Analysis of HSF2 target genes using a global promoter ChIP-chip strategy on mouse wild-type testis
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE9289 | GEO | 2008/08/06
SECONDARY ACCESSION(S): PRJNA102927
REPOSITORIES: GEO
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Project description:Mapping of in vivo targets for HSF2 by using mammalian tissue material in a global ChIP-chip promoter screen. Chromatin was isolated from three wild-type (WT) mouse testes, crosslinked and sonicated into fragments of 100-500 bp, and the quality of the DNA was controlled before the immunoprecipitation, showing no signs of degradation. The DNA amplified from the HSF2 immunoprecipitation samples was labeled and hybridized against the total input DNA samples, on a 1.5 kb promoter tiling array (NimbleGen Systems Inc.), covering ~26000 promoters of the mouse genome. After hybridization and scanning, HSF2 hybridization signals were Lowess normalized to produce log2 ratios (HSF2 ChIP/Input DNA) for all replicate arrays. The promoter values were calculated as average log2 ratios over all probes for each promoter. To identify the HSF2 target population, we used a non-arbitrary analysis method called RankProd described in Breitling et al. 2004. RankProd has been published as an R/Bioconductor package. Keywords: 1.5 kb mouse promoter ChIP-chip (MM5, NimbleGen Systems Inc.), mouse testis HSF2 ChIP sample vs. Input sample (reference), biological replicates: 3, one replicate per array.
2008-08-05 | E-GEOD-9289 | biostudies-arrayexpress
Project description:Mapping of in vivo targets for HSF1 by using mammalian tissue material in a global ChIP-chip promoter screen. Chromatin was isolated from three wild-type (WT) mouse testes, crosslinked and sonicated into fragments of 100-500 bp, and the quality of the DNA was controlled before the immunoprecipitation, showing no signs of degradation. The DNA amplified from the HSF1 immunoprecipitation samples was labeled and hybridized against the total input DNA samples, on a 1.5 kb promoter tiling array (NimbleGen Systems Inc.), covering ~26000 promoters of the mouse genome. After hybridization and scanning, HSF1 hybridization signals were Lowess normalized to produce log2 ratios (HSF1 ChIP/Input DNA) for all replicate arrays. The promoter values were calculated as average log2 ratios over all probes for each promoter. To identify the HSF1 target population, we used a non-arbitrary analysis method called RankProd described in Breitling et al. 2004. RankProd has been published as an R/Bioconductor package.
2010-08-31 | GSE22492 | GEO
Project description:Mapping of in vivo targets for HSF1 by using mammalian tissue material in a global ChIP-chip promoter screen. Chromatin was isolated from three wild-type (WT) mouse testes, crosslinked and sonicated into fragments of 100-500 bp, and the quality of the DNA was controlled before the immunoprecipitation, showing no signs of degradation. The DNA amplified from the HSF1 immunoprecipitation samples was labeled and hybridized against the total input DNA samples, on a 1.5 kb promoter tiling array (NimbleGen Systems Inc.), covering ~26000 promoters of the mouse genome. After hybridization and scanning, HSF1 hybridization signals were Lowess normalized to produce log2 ratios (HSF1 ChIP/Input DNA) for all replicate arrays. The promoter values were calculated as average log2 ratios over all probes for each promoter. To identify the HSF1 target population, we used a non-arbitrary analysis method called RankProd described in Breitling et al. 2004. RankProd has been published as an R/Bioconductor package. HSF1 ChIP sample vs. Input sample (reference), biological replicates: 3, one replicate per array.
2010-08-31 | E-GEOD-22492 | biostudies-arrayexpress
Project description:A comparative ChIP-chip analysis of TFIIB and NC2 in human B cells reveals that basal core promoter architectures control the equilibrium between NC2 and preinitiation complexes. We conducted a comparative ChIP-chip and gene expression analysis of TFIIB in human B cells and analyze associated core promoter architectures. TFIIB occupancy relates well to gene expression, with the vast majority of promoters being GC-rich and lacking defined core promoter elements. TATA consensus and TATA-like motifs but not the previously in vitro defined TFIIB recognition elements (BREs) are enriched in approximately 5% of the genes. Further insight was obtained by performing a parallel ChIP-chip analysis of the TFIIB antagonist NC2. The latter identifies a highly related target gene set. Nonetheless, subpopulations show strong variations in TFIIB/NC2 ratios, with high NC2/TFIIB ratios correlating to promoters that show dispersed transcription start site patterns and lacking defined core elements. Conversely, high TFIIB/NC2 ratios select for conserved core promoter elements that include TATA and INR (initiator), the upstream TFIIB recognition element (BREu) and the downstream promoter element (DPE). Two biological samples from LCL 721 lymphoblastoid human B cells were subjected to ChIP-chip analysis of TFIIB and NC2 using a Nimblegen human promoter array (based on the HG17 genome build) covering 1.5 kb DNA around transcription start sites.
