Expression changes and chromatin architecture modifications in WBS cells.

<p>Changes in expression and chromatin structure in WBS (GM13472) versus Ctrl (GM07006) cells. Changes in histone marks are presented as the log2-fold ratio between WBS and Ctrl cells. Statistical analysis was performed by a 2-sample t-Test. Values in italics are not statistically different.</p><p>AREL  =  average relative expression level, BDL  =  below detection line, NS  =  no regions within gene were defined as significantly changed,</p><p>*most significant block according to SICER within the gene (FDR<1%).</p

Extensive chromatin interactions of seven genes flanking the WBSCR on human chromosome 7 (HSA7) in cells from a healthy control individual.

<p>(<b>A</b>) Windowed and normalized 4C signal of each of the seven viewpoints along the entire HSA7. The black ticks below each graph show the location of the Bricks (Blocks of Regulators In Chromosomal Kontext). The gene density across HSA7, as well as the windowed profiles of H4K20me1 and H3K27me3 marks in the same cell line are shown below. Some examples of strong correlation of gene-dense regions and high density of H4K20me1 marks with highly interacting regions are highlighted in blue. The mapping of the assessed genes/viewpoints and of the WBSCR is indicated at the bottom. The red box specifies the close-up shown in panel B. (<b>B</b>) Close-up of the windowed 4C signal of the seven viewpoints around the WBSCR for the region indicated with a red box on HSA7 (top panel). The windowed 4C signal is shown in grey, while the profile corrected 4C signal (after removal of the highly interacting neighboring background signal) is overlaid in black. The position of all genes are displayed at the bottom, and the mapping of the assessed viewpoints is highlighted by red and green arrows indicating if the corresponding genes are down- or upregulated in cells from WBS patients, respectively. Black arrows underscore the mapping of the viewpoint that is not modified in gene expression (<i>ZNF107</i>) and the newly identified interacting partners <i>AUTS2</i> and <i>CALN1</i>. The location of the WBSCR is indicated by a purple horizontal bar. A close-up of interactions within this WBSCR is provided in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079973#pone.0079973.s004" target="_blank">Figure S4</a></b>.</p

Gene expression associated with EspR binding.

<p>(<b>A</b>) Growth of H37Rv::pMYespR without (−P) or with (+P) pristinamycin IA. Growth was monitored at the designated time points measuring OD₆₀₀. (<b>B</b>) Immunoblot analysis of EspR expression in the absence (−P) or presence (+P) of pristinamycin IA after 3 days growth using rat polyclonal antibodies specific for EspR. Equivalent amounts of total protein lysates were loaded. (<b>C</b>, <b>D</b>) Quantitative RT-PCR analysis of relative mRNA levels extracted from H37Rv::pMYespR cells treated with 0 µg/ml or 2 µg/ml of pristinamycin using primers specific for the following regions: (<b>C</b>) the <i>espR</i> coding region (annealing to both endogenous and vector copies of <i>espR</i>) or to the 5′-UTR region of <i>espR</i> (<i>PespR</i>) (specific to endogenous <i>espR</i>). (<b>D</b>) The coding regions of genes related to the top 10 EspR binding peaks as obtained in ChIP-Seq experiments (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002621#ppat-1002621-t001" target="_blank">Table 1</a>). Relative gene expression was normalized against <i>sigA</i> and displayed as fold-induction (log-2 scale) relative to the untreated sample (0 µg/ml pristinamycin). Shown are the mean ± s.d. of a minimum of duplicate measurements from the average of three independent experiments. Statistical significance was evaluated using Students T-test. * indicates P<0.05, ** P<0.01 and *** P<0.001.</p

Top 10 EspR binding loci from ChIP-Seq.

<p>Top 10 EspR binding loci from ChIP-Seq.</p

Binding of EspR to the <i>Mtb</i> chromosome.

<p>(<b>A</b>) Pie chart of EspR-binding peaks annotated relatively to the nearest gene translation start using the <i>ChIPpeakAnno</i> package <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002621#ppat.1002621-Zhu1" target="_blank">[38]</a>. (<b>B</b>) Bar chart displaying the percentage of genes of the EspR regulon compared to the <i>Mtb</i> H37Rv genome belonging to functional categories defined in the TubercuList database (<a href="http://tuberculist.epfl.ch/" target="_blank">http://tuberculist.epfl.ch/</a>). (<b>C</b>) Most significant motif derived from ChIP-Seq binding sequences returned by the MEME tool <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002621#ppat.1002621-Bailey1" target="_blank">[19]</a>. The height of each letter represents the relative frequency of each base at different positions in the consensus.</p

Proportion of different genomic elements in the captured samples with and without COT-I DNA as compared to their relative abundance in the human genome.

<p>Reads were mapped to the reference human genome using BLAT, allowing 2 mismatches and 1 indel with a minimal perfect match of 30 bp.</p

SNPs in the coding sequences of the genes from the target region.

<p>SNPs in the coding sequences of the genes from the target region.</p

Distribution of sequence features of regions and corresponding probes with low (<3 fold) and normal (15-29 fold) sequence coverage.

<p>A,B) Length of the regions/probes. C,D) Percentage of low complexity DNA inside the regions/probes. E,F) GC content of regions/probes. G) Hybridization temperature of the probes.</p

Enrichment of target regions.

<p>All sequencing tags that map uniquely to the reference human genome are displayed in blue. The height of peaks corresponds to the number of tags mapping to the same location. Red circles represent genomic areas plotted on the array. The image was generated using Genome Graphs (<a href="http://genome.ucsc.edu/cgi-bin/hgGenome" target="_blank">http://genome.ucsc.edu/cgi-bin/hgGenome</a>).</p

Number of sequence reads extracted from intensity files using Illumina and Rolexa analysis.

<p>Number of sequence reads extracted from intensity files using Illumina and Rolexa analysis.</p