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ChIPseq measurement of transcriptional engagement and replication stress at the Drosophila Mid-blastula Transition

ABSTRACT: These experiments measure genome-wide RNA Pol II binding in precisely staged wild-type embryos at five time points spanning the maternal to zygotic transition (nuclear cycles 12 through 14) of Drosophila melanogaster. In addition, RNA Pol II binding at nuclear cycle 13 is measured in embryos mutant for either mei-41/ATR or zelda. To correlate RNA Pol II binding with replication stress, genome-wide profiles of Replication protein A (70kDa subunit, RpA-70 EGFP) were generated in parallel with RNA Pol II for both wild-type and zelda at nuclear cycle 13. Two replicates each for 5 time points for wild type RNA Pol II. Two replicates for mei-41 RNA Pol II, nuclear cycle 13. Two replicates for zelda RNA Pol II, nuclear cycle 13. Two replicates each for Rpa70-EGFP in wild-type or zelda, nuclear cycle 13, matched to the corresponding RNA Pol II sample.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Shelby Blythe

PROVIDER: E-GEOD-62925 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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ChIPseq measurement of transcriptional engagement and replication stress at the Drosophila Mid-blastula Transition

Project description:These experiments measure genome-wide RNA Pol II binding in precisely staged wild-type embryos at five time points spanning the maternal to zygotic transition (nuclear cycles 12 through 14) of Drosophila melanogaster. In addition, RNA Pol II binding at nuclear cycle 13 is measured in embryos mutant for either mei-41/ATR or zelda. To correlate RNA Pol II binding with replication stress, genome-wide profiles of Replication protein A (70kDa subunit, RpA-70 EGFP) were generated in parallel with RNA Pol II for both wild-type and zelda at nuclear cycle 13.

2015-03-05 | GSE62925 | GEO

In situ Hi-C study of chromatin conformation during early Drosophila development

Project description:In this experiment we examined higher order chromatin structure during early Drosophila melanogaster development. We performed in situ Hi-C for hand-sorted non-mitotic embryos at nuclear cycle number 12, 13 and 14, and for embryos at 3-4 hours post fertilisation. During this time in development, the zygotic genome is activated and zygotic transcription is taking place for the first time. To assess the impact of transcription on chromatin structure we injected the transcription inhibitors alpha-amanitin or triptolide before zygotic genome activation and performed Hi-C and ChIP-seq for RNA Pol II. Furthermore, we used Hi-C to study genome architecture in embryos lacking the transcription factor Zelda.

2017-04-06 | E-MTAB-4918 | biostudies-arrayexpress

Integral nuclear pore proteins bind to Pol III genes and are required for Pol III transcript processing in C. elegans [seq]

Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-M-CM-^_ IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans. Genome-wide ChIP-seq and ChIP-chip were performed in mixed-stage C. elegans embryos for nuclear pore proteins NPP-13, NPP-3, IMB-1 and chromatin proteins Pol III (RPC-1), TBP-1, TFC-1 (SFC-1), TFC-4 (TAG-315), and Pol II (AMA-1). For RPC-1 and TBP-1 ChIP-seq, embryos depleted for NPP-13 were also used. Total RNAs from wild-type, NPP-13 RNAi, and IMB-1 RNAi embryos were analyzed by RNA-seq.

2013-09-05 | E-GEOD-42714 | biostudies-arrayexpress

Pol II chromatin immunoprecipitation in S. cerevisiae

Project description:In Saccharomyces cerevisiae short non-coding RNA (ncRNA) generated by RNA Polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3 and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is generally required for NRD-dependent transcription termination through the action of its CTD interacting domain (CID). Pcf11 localizes downstream of Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA and restricts Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar as both accumulate RNA:DNA hybrids and display Pol II pausing downstream of NRD terminators. We predict a mechanism whereby Nrd1 and Pcf11 exchange on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo. ChIP-seq with antibody against pol II in wild type and Pcf11 mutants: Pcf11-2, Pcf11-9 and Pcf11-13 grown at 25C and 37C along with input samples

2015-04-02 | E-GEOD-67483 | biostudies-arrayexpress

Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation [RNA-seq]

