Manhattan plot of interaction p-values derived from the MESA genome-wide interaction analysis.

<p>Linear regression models, including main effects for SNP and asthma, and an interaction term for SNP × asthma, were used. Models were also adjusted for age, sex, and the first principal component. The red (upper) line represents the genome-wide significant p-value (0.05/721,893 = 6.93×10⁻⁸). The blue (lower) line represents the genome-wide suggestive p-value of 5×10⁻⁷.</p

Forest plot showing the association of rs2107212 with obesity in MESA, FHS and meta-analysis.

<p>Asthma status-specific odds ratios (OR) of being obese for rs2107212 were calculated using logistic regression models adjusted for age, gender and the first principal component in MESA and FHS respectively. The minor allele A is the “risk” allele, and an additive genetic model was used. Meta-analysis was performed under a fixed effect model since the heterogeneity tests were not significant. Boxes indicate group-specific OR point estimates, and lines indicate the respective 95% confidence interval (CI). Diamonds indicate meta-analysis OR and 95% CI.</p

Association of rs2107212 genotypes and BMI, stratified by asthma status in MESA (A) and FHS (B), respectively.

<p>Body mass index (BMI, kg/m²) was compared across subjects carrying the AA, AG or GG genotypes for rs2107212 in asthmatics and non-asthmatics. Numbers of subjects are shown in each bar. Data are represented as the mean ± SEM. Significance was tested using Welch’s t-test (*p<0.05, **p<0.01 and ***p<0.001).</p

Association of rs2107212 genotypes and BMI change by asthma status in FHS.

<p>A: For a subset of FHS subjects who had BMI and asthma information at both exam 2 (1979) and exam 8 (2005) (n = 983), the means of BMI at both time points are shown according to the rs2107212 genotypes AA/AG and GG, stratified by asthma status. B: Net BMI change (BMI value in 2005—BMI value in 1979) with rs2107212 genotypes AA/AG and GG stratified by asthma status is shown. The numbers of subjects are shown in each bar. Data are represented as the mean ± SEM. *p<0.05 under Welch’s t-test.</p

Stage and Gene Specific Signatures Defined by Histones H3K4me2 and H3K27me3 Accompany Mammalian Retina Maturation In Vivo

<div><p>The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By <em>in silico</em> analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue <em>in vivo</em>.</p> </div

Genes with the same expression patterns show different histone signatures in retina.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes up-regulated during retina maturation. (<b>B</b>) H3K4me2 and H3K27 accumulation in examples of genes down-regulated during retina maturation. Closed arrowheads show TSS of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s006" target="_blank">Tables S4</a> &S5). (<b>C</b>) Cluster analysis of H3K4me2 and H3K27me3 occupancy around TSS (+/−2.5 Kb) at all developmental stages for the genes upregulated in mature retina. Tree-view shows 4 clusters (C1–C4) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). (<b>D</b>) Same analysis as in (<b>C</b>) for the genes downregulated in mature retina. Cluster analysis of H3K4me2 and Tree-view shows 3 clusters (C1′–C3′) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). For <b>A</b> and <b>B</b> y-axis is the number of reads in a 100 bp interval. For <b>C</b> and <b>D</b>, (upper panels) y-axis is the normalized occupancy, or the number of reads for a given histone modification in an interval +/−2.5 kb around the TSS of given gene, normalized for the total number of mapped reads in given experiment.</p

Gene-wide coverage by H3K4me2 and H3K27me3 as a landscape for maintenance of gene activities.

<p>(<b>A</b>) Comparison of average normalized occupancy per 1 Kb of H3K4me2 (upper panels) and H3K27me3 (lower panels) between promoter region (from −2.5 Kb to TSS) and whole gene body (from TSS to TES) for the clusters from Fig. 4c, C1 to C4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s014" target="_blank">Table S12</a>). (<b>B</b>) Same comparison as in (<b>A</b>) for the clusters from Fig. 4d, C1′ to C3′ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s015" target="_blank">Table S13</a>). (<b>C</b>) Same comparison as in (<b>A</b>) for genes specific expression in retina but not photoreceptors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s016" target="_blank">Table S14</a>). Y-axis is average occupancies for genes in given cluster, where occupancy is (number of reads in an interval×5,000,000 reads×1 kb)/(total number of reads in experiment × length of genome interval in kb).t-Test for two-sample assuming equal variances was done to compare occupancy on promoter and gene body for each stage and * marks those with statistically significant differences: (*) 0.05><i>P</i>>0.01, (**) 0.01><i>P</i>>0.001, (***) <i>P</i><0.001 (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a>).</p

