The Drosophila speciation factor HMR localizes to genomic insulator sites

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR's function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species

HMR localisation to repetitive elements.

<p><b>(A)</b> HMR ChIP tag enrichment at repetitive DNA elements. To identify enriched sequences the enrichment (log2-fold) over input is plotted against the RPKM of an individual repeat sequence from RepBase. Repeats with less than 2-fold enrichment are not displayed. <b>(B)</b> ChIP tag density of HMR and the <i>gypsy</i>-insulator proteins CP190, Mod(mdg4), Su(Hw) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref039" target="_blank">39</a>] across the repetitive <i>gypsy</i> retrotransposon sequence. The <i>gypsy</i> insulator sequence at the 5' end is highlighted in <i>green</i>.</p

HMR genomic localization to <i>gypsy</i>-like insulator sites is dependent on CP190.

<p><b>(A)</b> Western Blot of cell lysates after treatment with specific and control dsRNA shows an efficient knock-down of HMR and CP190. <b>(B)</b> Venn diagram of the overlap between HMR, CP190, Mod(mdg4) and Su(Hw) peaks [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref039" target="_blank">39</a>] classifying HMR peaks as <i>gypsy</i>-like (highlighted in <i>green</i>) and non <i>gypsy</i>-like (highlighted in <i>orange</i>). <b>(C)</b> Composite analysis of HMR ChIP signals and Histone H3 ChIP signals at genomic HMR peak positions according to the groups defined in <b>(B)</b>. <b>(D)</b> Quantification of the fold-change of HMR ChIP enrichment upon CP190 RNAi and HMR RNAi. Box plots represent the fold-change of normalized HMR ChIP tag number aligned to 200 bp wide HMR peak regions. Peak regions with less than 50 aligned tags were excluded from the analysis. Significance of difference was estimated with p-values calculated with Wilcoxon rank sum test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref072" target="_blank">72</a>].</p

HMR localizes to genomic insulators and the <i>gypsy</i> transposon.

<p><b>(A)</b> Sequence motifs identified within HMR peak regions. The corresponding motif logo, p-value of enrichment, percentage of regions with this motif and putative binding factors are indicated. Dashed arrows mark the sequence that matches the published binding sites of Su(Hw) and BEAF-32 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.s002" target="_blank">S2A Fig</a>) <b>(B)</b> Peak overlap of HMR with peaks of the insulator proteins CP190, Mod(mdg4), Su(Hw), CTCF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref039" target="_blank">39</a>] and BEAF-32 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref040" target="_blank">40</a>]. The number of HMR peaks is indicated depending on their colocalization with known boundary factors. Groups with less than 11 members are not displayed. Su(Hw)-containing <i>gypsy</i>-like groups are depicted in <i>green</i>, non <i>gypsy</i>-like groups in <i>orange</i>. Combinations that contain less than ten HMR peaks are not shown. <b>(C)</b> Genome browser view of the Su(Hw) binding region 1A-2. ChIP signals of HMR and known <i>gypsy</i> binding factors are shown. The 1A-2 insulator is highlighted in <i>green</i>.</p

HMR borders HP1a together with BEAF-32 at the TSS of actively transcribed genes and enhances their transcription.

<p><b>(A)</b> Heatmaps of HMR, HP1a, BEAF-32 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref040" target="_blank">40</a>], Su(Hw), Mod(mdg4), CP190 and CTCF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref039" target="_blank">39</a>] ChIP signals. All signals are centered around the HMR binding sites, clustered according to adjacent HP1a signals and sorted by HMR intensity. <b>(B)</b> Sequence motifs identified within HMR peak regions from class 1 and class 2 based on HOMER motif analysis. The corresponding motif logo, p-value of enrichment, percentage of regions with this motif, and putative binding factors are indicated. Dashed arrows mark the sequence that matches the published binding sites of Su(Hw) and BEAF-32 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.s002" target="_blank">S2A Fig</a>). <b>(C)</b> Distribution of class 1 and class 2 HMR peaks among various genomic landmarks. <b>(D)</b> Box plot showing the normalized RNA expression of all genes and HMR-bound genes (promoter/TSS annotated) in class 1 and in class 2. S2 cells RNA expression levels were used according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref073" target="_blank">73</a>]. Significance of difference was estimated with p-values calculated with Wilcoxon signed rank test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref072" target="_blank">72</a>]. <b>(E)</b> Box plot showing the log2 fold change of protein coding gene transcripts of all analyzed genes and HMR-bound genes (promoter/TSS annotated) in class 1 and in class 2 comparing <i>Hmr</i> mutant against wild type flies. The RNA-Seq data comes from experiments done in <i>D</i>. <i>melanogaster</i> ovaries [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref004" target="_blank">4</a>]. Significance of difference was estimated with p-values calculated with Wilcoxon rank sum test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171798#pone.0171798.ref072" target="_blank">72</a>]. For both box plots the box represents the interval that contains the central 50% of the data with the line indicating the median. The length of the whiskers is 1.5 times the interquartile distance (IQD). <b>(F)</b> Histogram showing HMR peak density across the annotated <i>D</i>. <i>melanogaster</i> genome. Class 1 HMR binding sites are enriched at region 31, centromere-proximal regions and the 4th chromosome.</p

Data and Results (per site, etc.)

Data from Many Labs 2 Replication Projec