Project description:Immediately after fertilization the genome is transcriptionally quiescent. Maternally encoded pioneer transcription factors reprogram the chromatin state and facilitate the transcription of the zygotic genome. In Drosophila, transcription is initiated by the pioneer factor Zelda. While Zelda-occupied sites are enriched with histone acetylation, a post-translational mark associated with active cis-regulatory regions, the functional relationship between Zelda and histone acetylation in zygotic genome activation remained unclear. We show that Zelda-mediated recruitment of the histone acetyltransferase CBP is essential for zygotic transcription and embryonic development. CBP catalytic activity is necessary for release of RNA Polymerase II (Pol II) into transcription elongation. However, CBP largely activates zygotic transcription independent of acetylation through Pol II recruitment. Neither acetylation nor CBP are required for the pioneering function of Zelda. Our data suggest that pioneer factor-mediated recruitment of CBP is a conserved mechanism required to activate zygotic transcription but that this role is separable from the function of pioneer factors in restructuring chromatin accessibility.

2024-10-15 | GSE274629 | GEO

Integral nuclear pore proteins bind to Pol III genes and are required for Pol III transcript processing in C. elegans [array]

2013-09-05 | E-GEOD-42651 | biostudies-arrayexpress

Genome-wide profiles of RNA polymerase II (Pol 2) DNA occupancy and the histone modifications H3K4me3 and H3K36me3 around the circadian cycle

Project description:To understand the transcriptional basis of circadian rhythms in mouse liver, we generated genome-wide profiles of RNA polymerase II (Pol 2) DNA occupancy and the histone modifications H3K4me3 and H3K36me3 around the circadian cycle. We found that Pol 2 occupancy at promoters and in gene bodies cycle in synchrony, suggesting that Pol 2 recruitment at promoters underlies circadian gene transcription. On average, Pol 2 occupancy precedes mRNA accumulation by about three hours. Promoters of transcribed genes have tri-methylated H3K4 residues even at their trough activity times, and the tri-methylation levels increase in phase with Pol 2 loading. In contrast, the tri-methylation of H3K36 residues lags transcription by three hours with amplitudes that are markedly shallower, indicating that this mark is less dynamic on the circadian time scale. The comparison of Pol 2 occupancy and mRNA accumulation revealed that both transcriptional and post-transcriptional regulatory mechanisms can account for diurnal mRNA profiles. Additional processed data and visualization tools are available at http://cyclix.vital-it.ch/ Contributed by members of CycliX consortium (http://cyclix.vital-it.ch/) 28 samples examinded, 7 Polr2b, 7 H3K4me3, 7 H3k36me3, 7 input samples.

2012-12-06 | E-GEOD-35788 | biostudies-arrayexpress

Genome-wide RNA Pol II-CTD occupancy in wild type and mutant worms

Project description:We used RNA Pol II-CTD occupancy assay to identify mRNA isoform switch in mutant animals compared to wild type animals. Examination of RNA Pol II-CTD Ser5P recognized by 4H8 in wild type, sydn-1(0), ssup-72(0) and ssup-72(0);sydn-1(0)

2015-11-01 | E-GEOD-68203 | biostudies-arrayexpress

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

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.

2022-08-09 | PXD035866 |

Mitotic Transcriptional Clearance of Actively Engaged Pol II Through Transcriptional Elongation Control

Project description:Chromatin is highly condensed and transcriptionally repressed during mitosis. Although it is established that some general transcription factors are inactivated by phosphorylation at mitosis, many details of mitotic transcriptional repression and its underlying mechanisms are largely unknown. Here, we provide evidence that as cells enter mitosis, genes with transcriptionally engaged RNA Polymerase II (Pol II) can continue transcription until the end of the gene to clear Pol II from mitotic chromatin. Using ChIP-Seq, we find that the transcriptional reinitiation process is globally impaired in early mitosis (prophase/prometaphase), with loss of TFIIB occupancy and nucleosome-free regions at promoters. Pretreatment of nocodazole-arrested mitotic cells with the P-TEFb inhibitor flavopiridol prevents the release of promoter-proximal engaged Pol II. Global nascent RNA sequencing and RNA fluorescence in situ hybridization (FISH) of individual genes demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Chemical and mutational inhibition of P-TEFb in mitosis leads to delays in the progression of cell division. Together, our study reveals a novel mechanism for mitotic transcriptional repression whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. ChIP-Seq of Pol II of different forms, TFIIB, H3K4me3 in human HeLa cells at different cell cycle stages. ChIP-Seq of Pol II in HeLa mitotic cells with or without CDK9 inhibitor flavopiridol pretreatment. Nascent RNA-seq in asynchronous and arrested mitotic cells.

2015-10-30 | E-GEOD-71848 | biostudies-arrayexpress

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