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Genome-wide Analysis of Vitamin D Receptor Binding By ChIP-Seq

ABSTRACT: We used ChIP-Seq to identify the genomic locations bound by the vitamin D receptor (VDR) in two lymphoblastoid cell lines (LCLs) (CEPH individuals GM10855 and GM10861 from the International HapMap Project) before and after calcitriol treatment for 36 hours. Immunoprecipitated DNA was sequenced using the Illumina Genome Analyzer II. Sequence reads (35 bases; 10-19 million quality-filtered reads/sample) were aligned to the human genome (NCBI Build 36.3) using ELAND software. The number of unique alignments ranged from 7.73 million to 14.32 million. Peaks were called in the aligned sequence data using a model-based analysis of ChIP-Seq (MACS) and compared with sequenced sonicated and amplified input DNA. In the samples not treated with calcitriol, the number of peaks ranged from 468 to 4538 (median 975, mean 1587), while in the calcitriol-treated samples the number of peaks was between 2560 and 7244 (median 4546, mean 4560). 65-75% of peaks of untreated samples were in promoters, while only 24-50% of peaks of calcitirol-treated samples were in promoters. This study provides a comprehensive map of VDR binding in lymphoblastoid cell lines.

ORGANISM(S): Homo sapiens

PROVIDER: GSE22484 | GEO | 2010/06/22

SECONDARY ACCESSION(S): PRJNA128697

REPOSITORIES: GEO

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Genome-wide Analysis of Vitamin D Receptor Binding By ChIP-Seq

Project description:We used ChIP-Seq to identify the genomic locations bound by the vitamin D receptor (VDR) in two lymphoblastoid cell lines (LCLs) (CEPH individuals GM10855 and GM10861 from the International HapMap Project) before and after calcitriol treatment for 36 hours. Immunoprecipitated DNA was sequenced using the Illumina Genome Analyzer II. Sequence reads (35 bases; 10-19 million quality-filtered reads/sample) were aligned to the human genome (NCBI Build 36.3) using ELAND software. The number of unique alignments ranged from 7.73 million to 14.32 million. Peaks were called in the aligned sequence data using a model-based analysis of ChIP-Seq (MACS) and compared with sequenced sonicated and amplified input DNA. In the samples not treated with calcitriol, the number of peaks ranged from 468 to 4538 (median 975, mean 1587), while in the calcitriol-treated samples the number of peaks was between 2560 and 7244 (median 4546, mean 4560). 65-75% of peaks of untreated samples were in promoters, while only 24-50% of peaks of calcitirol-treated samples were in promoters. This study provides a comprehensive map of VDR binding in lymphoblastoid cell lines. The GM10855 and GM10861 cell lines were either stimulated for 36 hours with calcitriol or unstimulated, and crosslinked with formaldehyde to generate a snapshot of all protein-DNA interactions occurring in the nucleus at that particular point in time. Antibodies were then used to immunoprecipitate the VDR protein together with the crosslinked DNA fragments. Following reversal of crosslinks and digestion of protein, adaptors were ligated to immunoprecipitated DNA, and bridge PCR was used to generate clonally amplified amplicons before sequencing by synthesis using the Illumina Solexa Genome Analyzer.

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Project description:Vitamin D induces anti-proliferative and differentiating effects in prostate cancer. Thus calcitriol, the hormonally active form of Vitamin D, and its analogs have been extensively studied in prostate cancer cells. Yet despite its importance, relatively little is known about the genome-scale mechanisms by which Vitamin D, through its cognate nuclear vitamin D receptor (VDR), exerts its regulatory functions at the genomic level. In this study, we defined VDR transcriptional networks in the LNCaP prostate cancer cell line by mapping the genomic binding sites of VDR and by identifying differentially expressed genes upon calcitriol treatment. We found that VDR and androgen receptor (AR) antagonistically regulate a subset of cell cycle-related genes that are over-expressed in prostate cancer tumors. The expression balance of these genes is partially regulated through the competition dynamics between AR and VDR binding to shared cis-regulatory elements. On such shared elements, we found that FOXA1 mediates this competition by serving as a pioneering factor for both AR and VDR binding. We also found significant enrichment of AR-, VDR-, and AR/VDR overlapping binding sites in prostate cancer-associated single-nucleotide polymorphism (SNP) intervals identified from genome-wide association studies (GWAS), providing genetic evidence to link AR, VDR and their crosstalk to prostate cancer susceptibilities. In particular, we found that in a cis-regulatory element of the RFX6 gene implicated in prostate cancer progression, an allelic variant increases prostate cancer risk by switching the antagonism between AR and VDR into a synergistic interaction. Gene expression profiling in LNCaP cells after 24hr treatment with different nuclear recpetor ligrands, with time-matching control, in triplicates Expression is assayed with Agilent G4112F.

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