CDK8 ChIP-seq from HCT116 cells in normoxia and hypoxia
ABSTRACT: To test if CDK8 acts directly at HIF1A target genes, we performed ChIP-seq experiments in HCT116 cells under normoxic and hypoxic conditions. ChIP-seq for CDK8 versus Input under normoxia and 24hrs hypoxia (1% O2).
Project description:First lineage specification in the mammalian blastocyst embryo leads to formation of the inner cell mass (ICM) and trophectoderm (TE), which respectively give rise to the embryo proper and extraembryonic tissues. We show histone methylation asymmetry on promoters in the first two developmental lineages, and highlight epigenetic skewing associated with derivation of embryonic stem (ES) cells. Comparison of histone methylation patterns on promoters in the ICM, TE and in ES cells derived from ICM. ChIP-chip experiments using anti-H3K4me3 or anti-H3K27me3 antibodies. Two or three replicates per sample.
Project description:We report the application of ChIP bisulfite sequencing (ChIPbisSeq) to establish the methylation state of DNA bound to RNApol2 phosphorylated in Ser5 (RNAPol2Ser5) in the mouse cortex. We first profiled the RNAPol2Ser5 binding using regular ChIPSeq from 45 million raw read pairs (31 million unique pairs were aligned to the genome). Then we used bisulfite converted DNA immunoprecipitated with an antibody against RNAPol2Ser5 (87 million raw read pairs, 38 million unique read pairs aligned to the genome) to map RNAPol2Ser5 sites and to establish the methylation state at these sites. Examination of methylation state at RNAPol2Ser5 binding sites in the mouse cortex.
Project description:To identify the MYCN transcription factor binding sites across the genome, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-MYCN and anti-IgG antibodies on a MYCN-amplified NB cell line, SK-N-BE(2)-C. Identification of MYCN binding in neuroblastoma cells.
Project description:Nuclear lamins contact the genome at the nuclear periphery through large domains and are involved in chromatin organization. Among broad peak calling algorithms available to date, none are suited for mapping lamin-genome interactions genome-wide. We disclose a novel algorithm, Enriched Domain Detector (EDD), for analysis of broad enrichment domains from ChIP-seq data. EDD enables discovery of genomic domains interacting with broadly distributed chromatin-associated proteins such as lamins. The main advantage of EDD over existing broad peak callers is sensitivity to domain width rather than enrichment strength at a particular site, and robustness against local variations. EDD is downloadable from http://github.com/eivindgl/edd. LMNA ChIP-seq experiments in human normal dermal fibroblasts (Lonza CC-2511; LDFs) and human normal primary dermal fibroblasts (Norwegian Stem Cell Center AD04DFs).
Project description:We found that MCF7 and ZR751 Sox2-expressing breast cancer cell lines comprise of cells with heterogeneous Sox2 transcription activity reporter response. A small subset of Sox2 reporter responsive cells are more tumourigenic than the bulk Sox2 reporter unresponsive cells. We questioned whether Sox2 exhibit differential gene promoter occupancies in the two cell subsets to govern differential gene expression patterns. Sox2 ChIP in reporter unresponsive (RU) and reporter responsive (RR) cells (duplicate samples) were compared. IgG ChIP in RU and RR cells served as the negative controls.
Project description:Miz1 is a zinc finger protein that regulates expression of cell cycle inhibitors as part of a complex with Myc. Cell cycle-independent functions of Miz1 are poorly understood. Here, we use a Nestin-Cre transgene to delete an essential domain of Miz1 in the central nervous system (Miz1ΔPOZNes). Miz1ΔPOZNes mice display cerebellar neurodegeneration characterized by the progressive loss of Purkinje cells. Chromatin immunoprecipitation sequencing and biochemical analyses show that Miz1 activates transcription upon binding to a non-palindromic sequence present in core promoters. Target genes of Miz1 encode regulators of autophagy and proteins involved in vesicular transport that are required for autophagy. Miz1ΔPOZ neuronal progenitors and fibroblasts show reduced autophagic flux. Consistently, polyubiquitinated proteins and p62/Sqtm1 accumulate in the cerebella of Miz1ΔPOZNes mice, characteristic features of defective autophagy. Our data suggest that Miz1 may link cell growth and ribosome biogenesis to the transcriptional regulation of vesicular transport and autophagy. ChIP-Seq with H190 and G18 on an Illumina Genome Analyzer IIx.
Project description:Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been studied extensively in model organisms, little is known about these “first responders” in humans, especially their lineage and functional kinships to cytokine-secreting T helper cell (Th) counterparts. Here, we report gene regulatory circuitries for four human ILC–Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells, but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell identity genes in each lineage, uncovering new modes of regulation for signature cytokines, novel molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC–Th subsets. Molecular profiling of innate lymphoid and T helper cells subsets purified from tonsils and NK cells purified from peripheral blood using Assay for Transposase-Accessible Chromatin (ATAC) and chromatin immunoprecipitation (H3K4me3 and H3K27ac).
Project description:The model describing that aberrant CpG island (CGI) methylation leads to transcription repression of tumor suppressor genes and thereby is implicated in tumor progression has been established in many cancers. However, recent studies indicated aberrantly hypermethylated genes in multiple cancers are already repressed in pre-cancerous tissues despite their promoters are hypomethylated. Here, we hypothesized that the occurrence of CGI promoter hypermethylation in cancers are associated with Polycomb-repressive complex and the associated H3K27me3 mark in pre-cancerous tissues. By using a ChIP-BS-seq technology that examines methylation of the DNA fragments precipitated by the antibodies to histone modifications, we provided direct evidences showing that genes highly enriched with H3K27me3 marks both in cancer and normal cells became aberrantly hypermethylated in CGI promoters in cancer cells in comparison with normal cells. Furthermore, we confirmed that these genes consistently were significantly hypermethylated in TCGA primary cancer in comparison with normal tissues. Thus, we provided direct evidences supporting that the presence of H3K27m3 may serve as a guide to promoter hypermethylation. This will spur future work on epigenetic signature of combined histone and DNA methylation that could define a cancer’s epigenetic abnormalities, therefore helping distinguish subtypes of cancers and aiding future diagnosis and therapeutics of cancers. We applied ChIP-BS technology to examine H3K27me3 marks, which are catalyzed by the SET domain histone methyltransferase EZH2 and have a repressive function with 50bp pair-end sequencing. found H3K27me3 marks were enriched preferentially at CpG islands, (+/-500) transcription start sites (TSSs) and exons in two GC cell lines (BGC-823 and AGS). In YH cells, H3K27me3 marks were only preferentially enriched at CpG islands. In contrast, Hela cells presented a reverse pattern with highest H3K27me3 enrichment in intergenic regions. To confirm this result in Hela cells, we performed two independent replicates of ChIP-Seq and ChIP-BS-seq. Cause of useful was relative small. we still sequenced one 100bp pe reads replicate for H3K4me3 and two replicate for H3K27me3 ChIP-BS-seq.
Project description:The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mistargeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report the surprising finding that RAG1 binds to thousands of sites in the genome of developing lymphocytes, primarily at active promoters and enhancers. The genome has responded by reducing the abundance of "cryptic" recombination signals near sites of RAG1 binding. This depletion operates specifically on the RSS heptamer, with nonamers enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes. RAG1,RAG2 and H3K4me3 ChIP-seq profiles of human thymocytes, mouse thymocytes and preB cells, and Abelson pre-B cell line treated with STI-571