PRO-cap in Drosophila melanogaster embryo at 3-4h and 6-8h after egg laying
Ontology highlight
ABSTRACT: Mapping of active transcription start sites using PRO-cap in whole-embryo at 3-4h and 6-8h AEL. Two biological replicates were performed at each time point.
Project description:Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is not fully understood. Here we identify a novel activity of the co-regulator and histone acetyltransferase p300/CBP in positioning promoter-proximal paused Pol II. We find that CBP inhibition impedes transcription through the +1 nucleosome, causing âdribblingâ of Pol II from the canonical pause site genome-wide. We further discovered that promoters strongly occupied by Drosophila CBP and GAGA-factor have high levels of paused Pol II, a unique chromatin signature and strong expression regardless of cell type. Interestingly, CBP activity is rate-limiting for Pol II recruitment to these highly-paused promoters but for transit into elongation at other genes. Thus, we uncover a key role for CBP during transcription in directly controlling different rate-limiting steps depending on promoter features. Examination of transcriptional regulation with and without CBP inhibition for 10 minutes in Drosophila S2 cells. Two biological replicates for each condition.
Project description:Heat shock response (HSR) is critical for survival of organisms undergoing proteotoxic stress. Heat shock factor 1 (HSF1) is widely believed to be the master regulator of this response. Here, we examined the kinetics of the transcriptional response and HSF1 binding changes genome-wide after heat shock (HS) with high sensitivity and high spatial and temporal resolution using PRO-seq and ChIP-seq assays respectively in wild type and in hsf1-deletion mouse embryonic fibroblasts. The transcriptional response is rapid, dynamic, and extensive with activation of several hundred genes and repression of several thousands of genes. Although HSF1 is critically required for classical inducible Heat Shock Protein (HSP) genes, it is not required for the majority of gene activation. HSF1 acts mechanistically to promote RNA polymerase II (Pol II) release from the promoter-proximal pause, while recruitment and initiation of Pol II are predominantly HSF1-independent. Surprisingly, a major class of cytoskeleton genes is immediately (within 2.5 minutes) and transiently activated in an HSF1-independent manner. A second wave of regulation is characterized by robust activation or repression of new sets of genes. The broad repression of thousands of genes, a quarter of which are repressed immediately upon HS and the rest are repressed in the second wave of regulation, is mediated at the level of decreasing Pol II pause release and not at prior steps of Pol II recruitment or initiation. Notably, heat shock does not induce transcription of some previously defined HSP genes, and HSF2 â a homolog of HSF1 â does not compensate for the lack of HSF1. Together, these findings indicate that mammalian cells cope with stress by rapidly inducing pervasive and dramatic changes in transcription that are largely modulated at the step of pause release, and only a fraction of this regulation is HSF1-dependent. Examination of transcriptional regulation before and after heat shock in WT, HSF1-/-, and HSF1&2-/- MEFs
Project description:Global nuclear run-on sequencing analysis was performed to determine transcription elongation rates across the genome of three human cell lines.
Project description:Transcriptional regulatory elements (TREs), including enhancers and promoters, determine the transcription levels of associated genes. We have recently shown that global run-on and sequencing (GRO-seq) with enrichment for 5'-capped RNAs reveals active TREs with high accuracy. Here, we demonstrate that active TREs can be identified by applying sensitive machine-learning methods to standard GRO-seq data. This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Our prediction method, called discriminative Regulatory Element detection from GRO-seq (dREG), summarizes GRO-seq read counts at multiple scales and uses support vector regression to identify active TREs. The predicted TREs are more strongly enriched for several marks of transcriptional activation, including eQTL, GWAS-associated SNPs, H3K27ac, and transcription factor binding than those identified by alternative functional assays. Using dREG, we survey TREs in eight human cell types and provide new insights into global patterns of TRE function. We analyzed GRO-seq or PRO-seq data from eight human cell lines. Please note that this study comprises new sample data plus reanalysis of old Sample data submitted by another user. Existing PRO-seq or GRO-seq data was combined as detailed in the GSE66031_readme.txt. See GSM1613181 and GSM1613182 Sample records for data processing information.
Project description:Global run-on sequencing analysis of human fetal lung fibroblasts uninfected and infected with Sendai virus for 6 hour was performed to define key transcriptional changes that were induced upon infection at 6 hour time point.
Project description:The majority of transcription studies examine steady-state RNA . However steady-state RNA is not a true reflection of the transcriptome, because the RNA levels are affected by both transcription rate and degradation rate. In this experiment we measured the amount of transcription occurring in HCT116 colon cancer cells, regardless of degradation, using GRO-seq (global nuclear run-on sequencing). This information demonstrates that many genes have a pile-up of transcriptionally-engaged polymerase near their 5'-end. Nuclei were prepared from HCT116 cells (treated for 1hr with DMSO as control for additional GRO-seq experiments to be reported separately). Transcription run-on was performed (as per Core, L.J., Waterfall, J.J., and Lis, J.T. (2008). Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845-1848) and nascent RNAs were purified and sequenced.
Project description:Precision Run-On Sequencing (PROseq) performed as previously described (Kwak et al., 2013; Judd et al., 2020) with slight modifications. Data is obtained from 1) DNA methyltransferase triple knockout mouse embryonic stem cells (DNMT TKO mESCs) and 2) Drosophila Schneider's S2 cell line. For the Drosophila S2 cell samples, 10 million Drosophila S2 cells were used per sample with a spike-in of 0.1 million (1%) TKO mESCs. For the TKO mESC samples, 5 million TKO mESCs were used per sample with a spike-in of 0.05 million (1%) Drosophila S2 cells. Treatment samples were treated with 10 microM triptolide for various time points (2.5, 5, 10, or 20 minutes). Control samples were treated with DMSO for 20 minutes. PROseq data was obtained by permeabilising the cells, performing a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP and subsequent enrichments steps for biotinylated RNA during the library preparation