Dynamic Epigenetic Control of Highly Conserved Noncoding Elements

<div><p>Background</p><p>Many noncoding genomic loci have remained constant over long evolutionary periods, suggesting that they are exposed to strong selective pressures. The molecular functions of these elements have been partially elucidated, but the fundamental reason for their extreme conservation is still unknown.</p><p>Results</p><p>To gain new insights into the extreme selection of highly conserved noncoding elements (HCNEs), we used a systematic analysis of multi-omic data to study the epigenetic regulation of such elements during the development of <i>Drosophila melanogaster</i>. At the sequence level, HCNEs are GC-rich and have a characteristic oligomeric composition. They have higher levels of stable nucleosome occupancy than their flanking regions, and lower levels of mononucleosomes and H3.3, suggesting that these regions reside in compact chromatin. Furthermore, these regions showed remarkable modulations in histone modification and the expression levels of adjacent genes during development. Although HCNEs are primarily initiated late in replication, about 10% were related to early replication origins. Finally, HCNEs showed strong enrichment within lamina-associated domains.</p><p>Conclusion</p><p>HCNEs have distinct and protective sequence properties, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. These observations indicate that such elements are likely to have essential cellular functions, and offer insights into their epigenetic properties.</p></div

Dynamic histone modification at HCNEs during <i>Drosophila</i> development.

<p>Table shows the percentage of HCNEs that were positive for any of the six analyzed histone modification markers or CBP binding across 12 developmental stages. A dash (−) indicates missing data. An HCNE is considered to have a given marker when at least 10% of its length overlapped with the marker. We observed a prominent presence of H3K27me3 and H3K9me3 among the HCNEs throughout development. Markers associated with the active chromatin state (H3K27ac, H3K9ac, and H3K4me3) displayed stage-specific patterns.</p><p>Dynamic histone modification at HCNEs during <i>Drosophila</i> development.</p

Transcriptional activity of HCNE-proximal genes and their associations with histone modifications of HCNEs.

<p>(A) (upper) Histogram showing the bimodal distribution of stage specificity amongst HCNE-proximal genes, measured as 1-entropy; 0 indicates that genes were expressed evenly across different stages, while 1 indicates that genes were expressed during only one stage. (lower) Violin plots showing the stage specificity of HCNE-proximal genes grouped by their core promoter type. Genes with Inr-motif promoters are more stage-specific than genes of the other core promoter types. (B) Same as in (A), but assessing tissue specificity. (C) Heatmaps illustrating the expression levels of stage-specific HCNE-proximal genes across 30 developmental stages (from FlyBase); E, L, P, AdF and AdM refer to Embryonic, Larva, Pupa, Adult Female and Adult Male stages. Genes were grouped by their promoter type (Color key for promoter type is shown on the top of each Heatmap; Inr only and Inr/DRE are grouped together for visualization purposes), and expression values greater than 100 were rounded to 100. Complete linkage hierarchical clustering is performed with Euclidean distance as the distance metric. Clusters of genes with Inr-motif promoters exhibit high levels of expression throughout development, whereas the genes having other promoter types are predominantly expressed during the later stages. (D) Boxplot showing differences in the expression levels of HCNE-proximal genes grouped by the presence (sharp color) or absence (faded color) of active markers at the nearest HCNEs. Expression levels were examined for all 12 developmental stages (from modEncode). Symbol ‘*’ indicates P<0.05.</p

Nucleosome landscape at HCNEs.

<p>(A) Mean nucleosome density of sequences aligned with respect to their centers. Nucleosomes are enriched at the centers of HCNEs compared to the flanking regions. (B) Mononucleosome enrichment was calculated from sequences aligned as described in (A). Mononucleosomes are depleted in HCNEs compared to flanking regions. (C) H3.3 enrichment, calculated from sequences aligned as described in (A). H3.3 is depleted at HCNEs compared to the flanking regions. (D) Same as (C) but for H3.3dN.</p

Genomic and epigenomic properties of HCNEs.