Genes with the same expression patterns show different histone signatures in retina.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes up-regulated during retina maturation. (<b>B</b>) H3K4me2 and H3K27 accumulation in examples of genes down-regulated during retina maturation. Closed arrowheads show TSS of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s006" target="_blank">Tables S4</a> &S5). (<b>C</b>) Cluster analysis of H3K4me2 and H3K27me3 occupancy around TSS (+/−2.5 Kb) at all developmental stages for the genes upregulated in mature retina. Tree-view shows 4 clusters (C1–C4) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). (<b>D</b>) Same analysis as in (<b>C</b>) for the genes downregulated in mature retina. Cluster analysis of H3K4me2 and Tree-view shows 3 clusters (C1′–C3′) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). For <b>A</b> and <b>B</b> y-axis is the number of reads in a 100 bp interval. For <b>C</b> and <b>D</b>, (upper panels) y-axis is the normalized occupancy, or the number of reads for a given histone modification in an interval +/−2.5 kb around the TSS of given gene, normalized for the total number of mapped reads in given experiment.</p

Changes in histone modifications during retina maturation.

<p>(<b>A</b>) Immunofluorescence microscopic images of sagittal sections of developmental mouse retina tissue array stained with anti-H3K4me2 (<b>A</b>, green, upper panels) and anti-Rhodopsin (<b>A</b>, red, upper panels), anti-H3K27me3 (<b>A</b>, green, lower panels) and anti-SVP38 (<b>A</b>, red, lower panels), and counterstained with Hoechst in cell nuclei (<b>A,</b> blue). ONBL, outer neuroblast layer; INBL, inner neuroblast layer; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar represents 25 µm. (<b>B</b>) H3K4me2 labeling with high magnification for ONL from adult retina (Scale bar = 6 µm). (<b>C</b>) H3K27me3 labeling with high magnification for ONL from adult retina (Scale bar = 6 µm). (<b>D</b>) Averaged and normalized intensity profiles for fluorescence of each specific antibody or Hoechst staining across the nuclear centers (e.g. white bars in <b>B</b> and <b>C</b>) of rod photoreceptor nuclei (n = 5). (<b>E</b>) Adult retina outer nuclear layer labeled with an antibody recognizing Crx. (<b>F</b>) Control labeling of adult ONL showing lack of staining with secondary antibody alone. (<b>G</b>) Averaged and normalized intensity profile for Crx labeling (green) and Hoechst (blue) across rod nuclear centers (bar in <b>E</b>). (<b>H, I</b>) Confocal images with high magnification for RGCL from adult retina (Scale bar = 15 µm). Cells marked * or # show distinct cellular distribution with H3K4me2 antibody (<b>H</b>, green) and a′ or b′ show distinct cellular distribution with H3K27me3 antibody (<b>I</b>, green).</p

Histone modifications show an association with genes and their TSS.

<p>(<b>A–B</b>) Histone H3K4me2 (<b>A</b>) and H3K27me3 (<b>B</b>) modification patterns at part of chromosome 19. Peaks of normalized sequenced tags from ChIP-Seq analysis of mouse retina at 4 developmental stages (E17, PN1, PN7, and PN15) were mapped to mouse genome (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a>). Scale bar represents 20 Mb. (<b>C–D</b>) High-resolution view of the part of chromosome 19 patterns shown in (<b>A</b>) and (<b>B</b>) with gene poor or rich regions. Scale bar represents 1 Mb. Y-axis in <b>A–D</b> represents the number of reads in a 100 bp interval. (<b>E–F</b>) Normalized tag counts of histone modifications by H3K4me2 (<b>E</b>) and H3K27me3 (<b>F</b>) for all NCBI RefSeq genes around TSS (+/−5 Kb) at E17.5 and PN15. Red lines are average tag counts around TSS.Y-axis in <b>E</b> and <b>F</b> represents the number of reads in a 100 bp interval.</p