Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates
ABSTRACT: Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation. In lower vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. Bio-CAP was used to identify non-methylated regions of the genome in seven diverse vertebrates (human, mouse, platypus, chicken, lizard, frog and zebrafish) across a number of tissues.
Project description:This study aims to investigate whether the passage of human chromosome 21 through the mouse male germline results in changes in the transcriptional deployment of the exogenous chromosome in the offspring generation. We used the Tc1 mouse model that stably carries almost an entire copy of human chromosome 21 and profiled the genome-wide pattern of non-methylated DNA using BioCAP-sequencing (doi: 10.1093/nar/gkr1207) in the livers of male- and female-germline derived Tc1 mice. This dataset contains only the samples for male-germline derived animals, BioCAP-Seq data for female-germline derived animals have already been deposited in Gene Expression Omnibus with the accession number GSE72208.
Project description:ChIP-seq to identify sigma38 binding sites in wild-type and delta ssrS (6S RNA knockout) strains of E. Coli K-12 MG1655, during stationary phase ChIP-seq using antibody against sigma38 in wild-type and ssrS deletion strain. Two replicates for wild type and one replicate for ssrS deletion.
Project description:Tissue-specific methylation patterns suggest a role for CpG island methylation in differentiation and cell-type-specific gene regulation. We have profiled CpG island methylation in different cells of the immune cell lineage to investigate this role. MBD-affinity purification combined with next generation sequencing was used to analyse CpG island methylation in dendritic cells, B cells, Th1, Th2 and naïve T cells. ChIP-seq was carried out to determine RNA polymerase II binding sites in these cell types and this was compared to the methylation profiles obtained. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ Abstract: We have profiled CpG island methylation in various immune system cell types and related this to gene expression in these cells.
Project description:The genome wide ChIP-on-chip analysis to identify the DNA binding sites for the M.tuberculosis sigma factor Rv3286c (SigF) SigF binding sites were determined by microarray analysis of anti-sigF immunoprecipitated DNA from H37Rv(ΔrsbW/sigF)::pmySigF compared to H37Rv(ΔrsbW/sigF)::pMY769 (both cultured with pristinamycin for 3 days). 4 biological replicates were performed.
Project description:We performed chromatin immunoprecipitation on microarray (ChIP-chip) transcriptional profiling of the SsrB regulator of Salmonella enterica Typhimurium to better characterize its regulon and to identify the DNA-recognition element coordinating its specific interaction at cis-regulatory sites. SsrB, the response regulator of the two component regulatory system SsrA-SsrB encoded within the Salmonella Pathogenicity Island (SPI-2), directs transcriptional activation of the closely associated type three secretion system (T3SS) also encoded at this locus. Immunoprecipitaiton of SsrB translationally fused to a C-terminal FLAG-tag from formaldehyde cross-linked genomic DNA was performed under SPI-2 activating and non-activating conditions. Downstream analysis and experimental work identified specific interactions within SPI-2 at the previously identified ssrA, ssaB, sseA, ssaG, ssaM promoters and identified an additional cryptic promoter driving expression upstream of ssaR. Additionally, a previously unknown SsrB interaction site within ssaE was shown to be the major contributor driving the expression of the downstream genes and not the previously identified interaction site within the intergenic region of ssaE-sseA. Interactions were also identified outside of SPI-2 upstream of other T3SS-associated genes encoded within other genomic islands, and for other uncharacterized genes. Integration of this data with transcriptional microarray work, previously published DNase I footprinting data, bacteria 1-hybrid investigations and comparative genomics analyses of cis-regulatory regions within the orthologous Sodalis symbiosis region 3 (SSR-3) of Sodalis glossinidius enabled identification of a conserved 18bp palindrome which was experimentally validated as being required for transcriptional activation of SsrB dependant genes. SsrB-FLAG immunoprecipitations were performed under SsrB activating and non-activating conditions from formaldehyde cross-linked bacterial lysates. Nine immunoprecipitation reactions were pooled into three replicate samples for the activating condition and three reactions were pooled into one sample for the non-activating control condition. The immunoprecipitated DNA in addition to the non-immunoprecipitated control DNA for each of the four samples were labeled with Cy3 or Cy5 fluorophores respectively and were concurrently hybridized to four arrays on a single slide.
