preloading fit within a unified model of gene regulation is unclear. In addition, the mechanisms through which cellular signals activate preloaded Pol II across mammalian genomes remain largely unknown. Here we show that the predominant genomic outcome of estrogen signaling is the post-recruitment regulation of Pol II activity through phosphorylation, rather than recruitment of Pol II. Furthermore, we show that negative elongation factor (NELF) binds to estrogen target promoters in conjunction with preloaded Pol II and represses gene expression until the appropriate signal is received. Finally, our studies reveal that the estrogen-dependent activation of preloaded Pol II facilitates rapid transcriptional and post-transcriptional responses which play important physiological roles in regulating estrogen signaling itself. Our results reveal a broad use of post-recruitment Pol II regulation by the estrogen signaling pathway, a mode of regulation that is likely to apply to a wide variety of signal-regulated pathways. ChIP-chip analysis for RNA Pol II, Ser5 phosphorylated RNA Pol II and NELF-A in MCF7 breast cancer cells.
Project description:Control of RNA transcription is critical for the development and homeostasis of all organisms, and can occur at multiple steps of the transcription cycle, including RNA polymerase II (Pol II) recruitment, initiation, promoter-proximal pausing, and elongation. That Pol II accumulates on many promoters in metazoans implies that steps other than Pol II recruitment are rate-limiting and regulated 1-6. By integrating genome-wide Pol II chromatin immunoprecipition (ChIP) and Global Run-On (GRO) genomic data sets from Drosophila cells, we examined critical features of Pol II near promoters. The accumulation of promoter-proximal polymerase is widespread, occurring on 70% of active genes; and unlike elongating Pol II within the body of genes, promoter Pol II are held paused by factors like NELF, unable to transcribe unless nuclei are treated with strong detergent. Notably, we find that the vast majority of promoter-proximal Pol II detected by ChIP are paused, thereby identifying the biochemical nature of this rate-limiting step in transcription. Finally, we demonstrate that Drosophila promoters do not have the upstream divergent Pol II that is seen so broadly and prominently on mammalian promoters. We postulate this is a consequence of Drosophila’s extensive use of directional core promoter sequence elements, which contrasts with mammals’ lack of directional elements and prevalence of CpG island core promoters. In support of this idea, we show that the fraction of mammalian promoters containing a TATA box core element is dramatically depleted of upstream divergent transcription. ChIP-seq data set for Pol II (rpb3) (2 replicates).
Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-seq data for Pol II and additional factors controlling pause release in mouse ES cells.
Project description:Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ~3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1,000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes. We isolated replicates of nuclei of untreated mESCs and cells treated for 2, 5, 12.5, 25 and 50 min with 300nM flavopiridol, as well as nuclei treated for 12.5, 25, and 50 min with 500nM triptolide and performed GRO-seq with these.
Project description:The analysis of interactome of different DIDO isoforms and mutants upon transient overexpression of FLAG-tagged constructs in HEK293T cells.
Project description:We examined genome-wide variation in transcription factor binding in different individuals and a chimpanzee using chromatin immunoprecipitation followed by massively-parallel sequencing (ChIP-Seq). The binding sites of RNA Polymerase II (Pol II) as well as a key regulator of immune responses, NFkB, were mapped in ten HapMap lymphoblastoid cell lines derived from individuals of African, European, and Asian ancestry, including a parent-offspring trio. We also mapped gene expression in all ten human cell lines for two treatment conditions: a) no treatment and b) following induction by TNF-alpha. Genome-wide comparison of Pol II and NF-KappaB binding in eleven individuals. ChIP-seq study with Pol II.