Project description:Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 show an increase of relative 5meC levels at the TSS and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. Mouse germ-cells ChIPseq
Project description:DNA Double Strands Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the role of senataxin, a RNA:DNA helicase involved in the regulation of transcription and the maintenance of genome integrity, at sites of DNA Double Strand Breaks. We performed ChIP-Seq mapping of senataxin before and after damage, genome-wide DNA:RNA hybrids (DRIP) mapping before and after damage. We also performed RNA-Seq and RNA pol II mapping (total and phosphorylated on serine 2 of the CTD) by ChIP-Seq in undamaged cells to discriminate between damage in active versus inactive regions.
Project description:iMUT-seq profiles double-strand break (DSB) induced mutations at extremely high sensitive with single nucleotide resolution, allowing for the quantification of all mutation types, including chromosomal rearrangements, around endogenous DSBs. AID-DIvA cells are treated with or without 4-hydroxytamoxifen to induce DSBs, then with IAA to induce degradation of the DSB inducing AsiSI enzyme and are then incubated to allow the breaks to be repaired. The DSB loci are then PCR amplified from the genomic DNA and subjected to NGS to provide high depth mutation profiling. All samples are either untreated (m/0) or treated (p/4)with OHT and have three independent biological replicates, with the exception of ATRi which has 2 replicates and controlsi which has 6 replicates. This is because the siRNA were divided into two batches to create replicates 1-3 and 4-6 and each batch contained a controlsi sample for reference causing it to be in all 6 replicates.
Project description:Acute myeloid leukemia (AML) is characterized by a block in myeloid differentiation the stage of which is dependent on the nature of the transforming oncogene and the developmental stage of the oncogenic hit. This is also true for the t(8;21) translocation which gives rise to the RUNX1/ETO fusion protein and initiates the most common form of human AML. To understand the molecular principles governing this differential action, we used the differentiation of mouse embryonic stem cells expressing an inducible RUNX1/ETO protein into blood cells as a traceable model combined with genome-wide analyses of transcription factor binding and gene expression. We found that RUNX1/ETO interferes with both the activating and repressive function of its normal counterpart, RUNX1, at early and late stages of blood cell development. However, the response of the transcriptional network to RUNX1/ETO expression is stage-specific, highlighting the molecular mechanisms determining specific target cell expansion after an oncogenic hit. High throughput sequencing data have been used to study RUNX1/ETO role in hematopoietic system
Project description:A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4-binding and Sox2-binding sites. However, little is known regarding the components of the complex. In this study, we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Sall4 knockdown led to differentiation of ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4- and Sox2-double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4-triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2-double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells, together with Oct-3/4 and Sox2. ChIP-on-chip experiments using anti-Sall4 or anti-Sox2 antibody 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:We characterize the acetylation of H3K122 for the first time. Towards this we mapped the genomic distribution of H3K122Ac, identified the enzyme introducing H3K122Ac, and addressed the functional contribution H3K122Ac to transcription. We found that H3K122Ac is associated with chromatin marks and genomic regions associated with active transcription and is catalysed by p300/CBP and can be regulated by estrogen signaling in MCF-7. Moreover H3K122Ac stimulates transcription as dermined by in vitro transcription assays ChIP seq study
Project description:Chromatin immunoprecipitation of Sir3 applied with tilling array chip ( ChIP on chip of Sir3) analysis demonstrated that Changes of conventional and dynamical Genome-wide localization of Sir3 in Saccharomyces cerevisiae during different growth stages comparison the genome-wide localization of Sir3 of logarithmic phase younger cells vs of stationary phase older cells
Project description:O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. chromatin affinity-precipitation (ChAP) method was used to detect the chromatin fragments at which small molecules interact with binding partners. Chromatin immunoprecipitation of Sir3 and of Sir2, respectively, applied with tilling array chip (ChIP on chip of Sir3 and of Sir2, respectively) and Chromatin affinity-precipitation of AAR applied with tilling array chip (ChAP on chip of AAR ) analysis demonstrated that an extended spreading of Sir3 and of AAR, but not Sir2 in Saccharomyces cerevisiae Ysa1 deleted cells compared with those in wild type cells Comparison the distributions of Sir3, of Sir2 and of AAR on silent heterochromatin of Ysa1 deletion cells vs those of wild type cells