Project description:Somatic cell reprogramming towards induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine, however, the reprogramming process mediated by the traditional defined factors (OSNK) is slow and extremely inefficient. Here we show that a combination of modified reprogramming factors (OySyNyK), in which the transactivation domain of the Yes-associated protein is fused to OCT4, SOX2 and NANOG respectively, could be used to establish a highly efficient and rapid reprogramming system for iPSC generation. We show that the efficiency of OySyNyK-induced iPSCs was up to 100-fold higher than that of induction by traditional OSNK. Moreover we show that the reprogramming by OySyNyK is very rapid (initiated at 24 h versus 5 d by OSNK). Compared with OSNK, we found that OySyNyK factors significantly increased the expression of Tet1/2 at the early stage, which will interact with OSNK factors, and co-occupy pluripotency loci in the genome for somatic cell reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system, but also uncover a novel mechanism by which OSNK factors coordinate with TET proteins to regulate 5hmC-mediated epigenetic control, thereby promoting somatic cell reprogramming. We examined DNA hydroxymethylation progression of reprogramming intermediates. To this end, we profiled the genome-wide 5hmC distribution in MEFs, the reprogramming intermediates at different stages induced by either the OSNK or OySyNyK methods, and iPSCs using the chemical capture approach
Project description:Despite serving as a central experimental technique in epigenetics research, chromatin immunoprecipitation (ChIP) suffers from several serious drawbacks: it is a relative measurement untethered to any external scale that obviates fair comparison amongst experiments; it employs antibody reagents that have differing affinity and specificity for target epitopes, which are in turn variable in abundance; and it is frequently not reproducible. To address these problems, we developed internal standard calibrated ChIP (ICeChIP), a method of spiking a native chromatin sample with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and revealing a correlation between the apparent symmetry of H3K4me3 in promoter nucleosomes and gene expression. Direct in situ assessment of immunoprecipitation accommodates for a number of experimental pitfalls, and provides a critical examination of untested assumptions inherent in conventional ChIP. Examination of spiked-in semi-synthetic nucleosomes in ICeChIP-seq experiments performed for HEK293, mESC E14 and DM S2 cell line
Project description:While the regulation of metabolic enzymes by oncogenic drivers or tumor suppressors has been intensively studied over recent years, our understanding of how metabolic processes directly regulate cell proliferation has remained fragmentary. Here we show how the alteration of metabolism directly affects cell cycle progression in cancer cells. We found that activation of the nuclear receptor peroxisome-proliferation activated receptor gamma (PPARγ), a transcriptional master regulator of lipid metabolism, inhibits the growth of lung adenocarcinoma cells by triggering a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell cycle arrest. Both of these changes can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids. Thus, we provide a mechanism that directly links metabolic changes to inhibition of cancer cell cycle progression by altering ROS levels. We generated PPARG-LAP BAC transgenic NCI-H2347 and NCI-H1993 cell lines using the BAC-transgenesis approach. Cells at 80% confluency (~1-1.5x107) were cross-linked with 1% formaldehyde for 10 minutes at 37°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4°C, the cell pellets were resuspended in 100 μl ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 12 minutes). The sheared chromatin with a fragment length of ~200 – 600 bp) was centrifuged at 10,000 g for 10 minutes at 4°C). 100 μl of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4°C overnight with 6 μg/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitations were carried out by incubating with 40 μl pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 μl ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (Qiagen) and eluted into 30 μl H2O according to the manufacturer’s protocol after treatment with RNase A and Proteinase K.
Project description:Mono-methylation of histone H3 on lysine 4 (H3K4me1) and acetylation of histone H3 on lysine 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and enhancers. Although in yeast all H3K4 methylation patterns including H3K4me1 are implemented by Set1/COMPASS, there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of mammalian Mll3/4, can function as a major H3K4 mono-methyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac in various tissues. Assays with the cut wing margin enhancer imply a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrates that Trr is required for H3K4me1 and H3K27ac on chromatin signatures that resemble the histone modification patterns described for enhancers. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 mono-methyltransferase Trr, and the H3K27 demethylase, UTX, cooperate to regulate the transition from inactive/poised to active enhancers. ChIP-seq of Trr, LPT, UTX in Drosophila S2 Cells. ChIP-seq of H3K4me1, H3K4me3, H3K27ac, H3K27me3 in WT and Trr knock-down Drosophila S2 cells. ChIP-seq of H3K4me1, H3K27me3 in LPT knock-down Drosophila S2 cells. ChIP-seq of LPT and UTX in Trr knock-down Drosophila S2 cells. ChIP-seq of H3K4me1 and H3K27me3 in MLL1(+/+), MLL1(-/-), MLL3(+/+), and MLL3(-/-) Mouse Embryonic Fibroblasts (MEFs).
Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4°C, the cell pellets were resuspended in 100 μl ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 – 600 bp) was centrifuged at 20,000 g for 15 minutes at 4°C). 100 μl of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4°C overnight with 6 μg/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 μl pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 μl ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 μl H2O according to the manufacturer’s protocol after treatment with RNase A and Proteinase K.