<p>(A) Heatmap demonstrating the clustering of HCNEs based on the studied features (see below). (B) Heatmap showing correlation between features. The studied features include: the levels of nucleosomes, mononucleosomes, H3.3 and H3.3dN; the summed occurrence of each histone modification and the CBP binding for each HCNE during development; the replication timing in the three studied cell lines (columns labeled as RepTimeBg3, RepTimeS2 and RepTimeKc); and the LAD scores. To facilitate visualization, the values of each feature were scaled to between 0 and 1 using the equation(<i>value – min)/(max – min)</i>, where <i>max</i> and <i>min</i> were the maximum and minimum values of each feature, respectively; complete linkage hierarchical clustering is performed with Euclidean distance as the distance metric.</p

Genomic properties of HCNEs.

<p>(A) Violin plot illustrating that HCNEs have a higher GC content versus 10,000 random non-coding sequences. (B) Per-base A+T frequencies within 200 bp of HCNE-flanking regions and 15 bp of HCNEs aligned at their boundaries (region on downstream boundaries were reversed). (C) Sequence logos of four TFBSs that were among the most significant matches to overrepresented heptamers; red marks TFs reported to be involved in developmental processes; for complete list of TFBSs matches to top 50 overrepresented heptamers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109326#pone.0109326.s005" target="_blank">Table S2</a>. (D) Bar plot showing the fraction of core promoter predictions among the HCNE-proximal genes. HCNE-proximal genes are enriched in the Inr-motif and depleted of Motif1/6 and DRE core promoters, to a higher degree than expected by chance. (E) GO enrichment among HCNE-proximal genes grouped by their core promoter type. The top 10 significantly enriched terms (Holm-Bonferroni adjusted P-values <0.05) are shown for each promoter-type group. Colors represent –log₁₀ (Holm-Bonferroni adjusted P-values).</p

The statistics of reads used for the miRNA identification.

<p>The statistics of reads used for the miRNA identification.</p

Number of reads present in <i>S</i>. <i>carteri</i> and <i>X</i>. <i>testudinaria</i> that matches known sequences in <i>A</i>. <i>queenslandica</i>.

<p>miRNAs in bold are the mature miRNA, while the unbolded ones are star sequences.</p

List of 11 <i>bona fide</i> miRNAs in <i>S</i>. <i>carteri</i> and 19 in <i>X</i>. <i>testudinaria</i>.

<p>Abbreviations used are ‘aqu’: <i>A</i>. <i>queenslandica</i>; ‘sca’: <i>S</i>. <i>carteri</i>; ‘xte’: <i>X</i>. <i>testudinaria</i>.</p

Maximum-likelihood phylogenies of the core proteins involved in small RNA biogenesis.

<p>Trees were constructed for (A) Argonaute, (B) Piwi, (C) Dicer, (D) Drosha, (E) Pasha, and (F) HEN1. (A), (D), and (F) were constructed using the LG+G amino acid substitution model, while (B), (C), and (E) were constructed using the LG+I+G model. Bootstrap support values are indicated above the branches. Species abbreviations: Ath, <i>Arabidopsis thaliana</i>; Aqu, <i>Amphimedon queenslandica</i>; Cel, <i>Caenorhabditis elegans</i>; Dme, <i>Drosophila melanogaster</i>; Efl, <i>Ephydatia fluviatilis</i>; Hsa, <i>Homo sapiens</i>; Hvu, <i>Hydra vulgaris</i>; Nve, <i>Nematostella vectensis</i>; Sca, <i>S</i>. <i>carteri</i>; Spi, <i>Stylophora pistillata</i>; Spu, <i>Strongylocentrotus purpuratus</i>; and Xte, <i>X</i>. <i>testudinaria</i>. <i>A</i>. <i>thaliana</i> sequences were selected as the outgroup when available. For Piwi, Drosha, and Pasha (whose protein families are not found in plants), sponge sequences were chosen as the outgroup, as they phylogenetically form the most basal clade in the tree.</p