2010-05-16 | E-GEOD-19562 | biostudies-arrayexpress
Project description:We performed a genome-scale ChIP-chip comparison of two modifications (trimethylation of lysine 9 and trimethylation of lysine 27) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the PRC2 complex is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the PRC2 complex, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 co-repressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5kb promoter arrays. We found that a large number of promoters of zinc finger transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38 array tiling set, identifying ~7000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 zinc fingers, especially those containing KRAB domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1. Keywords: Human whole genome tiling array Amplicons were applied either to ENCODE arrays, 5 kb promoter arrays, or to the human genome tiling array set consisting of 38 arrays (see www.nimblegen.com for details). The labeling and hybridization of DNA samples for ChIP-chip analysis was performed by NimbleGen Systems, Inc, except that ENCODE arrays were hybridized at UC Davis. Briefly, each DNA sample (1 μg) was denatured in the presence of 5'-Cy3- or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water. Then, 13ug of the Cy5-labeled ChIP sample and 13ug of the Cy3- labeled total sample were mixed, dried down, and resuspended in 40 μl of NimbleGen Hybridization Buffer (NimbleGen Systems) plus 1.5 ug of human COT1 DNA. After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems), dried by centrifugation, and scanned at 5-μm resolution using the GenePix 4000B scanner (Axon Instruments). Fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NIMBLESCAN 2.0 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure is approximately equivalent to mean-normalization of each channel. Total RNA was prepared from 5x106 Ntera cells using RNAeasy Kit (Qiagen) following the manufacturer’s instructions. RNA quality was ensured using the Agilent Systems Bioanalyzer. The RNA was hybridized to human whole genome expression microarrays from Nimblegen, which contain probes for every human gene based on genome build HG18 from the UCSC database (more details at http://www.nimblegen.com). 10 ug total RNA were used to synthesize cDNA using the SuperScript Double-Stranded cDNA synthesis kit (Invitrogen). The labeling of RNA samples, array hybridization and preliminary RNA expression analysis (data normalization) was performed by NimbleGen Systems, Inc.
2007-09-29 | E-GEOD-8667 | biostudies-arrayexpress
Project description:We used NimbleGen Mouse ChIP-chip 3 × 720K RefSeq Promoter Arrays (NimbleGen Roche, Madison, WI). Briefly, we isolated three independent samples of gender and age matched HDAC6KO and WT control CD4+CD25+ Treg, isolated, cross-linked and processed genomic DNA as above. DNA-protein complexes were bound with Foxp3 mAb, and enriched through immunoprecipitation, which a control sample is retained without immunoprecipitation (input). Both input and Foxp3-IP enriched samples underwent whole-genome amplification or ligation mediated-PCR to obtain adequate amounts of DNA for labeling. Foxp3-IP enriched samples were labeled with Cy5, and input samples with Cy3, and the arrays submitted to NimbleGen for analysis. Data were analyzed using Partek GS (Partek Inc., St Louis, MO) and the Broad Institutes Interactive Genome Viewer (Version 2.3.32) (Robinson et al., 2011). Raw data underwent Loess normalization, and Student t-testing with a false discovery rate of 0.1 (p-value cutoff 0.0029). For visualization, we generated IGV files using NimbleGen mapping information from the SignalMap GFF files.
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Project description:For each sample a matched total was prepared in parallel. For each biological replicate the cells were crosslinked on different days. Amplicons were prepared from each sample and hybridized on mouse 1.5 kb promoter arrays from Nimblegen. Keywords: ChIP-chip
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Project description:Samples from 20 children with B-cell precursor ALL, comprising ten with high hyperdiploidy and ten with the ETV6/RUNX1 fusion, were included in the study. Four µg of methylated and input fraction DNA were analyzed with a classic MeDIP assay and subsequent microarray hybridization using the NimbleGen CpG Island-Plus-Promoter Array (Roche NimbleGen, Madison, WI).<br><br>This array platform covers all 28,226 UCSC-annotated CpG islands and promoter regions for all RefSeq genes, including 385,000 probes of 50-75mer length per array, with an upstream and downstream promoter tiling of 800 bp and 200 bp, respectively. Signal intensity data were extracted from the scanned images. Each feature on the array has a corresponding log2 ratio, which is the ratio of the input signals for the methylated DNA and the input fraction DNA. The log2 ratio was scaled in order to center the ratio data around zero by subtracting the bi-weight mean for the log2 ratio values for all features on the array from each log2 ratio value. A modified ACME algorithm, where a fixed-length window (750 bp) is placed around each consecutive probe, and the one-sided Kolmogorov-Smirnov test were then applied on the scaled log2 ratio data to determine whether the probes were drawn from a significantly more positive distribution (peak) of intensity log2 ratios than the other probes in the array. The resulting score for each probe is the -log10 p-value from the windowed Kolmogorov-Smirnov test around that probe. Peak data were generated by searching for probes above a p-value minimum cut-off (-log10) of 2.<br><br>Peaks within 500 bp of each other were merged. Each annotated gene was subsequently searched for peaks appearing in a specified promoter region around the transcription start site. The region searched was design-specific, spanning from 5 kb upstream to 1 kb downstream of the transcription start site. Genes that had at least two probes with peaks above the p-value minimum cut-off were considered to have significant CpG island methylation scores for hypermethylation.
2009-09-19 | E-MTAB-144 | biostudies-arrayexpress