[Show abstract][Hide abstract] ABSTRACT: OmpR is a multifunctional DNA binding regulator with orthologues in many enteric bacteria, that exhibits classical regulator activity as well as nucleoid-associated-protein-like characteristics. In the enteric pathogen Salmonella enterica, using chromatin immunoprecipitation of OmpR::FLAG and nucleotide sequencing, 43 putative OmpR-binding sites were identified in S. enterica serovar Typhi, 22 of which were associated with OmpR-regulated genes. Mutation of a sequence motif (TGTWACAW) that was associated with the putative OmpR-binding sites abrogated binding of OmpR::6xhis to the tviA upstream region. A core set of 31 orthologous genes were found to exhibit OmpR-dependent expression in both S. Typhi and S. Typhimurium. S. Typhimurium-encoded orthologues of two divergently transcribed OmpR regulated operons (SL1068-71 and SL1066-67) had a putative OmpR binding site in the inter-operon region in S. Typhi, and were characterized using in vitro and in vivo assays. These operons are widely distributed within Salmonella enterica but absent from the closely related Escherichia coli. SL1066 and SL1067 were required for growth on N-acetyl muramic acid as a sole carbon source. SL1068-71 exhibited sequence similarity to sialic acid uptake systems and contributed to colonization of the ileum and cecum in the streptomycin pretreated mouse model of colitis.
Full-text · Article · Nov 2012 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Proportional relationship between CpG density and H3K4me3 at Human CGIs. Box plots of H3K4me3 reads per base (averaged across 500 bp with a 100 bp slide) spanning 5 kb of all human CGIs at different CpG densities (CpGs per 100 bp). CpG density categories applied are ≤5, 5–6, 6–7, 7–8, 8–9 and >9 CpGs per 100 bp, arranged in ascending order from top to bottom. Box plots represent the distribution of the central 50% of the data (filled box) and the median (black bisecting line). The numbers of islands in each category (n) is noted in parenthesis.
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[Show abstract][Hide abstract] ABSTRACT: Global scatter plots reveal a reciprocal relationship between CAP- and MAP-seq data for human sperm, blood, and cerebellum. Scatter plots display pairwise comparisons of CAP- and MAP-seq data for every contiguous 1 kb window in the human genome using normalised data for human sperm, blood and cerebellum. Plots are represented as for Figure S2.
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[Show abstract][Hide abstract] ABSTRACT: CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are "orphans" that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development.
[Show abstract][Hide abstract] ABSTRACT: Tumour-specific CGI methylation associated with PDX1. MAP-seq profiles (red) for five colon mucosa (C3, C5, C6, C9 and C10) and five matched colorectal tumour (T3, T5, T6, T9 and T10) biopsy samples for human chr13: 27,325,000–27,402,000. CGIs (blue bars) and sites of hES H3K27 trimethylation (hES H3K27me3; black bars;  are represented).
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[Show abstract][Hide abstract] ABSTRACT: Preliminary characterisation of Human and Mouse CAP-seq results. (A) Sperm CAP-seq read density profiles (blue) for human and mouse sperm generated by washing DNA bound to the CXXC column with 600 mM NaCl prior to elution. CpG density (black; 300 bp windows with a 10 bp slide) at 4 human and mouse syntenic chromosomal locations is shown below the read profiles. Genes (Refseq) are annotated below the CAP-seq profiles with those mapped to the positive and negative strand displayed above and below the chromosome (grey line) respectively. The CpG density of 5 CpGs per 100 bp (dashed black line) is indicated for reference. Mouse regions assessed by bisulfite sequencing are indicated (bisulfite; grey bars). (B) Bisulfite sequencing of four putative mouse CGI island promoters. Open circles represent unmethylated CpG sites. Each column represents a single PCR amplicon and horizontal lines represent single sequenced DNA clones. Vertical strokes represent the relative CpG position within each amplicon. (C) Histogram depicting the CpG observed/expected (o/e) values for all human (pink) and mouse (black) CGIs identified by CAP with washing at 600 mM NaCl. Statistical significance (**) was determined using a Welch Two Sample t-Test and CpG o/e values of 0.21 (broken red line; human genome average) and 0.6 (broken black line; standard CGI prediction parameter) are indicated. (D) Sperm CAP-seq read density profiles (blue) for mouse sperm generated by washing with the optimised NaCl concentration (560 mM) in comparison with CpG density (black; 300 bp windows with a 10 bp slide).
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[Show abstract][Hide abstract] ABSTRACT: Pairwise comparisons of MAP-seq data reveal consistent tumour-specific methylation. Scatter plots displaying pairwise comparisons of MAP-seq data for every colon (C) and colorectal tumour (T) sample screened by MAP-seq. Data represents the mean sequence depth for every 1 kb window in the human genome. Data is presented as for Figure S2.
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[Show abstract][Hide abstract] ABSTRACT: Pairwise analysis of mouse CAP-seq data. Scatter plots of CAP-seq data representing the mean sequence read depth for every contiguous 1 kb window in the mouse genome. Each pairwise comparison was assessed by calculating a Pearson correlation coefficient, which is presented above each plot. Tissue and replicate status for pairwise comparisons are noted above and to the left of the plots.
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[Show abstract][Hide abstract] ABSTRACT: Characterisation of MAP enrichment. Histograms representing the CpG density of MAP-enriched genomic loci in human (hMAP) and mouse (mMAP). The vertical dashed red line represents the lower tenth percentile of the data indicating that the majority of characterised MAP enriched DNA fragments have a CpG density of at least 1 and 1.3 CpGs per 100 bp in human and mouse respectively.
