RNA-sequencing of human cervical cancer HeLa cells treated with splicing inhibitors Sudemycin C1, Sudemycin K, Spliceostatin A or control DMSO.
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ABSTRACT: HeLa cells were co-treated with the drugs or DMSO control and BrU. After 3 hours, cells were harvested. Both total RNA and recently transcribed RNA (isolated by BrU immunoprecipitation) were sequenced.
Project description:Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, miRNAs, and RNA-binding proteins. Here, we present Bru-Seq and BruChase-Seq to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory TNF. The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steadystate total RNA and should be useful in many biological settings. Analysis of the effect of TNF exposure in nascent gene expression and in transcript stability
Project description:Bru-seq nascent RNA sequencing (PubMed ID 23973811) was performed on two primary human fibroblast cell lines, mouse embryonic stem cells, and GM12878 human lymphoblastoid cells. Read data, which include both exon and intron signals, were used to identify transcription unit spans genome-wide, where a transcription unit is roughly correspondent to the longest expressed isoform of a gene. However, because algorithms were not constrained by annotated genes, transcription units need not and often do not correspond precisely to gene boundaries and include extragenic transcription. Transcription units were then compared to separate data sets that comprised induced copy number variants, common fragile sites, and Repli-seq replication timing. The objective was to discover the relationships between transcription unit span and size, local genomic instability, and replication timing. This GEO sample series provides the span and intensity of transcription units called genome-wide in the various samples. Correlations to genome stability and replication timing are provided in the associated manuscript. In addition, one human fibroblast line and the mouse embryonic stem cells had paired samples treated and untreated with low dose aphidicolin. Gene RPKM signal intensities are provided for these samples, although comparing these was not the principal objective of the study. Bru-seq single-read nascent RNA sequencing on human 090 fibroblasts +/- aphidicolin treatment, human UMHF1 fibroblasts (3 replicates), human GM12878 lymphoblastoid cells, and mouse embryonic stem cells+/- aphidicolin treatment.
Project description:The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5’-3’ direction with slow recovery and TC-NER at the 3’ end of long genes. RNA synthesis resumed fully at the 3’-end of genes after a 24-hour recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases. This study is composed of three identical experiments run in three different cell lines. For each experiment, there is one control (mock irradiated cells) and four test samples (0h, 2h, 6h and 24h after UV 10J irradiation).
Project description:The rate of transcription elongation plays important roles in the timing of expression of full-length transcripts as well as for the regulation of alternative splicing. In this study we coupled Bru-Seq technology with 5,6-dichlorobenzimidazole 1-M-NM-2-D-ribofuranoside (DRB) to estimate the elongation rates of over 2,000 individual genes in human cells. This technique, BruDRB-Seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with fast elongation rates showed higher densities of H3K79m2 and H4K20me1 marks compared to slower elongating genes. Furthermore, fast elongation rates had a positive correlation with gene length, low complexity DNA sequence and distance from nearest active transcription unit. Features that negatively correlated with elongation rate included exon density and the number of LINE sequences in the gene. The BruDRB-Seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation. Measurement of RNA Pol II elogation rate. Normal fibroblasts (HF1 and TM), Cockayne syndrome group B fibroblasts, K562 and MCF-7 cells were exposed to DRB for 60 minutes, after which a washout was performed. Nascent RNA was labeled using bromouridine for 10 minutes immediately after the washout. The genomic region extending from actice Trancription Start Sites was used to determine the gene's elongation rate. Please note that the nf_0h_3* samples are duplicated sample records of GSM1062445 and GSM1062446, for the convenient retrieval of the complete raw data from SRA.
Project description:Myotonic dystrophy type 1 (DM1) is a neuro-muscular disorder caused by CTG triplet expansion in the 3'-UTR of the DMPK gene. Mutated transcripts aggregate in muscle nuclei and increase CUGBP1 stability by a not-yet determined mechanism. To assess the involvement of CUGBP1 in DM1, we used Drosophila as a model of the disease and we performed genome wide analyses of gene expression of bru-3 (CUGBP1 orthologue) gain of function line (Mef>bru-3) vs. Mef>lacZ control line.
Project description:RNA-DNA hybrids are widespread epigenetic features of genomes that provide a growing range of activities in transcription, chromatin and DNA replication and repair. Understanding of these diverse functions has been advanced by characterising the proteins that interact with the hybrids, with all such studies revealing hundreds of potential interactors. However, all interaction analyses to date have focused on mammalian cells, and so it is unclear if a similar spectrum of RNA-DNA hybrid interactors found in other eukaryotes, thus limiting our understanding of the conserved and lineage-specific activities linked to these genetic structures. The African trypanosome is a compelling organism in which to address these questions. As a divergent single-cell eukaryotic parasite of the Discoba grouping, Trypanosoma brucei displays substantial divergence in several aspects of core biology from its mammalian host and, unusually for a protist, has well-developed tools for molecular genetic analysis. For these reasons, we used DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry to reveal 602 putative interactors in T. brucei mammal- or insect vector-infective stage cells. We show that the approach selects for a subset of the parasite proteome and reveals a range of predicted RNA-DNA hybrid associated activities, some overlapping with similar studies in mammals. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, impact on T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each affects the operation of the crucial parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as many conserved with mammals, and so likely fundamental to eukaryotic genome function.
Project description:The anti-cancer drug camptothecin inhibits replication and transcription by trapping DNA topoisomerase I (Top1) covalently to DNA in a M-bM-^@M-^\cleavable complexM-bM-^@M-^]. To examine the effects of camptothecin on RNA synthesis genome-wide we used Bru-Seq and show that camptothecin treatment affected transcription initiation, elongation, termination, splicing and enhancer activity. Following removal of camptothecin, transcription spread as a wave from the 5M-bM-^@M-^Y-end of genes with no recovery of transcription apparent from RNA polymerases stalled in the body of genes. As a result, camptothecin preferentially inhibited the expression of large genes such as proto-oncogenes, and anti-apoptotic genes while smaller ribosomal protein genes, pro-apoptotic genes and p53 target genes showed relative higher expression. In addition, a set of mitotic regulator genes and histone genes were inhibited in a size-independent manner. Cockayne syndrome group B fibroblasts showed a very similar RNA synthesis recovery profile to normal fibroblasts suggesting that transcription-coupled repair is not involved in the repair of transcription-blocking TOP1 lesions. These findings of the effects of camptothecin on transcription have important implications for its anti-cancer activities and may aid in the design of improved combinatorial treatments involving Top1 poisons. Analysis of the effect of Camptothecin (CPT) on transcription. Normal fibroblasts and Cockayne syndrome group B fibroblasts were exposed to CPT for 60 minutes, after which a washout was performed. Nascent RNA was labeled using bromouridine for 15 minutes starting at time points: 1) 15 minutes before the washout; 2) Immediately after the washout; 3) 15 minutes after the washout. Test samples are compared to control cells that were not exposed to CPT.
Project description:High throughput sequencing to derive function of cde-1 in endogenous RNAi in C. elegans Small RNAs were cloned from C. elegans adults, following removal of tri-phosphate groups from 5' end. Sequencing was performed using the Illumina 1G platform.
Project description:B cells from tonsils of human donors were extracted for combined RNAseq+ATACseq from the same cell. One sample was prepared separately ("old") and is of lower quality, but still included. It primarily holds ATACseq information.
Project description:In this work we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits PolII and robustly activates gene transcription. Chip-seq profiling of MyoD, Myf5, Histone H4 acetylation (H4Ac), and Pol II in MyoD-/-; Myf5-/- MEFs (M&M MEFs)