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Coilin ChIP-seq in cycling HeLa cells

ABSTRACT: To investigate location of Cajal bodies (CB) inside the cell nucleus, we created a stable cell line expressing the GFP version of CB-marker protein coilin at endogenous levels and performed ChIP-seq with anti-EGFP antibody. ChIP-seq revealed close association of CBs wih U3 genes, snRNA genes and histone genes in histone cluster 1 and 2. 1 biological replicate of coilin ChIP-seq and input sample

ORGANISM(S): Homo sapiens

SUBMITTER: Karla Neugebauer

PROVIDER: E-GEOD-58449 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Coilin ChIP-seq in cycling HeLa cells

Project description:To investigate location of Cajal bodies (CB) inside the cell nucleus, we created a stable cell line expressing the GFP version of CB-marker protein coilin at endogenous levels and performed ChIP-seq with anti-EGFP antibody. ChIP-seq revealed close association of CBs wih U3 genes, snRNA genes and histone genes in histone cluster 1 and 2.

2014-11-06 | GSE58449 | GEO

HeLa cell polyA- RNA-seq

Project description:Coilin iCLIP data revealed 42 novel human snoRNAs of intronic origin. To validate their expression and estimate abundance of novel and annotated snoRNAs, we performed RNA-seq on polyA- and rRNA-depleted RNA isolated from HeLa cells. Results show that expression of novel snoRNAs is comparable to the previously annotated snoRNAs. 1 replicate of RNA depleted of polyA and ribosomal RNA.

2014-11-06 | E-GEOD-58450 | biostudies-arrayexpress

Coilin and Pol II ChIP-seq in HeLa cells after RPL14 and RPL24 depletion

Project description:To investigate location of Cajal bodies (CB) inside the cell nucleus after RPL depletion, we performed ChIP-seq with an anti-Coilin antibody after siRNA knockdown of RPL14 and RPL24. ChIP-seq revealed a loss of association of CBs wih snRNA genes and histone genes in histone cluster 1 and 2 upon RPL KD. We also performed Pol 2 ChIP-seq to look at transcription of these gene loci upon RPL knockdown and found decreased Pol II binding along genes.

2024-03-27 | GSE199275 | GEO

iCLIP mapping of coilin-RNA interactions in HeLa and P19 cells

Project description:Our ChIP resuls suggested that coilin association with U3, snRNA and histone genes might be dependent on coilin-RNA interaction. We used iCLIP of coilin-GFP expressed in HeLa and P19 cell lines at endogenous levels to identify coilin RNA targets and investigate RNA-binding specificity. P19 cells expressing GFP fused to a nuclear localization signal (GFP-NLS) was used as a negative control. iCLIP results revealed that coilin binds several classes of ncRNA including snRNAs, U3 snoRNA and scaRNAs. Interestlignly the majority of coilin targets were intronic snoRNAs, suggesting a novel role of CBs in snoRNA biogenesis. 5 biological replicates from P19 and 2 biological replicates from HeLa cells after UV-crosslinking. Negative control samples prepared from GFP-NLS fusion protein are stored uder accession E-MTAB-747.

2014-11-06 | E-GEOD-58448 | biostudies-arrayexpress

Coilin's RNA, DNA and protein interactomes redefine the Cajal body as a hub for small non-coding RNAs

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2014-11-06 | E-GEOD-58451 | biostudies-arrayexpress

ChIP-MS reveals the local chromatin composition by label-free quantitative proteomics

Project description:Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versality of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.

2024-03-05 | PXD039627 | Pride

ChIP-MS reveals the local chromatin composition by label-free quantitative proteomics

2024-03-05 | PXD039701 | Pride

Atypical U3 snoRNA Suppresses the Process of Pterygia through Modulating 18S Ribosomal RNA Synthesis

Project description:Ribosome biosynthesis plays a crucial role in regulating protein translation and is essential for cell growth and development in physiological progress. The progression and recurrence of Pterygia mainly occur due to the abnormal proliferation and migration of stromal Pterygia fibroblasts. Small nucleolar RNA U3 (U3 snoRNA), harboring the atypical C/D boxes, is involved in 18S ribosomal RNA (18S rRNA) synthesis; however, the mechanism of U3 snoRNA in Pterygia remains unclear. Methods: Primary HCFs and HPFs were separated and cultured from fresh conjunctival grafts and Pterygia tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPF and HCF cells for further specific phenotypes analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in Pterygia promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5′ external transcribed spacer (5′ ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusion: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in Pterygia through modulating the 18S RNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of Pterygia.

2022-05-20 | PXD030801 | Pride

Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers

Project description:Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here we show that super-enhancers consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis. ChIP-Seq for H3K27ac and RNA Pol II in mouse embryonic stem cells with CRISPR-deleted enhancers

2014-12-15 | E-GEOD-64188 | biostudies-arrayexpress

In vivo live imaging of RNA polymerase II transcription factories in primary cells

Project description:Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as M-bM-^@M-^XM-bM-^@M-^Xtranscription factories,M-bM-^@M-^YM-bM-^@M-^Y but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9- mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments. Examination of genome occupancy of CDK9 and RNAPII that was performed by ChIP-seq in the MEL cell line as described (Soler et al. 2010, 2011) using CDK9 C20 antibody (Santa Cruz Biotechnology, C20, sc-484) and RNA Pol II antibody (Santa Cruz Biotechnology, N20, sc899),

2013-05-13 | E-GEOD-46849 | biostudies-arrayexpress

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