Project description:Study of the mechanisms of RecB mutant terminus DNA loss in Escherichia coli. FX158: WT MG1655 FX35: recB- FX37: ruvAB- FX51: matP- MIC18: recB- sbcD- sbcC- MIC20: recB- ruvAB- MIC24: matP- recB- MIC25: recA- recB- MIC31: sbcB- sbcD- MIC34: recA- recD- MIC40: linear chromosome MIC41: linear chromosome recB- MIC42: matP- ftsKC- MIC43: matP- ftsKC- recB- MIC48: recA- Cells were grown in M9 minimal medium supplemented with 0.4 % glucose to exponential phase (0.2 OD 650 nm). Chromosomal DNA was extracted using the Sigma GenElute bacterial genomic DNA kit. 5 μg of DNA were used to generate a genomic library according to Illumina's protocol. The libraries and the sequencing were performed by the High-throughput Sequencing facility of the I2BC (http://www.i2bc.paris-saclay.fr/spip.php?article399&lang=en, CNRS, Gif-sur-Yvette, France). Genomic DNA libraries were made with the ‘Nextera DNA library preparation kit’ (Illumina) following the manufacturer’s recommendations. Library quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit (Agilent technologies). Libraries were pooled in equimolar proportions. 75 bp single reads were generated on an Illumina MiSeq instrument, using a MiSeq Reagent kit V2 (500 cycles) (Illumina), with an expected depth of 217X. An in-lab written MATLAB-based script was used to perform marker frequency analysis. Reads were aligned on the Escherichia coli K12 MG1655 genome using BWA software. Data were normalized by dividing uniquely mapping sequence reads by the total number of reads. Enrichment of uniquely mapping sequence reads in 1 kb non-overlapping windows were calculated and plotted against the chromosomal coordinates.
Project description:We report the application of single molecule-based sequencing technology for high-throughput mapping of CFP1, RNA polymerase II and H3K4me3 in mouse brain. By obtaining sequence from chromatin immunoprecipitated DNA, we generated genome-wide binding / chromatin-state maps for mouse brain. We find a good correlation between CFP1 binding and H3K4me3 consistent with it presence in the SetD1 histone methylatransferase complex. Mapped RNA polymerase II colocalised with the majority of CFP1 / H3K4me3 positive CpG islands but not all. This study provides a comprehensive characterisation of the genome wide distribution of a previously uncharacaterised DNA binding factor and suggests a link between DNA base composition and chromatin state. Examination of H3K4me3, RNA PolymeraseII and CFP1 in mouse brain.
Project description:Discovery of the genome-wide location of proteins using ChIP-Seq has allowed global mapping of the key transcription factors and chromatin regulators that control gene expression programs in various cells. Many DNA-associated processes are targeted for disease therapy. This study investigates the functions of small molecule therapeutics that target DNA-associated processes involved with CDK9 and BRD4. Genomic DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. ChIP was performed using an antibody against Brd4, RNA Polymerase II, Med1, H3K27ac, and CDK8 as well as whole-cell extract (WCE) DNA controls
Project description:We report the application of single molecule-based sequencing technology for high-throughput mapping of CFP1, RNA polymerase II and H3K4me3 in mouse brain. By obtaining sequence from chromatin immunoprecipitated DNA, we generated genome-wide binding / chromatin-state maps for mouse brain. We find a good correlation between CFP1 binding and H3K4me3 consistent with it presence in the SetD1 histone methylatransferase complex. Mapped RNA polymerase II colocalised with the majority of CFP1 / H3K4me3 positive CpG islands but not all. This study provides a comprehensive characterisation of the genome wide distribution of a previously uncharacaterised DNA binding factor and suggests a link between DNA base composition and chromatin state.
Project description:We present a long-read, single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5-hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long single-molecule read-length of 100 kbp-1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data. In addition, optical 5-hmC mapping shows a strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression.
Project description:In Escherichia coli crosstalk between DNA supercoiling, nucleoid-associated proteins and major RNA polymerase σ initiation factors regulates growth phase-dependent gene transcription. We show that the highly conserved spatial ordering of relevant genes along the chromosomal replichores largely corresponds both to their temporal expression patterns during growth and to an inferred gradient of DNA superhelical density from the origin to the terminus. Genes implicated in similar functions are related mainly in trans across the chromosomal replichores, whereas DNA-binding transcriptional regulators interact predominantly with targets in cis along the replichores. We also demonstrate that macrodomains (the individual structural partitions of the chromosome) are regulated differently. We infer that spatial and temporal variation of DNA superhelicity during the growth cycle coordinates oxygen and nutrient availability with global chromosome structure, thus providing a mechanistic insight into how the organization of a complete bacterial chromosome encodes a spatiotemporal program integrating DNA replication and global gene expression.
Project description:We report the application of single molecule-based sequencing technology for high-throughput genom-wide mapping of MeCP2 binding and DNA methylation in mouse brain and cerebellum respectively. We find a good correlation between MeCP2 occupancy and methyl-CpG density and depletion of MeCP2 binding at CpG Islands, the majority of which are constitutively hypomethylated. This study provides a comprehensive characterisation of the genome wide distribution of MeCP2. Examination of MeCP2 occupancy in mouse brain and the distribution of methylation in mouse cerebellum.
Project description:Mapping the occupancy of FNR, HNS, and IHF throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anerobic growth conditions. We also mapped the binding of the M-CM-^_ subunit of RNA Polymerase under both aerobic and anaerobic growth conditions. As a control, we also performed ChIP-chip on FNR in a M-bM-^HM-^Ffnr mutant strain of Escherchia coli MG1655 K-12. We also examined FNR immunoprecipitation at various FNR concentrations using IPTG and Ptac::fnr (PK8263). The M-bM-^HM-^Fhns/M-bM-^HM-^FstpA strains were also used. Descirbed in the manuscript Genome-scale Analysis of E. coli FNR Reveals the Complexity of Bacterial Regulon Structure Mapping of occupancy of FNR, NNS, IHF and M-CM-^_ of RNAP in the genome of Escherchia coli MG1655 K-12 under aerobic or anaerobic growth conditions. Immunoprecipitated DNA compared to INPUT for each sample.