Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ChIP-seq of HCC1806 cells (H2Bub1, H3K79me3, H3K27ac), treated with siControl or siRNF40 for 48 hours

ABSTRACT: The analysis identifies differentially occupied genomic regions of H2Bub1, H3K79me3, and H3K27ac by RNF40 silencing in HCC1806 cells

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Homo sapiens

SUBMITTER: Evangelos Prokakis

PROVIDER: E-MTAB-12000 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A high percentage of potential oncology drugs fail in clinical trials, partly because preclinical models used to test them are inadequate. Breast cancer is the leading cause of cancer-related death among women worldwide but we lack appropriate in vivo models for the ER+ subtypes, which represent more than 75% of all cases. We address these issues by xenografting tumor cells to their site of origin, the milk ducts. All ER+ cell lines and patient-derived xenografts grow mimicking their clinical counterparts. Disease progresses with invasion and metastasis, which become amenable to study. The action of hormones, important in breast carcinogenesis, can now be studied in a relevant context. Importantly, these open opportunities for development and evaluation of therapies. Eight- to twelve-week-old female SCID Beige mice (Charles River) were injected with 5x10e5 BT20-GFP/luc2 cells (n=3) or 5x10e5 HCC1806-GFP/luc2 cells (n=3) either into the mammary fat pad or 2x10e5 BT20-GFP/luc2 cells (n=3) or 2x10e5 HCC1806-GFP/luc2 cells intraductally (n=3). Xenografted BT20 and HCC1806 basal breast cancer cells were sorted by FACS based on GFP expression; total RNA was extracted using Trizol Reagent (Invitrogen), purified with the miRNeasy Mini Kit (Qiagen), quantity and quality were assessed by NanoDropÂ®ND-1000 spectrophotometer and RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, USA). Only samples with RIN score >7.0 were included. For each sample, 300 ng of total RNA were amplified using the message amp II enhanced kit (AM1791, Ambion). 12.5 Î¼g of biotin-labelled cRNA were chemically fragmented. Affymetrix GeneChip Human Genome U133A 2.0 Arrays (Affymetrix, Santa Clara, CA, USA) were hybridized with 11Î¼g of fragmented target, at 45Â°C for 17 hours, washed and stained according to Affymetrix GeneChipÂ® Expression Analysis Manual (Fluidics protocol FS450_0007). Arrays were scanned using the GeneChipÂ® Scanner 3000 7G (Affymetrix) and raw data was extracted from the scanned images and analyzed with the Affymetrix Power Tools software package (Affymetrix). All statistical analyses were performed using R and Bioconductor packages (http://www.Bioconductor.org). Hybridization quality was assessed using the Expression Console software (Affymetrix). Normalized expression signals were calculated from Affymetrix CEL files using RMA. Differential hybridized features were identified using Bioconductor package â??limmaâ?? that implements linear models for microarray data (Smyth, 2004). P values were adjusted for multiple testing with Benjamini and Hochbergâ??s method to control false discovery rate (FDR) (Benjamini et al., 2001). Probe sets showing â?¥2-fold change and a FDR â?¤0.05 were considered significant.

Project description:Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification within transcribed regions, but it remains uncertain how these modifications influence, or are influenced by, properties of the elongation complex. Here we establish an intimate link between Cdk9, the kinase component of positive transcription elongation factor b (P-TEFb), and mono-ubiquitylation of histone H2B (H2Bub1), in the fission yeast Schizosaccharomyces pombe. Mutations that impair Cdk9 function reduce H2Bub1 levels in vivo. Conversely, mutations that prevent H2Bub1 decrease phosphorylation of elongation factor subunit Spt5, a sensitive and specific indicator of Cdk9 activity. Chromatin immunoprecipitation (ChIP) analysis suggests this is due to impaired Cdk9 recruitment to H2Bub1-deficient chromatin. P-TEFb and H2Bub1 pathways also interact genetically: mutation of the histone H2B ubiquitin-acceptor residue decreases the requirement for Cdk9 activity in vivo, and multiple cdk9 mutations suppress morphological defects of H2Bub1-deficient strains. Moreover, H2Bub1 loss causes redistribution of transcribing RNAPII on chromatin that is corrected by a hypomorphic cdk9 mutation. Therefore, whereas mutual dependence of Spt5 phosphorylation and H2Bub1 suggests positive feedback between P-TEFb and the ubiquitylation machinery, mutual suppression by cdk9 and H2Bub1-pathway mutations indicates an antagonistic relationship, whereby the activities must be balanced to properly regulate elongation. In order to study the genome-wide localization of H2Bub1 in Schizosaccharomyces pombe, H2Bub1, H2B-Flag as well as RNAPII (along with associated DNA sequences) were immunoprecipitated using repectively anti-H2Bub1, anti-Flag and anti-8WG16 antibodies. The ChIPs were performed in duplicate from WT cells as well as in the H2B-K119R mutant. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.

Dataset Information

ChIP-seq of HCC1806 cells (H2Bub1, H3K79me3, H3K27ac), treated with siControl or siRNF40 for 48 hours

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