Project description:Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution and newer techniques require significant experimental alterations and complex bioinformatics. Here we build upon our high-resolution crosslinking ChIP-seq (X-ChIP-seq) method and compare it to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing. Using High-resolution X-ChIP-seq we determined the genome-wide binding profile of various DNA binding proteins.
Project description:Kidney cancer accounts for more than 100,000 deaths per year world-wide. More than 80% are clear cell tumors (ccRCCs) and the majority are associated with loss of function of the von Hippel Lindau (pVHL) tumor suppressor resulting in upregulation of HIF-{alpha} subunits, and activation of HIF-dependent transcriptional pathways. Recent GWAS studies have discovered RCC-susceptibility loci both within EPAS1 (HIF-2{alpha}) and in an intergenic region of unknown function on 11q13.3. As part of an ongoing study to define the direct transcriptional targets of HIF-2 in renal cancer, we undertook a genome-wide analysis of HIF-2-binding sites in pVHL-defective 786-O cells (that lack functional HIF-1{alpha} due to a truncated transcript) using chromatin immunoprecipitation with antibodies directed against HIF-2{alpha} and its dimerization partner HIF-1{beta}, coupled to high-throughput sequencing (ChIP-seq). Amongst approximately 600 pangenomic HIF-2{beta} ChIP signals, we observed strong binding (ranked 12th by peak height) almost precisely coinciding with the RCC predisposition SNP rs7105934 on 11q13.3. We report binding of HIF-2{alpha} and HIF-1{beta} in 786-O clear cell renal carcinoma cells. 3 samples examined, HIF-2{alpha}, HIF-1{beta} and pre-immune control ChIP.
Project description:Genome-wide mapping of protein–DNA interactions is essential for a full understanding of transcriptional regulation. A precise map of binding sites for transcription factors, core transcriptional machinery is vital for deciphering the gene regulatory networks that underlie various biological processes. Chromatin immunoprecipitation followed by sequencing (ChIP–seq) is a technique for genome-wide profiling of DNA-binding proteins. However, our conventional ChIP–seq occasionally gives wider peaks which might be due to overlapping binding sites of two or more transcription factors. Therefore, to improve the resolution of our conventional ChIP–seq which have DNA-protein footprint of ~100 bp, we decreased the size of DNA-protein footprint to ~ 50 bp by DNaseI digestion of whole cell extract (WCE).
Project description:To identify genes directly regulated by HIF-1, we performed ChIP-seq on L4-stage egl-9(sa307); hif-1(ia4); odIs131[hif-1::gfp] animals, which have activated HIF-1 under aerobic conditions, using anti-GFP antibodies to precipitate DNA bound by HIF-1::GFP followed by high-throughput sequencing to identify peaks of reads corresponding to HIF-1 binding sites