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[Show abstract][Hide abstract] ABSTRACT: Determination of specificity of antibodies used in this study by dot blot analysis. Antibodies raised against histone modifications were each hybridized to a panel of relevant methyl-modified or acetyl modified peptides of histone H3 and histone H4 and their unmodified forms (see also Materials and Methods). Antibodies (Ab) and peptides (Pep) used are shown on the left and top of each panel respectively. Images in each panel are composites of different hybridizations denoted by the black lines dividing the sections of the panels. a. H3K9 methyl modifications. b. H3K36 methyl modifications. c. H3K27 methyl modifications. d. H3K79 methyl modifications. e. H3K9, 18, 27 acetyl modifications. f. H3K4 methyl modifications. g. H4K16 acetyl modification. In all panels, the results are shown for peptides spotted onto the immunoblot at a concentration of 25 ng/µl.
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[Show abstract][Hide abstract] ABSTRACT: Concordance of histone modification exon-intron marking biases between cell types. Table shows level of agreement in exon-intron marking biases for K562, U937 cell lines and CD14+ primary monocytes. Histone modifications are shown in columns along the top. Pairwise comparisons between cell lines are shown along the left-hand side. Each black box shows when the exon-intron marking bias for a histone modification (either an exon or intron bias) is in agreement between two cell types for either expressed (ON) (green boxes) or non-expressed (OFF) (red boxes) genes. White boxes represent discordance. Concordance was scored based on the data presented in Supplementary Figure S8. Overall concordance between all three cell types is shown in the bottom row of the table.
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[Show abstract][Hide abstract] ABSTRACT: Protocol for Sequential-ChIP (Seq-ChIP) used in this study. All reagents (including suppliers and catalogue numbers) used for Seq-Chip assays are shown at the top of the protocol. For details of hybridization of these samples to Sanger Institute tiling microarrays, refer to our previous publications1,2 1. Koch, C.M. et al. The landscape of histone modifications across 1% of the human genome in five human cell lines. Genome Res 17, 691-707 (2007). 2. Bruce, A.W., Lopez-Contreras, A., Flicek, P., Down, T.A., Dhami, P., Dillon, S.C., Koch, C.M., Langford, C.F., Dunham, I., Andrews, R.M. and Vetrie, D. Functional diversity for REST (NRSF) is defined by in vivo binding affinity hierachies at the DNA sequence level. Genome Res 19, 994-1005 (2009).
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[Show abstract][Hide abstract] ABSTRACT: Histograms show the levels of sequential-ChIP-chip enrichments for combinations of histone modifications (those described in Figure S9) spanning typical canonical/alternatively-spliced exons (CE and AE respectively) and introns (I) of expressed genes [n = 85, canonical exons:alternatively-spliced exons∶introns = 145∶68∶221 (5′ ends) or 796∶166∶976 (gene bodies)]. Blue bars show ChIP-chip enrichments after 1° antibody and red bars show ChIP-chip enrichment after the 2° antibody. Error bars are 95% confidence intervals. In all panels, histone modification ChIP-chip enrichment levels are expressed as mean Z-scores.
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[Show abstract][Hide abstract] ABSTRACT: Histone modification and chromatin accessibility (FAIRE) patterns track exons and introns across gene bodies of non-expressed genes and at 3′ ends of expressed genes. Histograms show the mean levels of ChIP-chip enrichments for histone modifications or FAIRE values (Z-scores) spanning the first ten exons and nine introns or last five exons and four introns of consensus genes (hypothetical gene structures are shown at the bottom of each panel of the figure). Data is derived from ENCODE regions in the K562 and U937 cell lines and CD14+ primary monocytes. a. Five histone modifications across first 10 exons and 9 introns of non-expressed genes with histone normalization (n = 92, exons∶introns = 393∶136). b. 14 histone modifications and FAIRE levels across last five exons and four introns of expressed genes with histone normalization (n = 268, exons∶introns = 848∶226). c. Five histone modifications across first ten exons and nine introns of non-expressed genes without histone normalization (n = 92, exons∶introns = 393∶136). d. 14 histone modifications and FAIRE levels across last five exons and four introns of expressed genes without histone normalization (n = 268, exons∶introns = 848∶226). Median P-values obtained from bootstrapping for exons and introns across all patterns shown were <1.0×10−15 (panel a), <1.0×10−15 (panel b), <1.0×10−15 (panel c), and <1.0×10−15 (panel d). Median P-values obtained for pair-wise t-tests between adjacent exon-intron pairs were 3.15×10−2 (panel a), 4.40×10−4 (panel b), 1.35×10−2 (panel c), and 1.49×10−7 (panel d).
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[Show abstract][Hide abstract] ABSTRACT: RNA polymerase II (Pol II) occupancy levels are not accounted for by nucleosome distributions. Histograms show the mean levels of ChIP-chip enrichments (Z-scores) for Pol II and histones across exons and introns of consensus expressed (ON) (green) or non-expressed (OFF) genes (red). a. Data derived from ENCODE regions in the K562 cell line: expressed genes (n = 76, exons/introns = 707/287), non expressed genes (n = 25, exons/introns = 133/76). b. Data derived from U937 cell line: expressed genes (n = 88, exons/introns = 797/325), non expressed genes (n = 20, exons/introns = 123/73). All exonic levels were determined for canonical exons only. Error bars are 95% confidence intervals.
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[Show abstract][Hide abstract] ABSTRACT: Expression levels of 9922 genes across the human genome in the K562 cell line. These genes gave consistent MAS5 values (all A or all P) in four bioreplicate Affymetrix GeneChIP® expression experiments and all had been assigned ENSEMBL identifiers. List shows their ENSEMBL IDs, MAS5 call and ranking based on RMA values.
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