Project description:We mapped the localization of insulator proteins in Drosophila S2 cells in the presence or absence of 12mM of 3AB. We found that inhibition of PARylation affects DNA binding of insulator proteins only at a small subsets of genomic sites. Examination of genomic occupancy for insulator proteins in S2 cells with or without 3AB inhibition.
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:Time course ChIP-seqwas performed to determine global changes in histone H3 lysine 27 acetylation (H3K27Ac) at enhancers upon Sendai Virus infection.
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:Proteins co-purifying with recombinantly purified and N-terminally GFP-3C-tagged CTCF N-terminus (amino acids 1-293) produced in bacteria were identified by incubating the tagged bait with soluble nuclear protein extracts from 0-12h hour-old wildtype (OregonR strain) Drosophila melanogaster embryos.
Project description:Chromatin insulators are DNA-protein complexes situated throughout the genome that contribute to higher order organization and demarcation into distinct transcriptional domains. Mounting evidence in different species implicates RNA and RNA-binding proteins as regulators of chromatin insulator activities. Here we identify the Drosophila hnRNP M homolog Rumpelstiltskin (Rump) as an antagonist of gypsy chromatin insulator enhancer-blocking and barrier activities. Despite ubiquitous expression of Rump, improvement of barrier activity is detected only in tissue outside of the central nervous system (CNS) when Rump levels are reduced. Furthermore, rump mutants restore insulator complex localization in an otherwise compromised genetic background only in non-CNS tissues. Rump associates physically with core gypsy insulator proteins, and ChIP-Seq analysis of Rump demonstrates extensive colocalization with a subset of gypsy insulator sites across the genome. The genome-wide binding profile and tissue-specificity of Rump contrast with that of Shep, a recently identified RNA-binding protein that antagonizes gypsy insulator activity exclusively in the CNS. Our findings indicate parallel roles for RNA-binding proteins in mediating tissue-specific regulation of chromatin insulator activity. ChIP-seq of Rump, Mod(mdg4)2.2, Shep, Su(Hw), and CP190 in Drosophila Kc167 cells
Project description:Mycobacterial HelD is a transcription factor that binds RNA polymerase (RNAP) and thereby rescues it from stalled transcription complexes. HelD also protects RNAP against the antibiotic rifampicin. HelD, however, must be released from the rescued RNAP to participate in the next round of the transcription cycle. The exact mechanism of this release is unknown. Here we show that HelD from Mycobacterium smegmatis can form a complex with RNAP, together with σA primary sigma factor, and RbpA, but not CarD, transcription factors. Using cryo-EM, we solved a series of RNAP-σA-RbpA-HelD structures with or without promoter DNA. These snapshots capture the involvement of HelD in transcription initiation, describes mechanistic aspects of HelD release, and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding/hydrolysis to/by HelD in the process, and functionally confirms the rifampicin-protective effect of HelD. Taken together, HelD assists an alternative pathway of transcription initiation.
Project description:ChIP-on-chip analysis of RNAP and RpoD binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between RNAP and RpoD binding and provided us with important insights into the global distribution of these factors. Furthermore this data was correlated with information on the location of 1873 transcription start sites identified by RNA-Seq technology, thereby providing a detailed transcriptional map of Salmonella Typhimurium. Analysis of RNAP, RNAP-Rifampicin and and RpoD binding in Luria Broth (LB)