Project description:The aim of the experiment is to determine if RNA pol II occupancy and abundance is altered human fibroblasts from a patient that has a mutation in the gene Taf8, which plays an important role in RNA pol II mediated transcription.
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:In order to idetify paused promoters in vivo, we performed tissue specific Pol II Chip-seq using mutant embryos for the dorsal gradient. We used two population of cells, either dorsal ectoderm cells (gd7 embryos) or mesodermal cells (Toll10b) embryos. ChIP-seq for Pol II in various Drosophila embryos
Project description:RNA Polymerase II transcribes protein-coding and many non-coding RNA genes in eukaryotes. The largest subunit of RNA Polymerase II, Rpb1, contains a hepta-peptide repeat on its C-terminal tail with three potential phosphorylation sites (Serine 2, Serine 5 and Serine 7). Mammalian Rpb1 contains 52 repeats. The phosphorylation events are catalyzed by specific protein kinases where the phosphorylation of specific residues is coupled to the transcription cycle. For example, the Cdk7 subunit of TFIIH phosphorylates both Serine 5 and Serine 7 during intiation and the Cdk9 subunit of P-TEFb phosphorylates Serine 2 during the transition into productive elongation. The dataset presented here is the genome-wide distribution of RNA Pol II with Serine 7 of the CTD phosphorylated in murine embryonic stem cells. This data, in addition to phospho-specific datasets generated in the same cell type in Rahl et al. Cell 2010 and Seila et al. Science 2008, represents the genome-wide distribution of multiple RNA Pol II isoforms in murine embryonic stem cells: total Pol II, hypophosphorylated CTD Pol II, Serine 2 phosphorylated CTD Pol II, Serine 5 phosphorylated CTD Pol II and Serine 7 phosphorylated CTD Pol II. An antibody specific to RNA Pol II Serine 7 phosphorylated CTD (gift of Dirk Eick; Chapman et al. Science 2008) was used to enrich for DNA fragments associated with this Pol II isoform in murine embryonic stem cells. DNA was purified and prepared for Illumina/Solexa sequencing following their standard protocol. This is a single dataset but together with datasets from Rahl et al. Cell 2010 and Seila et al. Science 2008, these datasets represent the genome-wide distribution of multiple RNA Pol II isoforms in murine embryonic stem cells: total Pol II, hypophosphorylated CTD Pol II, Serine 2 phosphorylated CTD Pol II, Serine 5 phosphorylated CTD Pol II and Serine 7 phosphorylated CTD Pol II.
Project description:RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits.
Project description:small RNA gene expression profiles of senescent human fibroblasts were compared to young proliferating fibroblasts. RNA-seq data comprises 5 groups: 16, 26, 46, 64 and 74 population doublings of HFF fibroblasts. Jena Centre for Systems Biology of Ageing - JenAge (www.jenage.de)
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.