Subgroups of CTRs based on associated proteins in <i>Drosophila</i> S2 cell line.

<p>(A) Heat-map of the hierarchical clustering analysis result. Each column denotes a single CTR, and each row represents one protein included in the association analysis. The red and blue bars denote the presence or absence of an association with the corresponding CTR, respectively. Capital letters within colored boxes highlight the different subgroups of CTRs. (B) Proportions of CTRs in each subgroup (identified in (A)) that are associated with individual protein. The width of the bar indicates the percentage of CTRs in each group that are bound by the respective protein.</p

Genome-Wide Identification of Chromatin Transitional Regions Reveals Diverse Mechanisms Defining the Boundary of Facultative Heterochromatin

<div><p>Due to the self-propagating nature of the heterochromatic modification H3K27me3, chromatin barrier activities are required to demarcate the boundary and prevent it from encroaching into euchromatic regions. Studies in <i>Drosophila</i> and vertebrate systems have revealed several important chromatin barrier elements and their respective binding factors. However, epigenomic data indicate that the binding of these factors are not exclusive to chromatin boundaries. To gain a comprehensive understanding of facultative heterochromatin boundaries, we developed a two-tiered method to identify the Chromatin Transitional Region (CTR), i.e. the nucleosomal region that shows the greatest transition rate of the H3K27me3 modification as revealed by ChIP-Seq. This approach was applied to identify CTRs in <i>Drosophila</i> S2 cells and human HeLa cells. Although many insulator proteins have been characterized in <i>Drosophila</i>, less than half of the CTRs in S2 cells are associated with known insulator proteins, indicating unknown mechanisms remain to be characterized. Our analysis also revealed that the peak binding of insulator proteins are usually 1–2 nucleosomes away from the CTR. Comparison of CTR-associated insulator protein binding sites vs. those in heterochromatic region revealed that boundary-associated binding sites are distinctively flanked by nucleosome destabilizing sequences, which correlates with significant decreased nucleosome density and increased binding intensities of co-factors. Interestingly, several subgroups of boundaries have enhanced H3.3 incorporation but reduced nucleosome turnover rate. Our genome-wide study reveals that diverse mechanisms are employed to define the boundaries of facultative heterochromatin. In both <i>Drosophila</i> and mammalian systems, only a small fraction of insulator protein binding sites co-localize with H3K27me3 boundaries. However, boundary-associated insulator binding sites are distinctively flanked by nucleosome destabilizing sequences, which correlates with significantly decreased nucleosome density and increased binding of co-factors.</p></div

Contrasting patterns of H3.3 enrichment and nucleosome turnover rate associated with subgroups of CTRs in <i>Drosophila</i> S2 cell line.

<p>(A) Composite plot for all CTRs. H3.3 (low salt) incorporation is enriched on the euchromatic side of CTRs (red arrow), while nucleosome turnover rate (CATCH-IT) is drops down sharply at the same region (green arrow). (B) H3.3 enrichment and CATCH-IT measurements of nucleosome turnover rate moves to the same direction for GAF (both CTR-associated and others). In contrast, for CTR-associated dCTCF binding sites, the enrichment of H3.3 is accompanied by decreased turnover rate. (C) Plots of H3.3 enrichment (red), nucleosome turnover rate (green, measured with CATCH-IT), and nucleosome density (purple) for each subgroup of the CTRs (for group F only those co-localized with GAF were included). Note the contrasting pattern between H3.3 enrichment and CATCH-IT profile in subgroups A, B, C, G, but not in subgroups D and E.</p

Binding intensity and patterns of insulator proteins and co-factors associated with CTRs in <i>Drosophila</i> S2 cell line.

<p>The enrichment levels of respective insulator proteins (A) and co-factors (B) around binding sites associated with CTR (solid lines) or located in H3K27me3-enriched region (dashed lines). For CTR-associated binding sites, negative and positive distances denote euchromatic and heterochromatic side. Box plots show the peak values for individual insulator proteins (A) and co-factors (B) at binding sites associated with CTR (open box) or in heterochromatic regions (shaded box). (C) Box plots of the width of insulator proteins binding patterns at binding sites associated with CTR (open box) or in heterochromatic regions (shaded box). P-values were all calculated by Wilcoxon rank sum test.</p

CTRs and the known insulator proteins in <i>Drosophila</i> S2 cell line.

<p>(A) Percentages of insulator protein binding sites are associated with a CTR. The x-axis shows the distance between insulator protein binding site and the nearest CTR, and y-axis shows the percentage of binding sites that are within a certain distance from the nearest CTR. The dashed line indicates the distance cutoff of 1 kb, which is used for association analysis. (B) A 200 kb region on chromosome 2L as an example. There are five Su(Hw) binding sites in this region, one is associated with a CTR (red bar, highlighted region), the others locate in regions enriched for H3K27me3. The intensities of co-factors (CP190, Mod(mdg4)) are relatively high at the CTR-associated binding site, and lower at the binding sites in the H3K27me3-enriched region. (C) Venn diagram shows the number of CTRs that are associated with four insulator proteins. Note that more than half (1203/2082) of the CTRs are not associated with any of the four insulator proteins. (D) Enrichment of insulator proteins in the <u>+</u>5 kb region around corresponding CTRs. The negative and positive distances also indicate the euchromatic and heterochromatic side of CTR, respectively. (E) An example illustrates the relative positions of a predicted CTR and the binding profiles of BEAF-32 and CP190. The peaks of the binding sites locate on the euchromatic side of the CTR, and the distance between the peaks of binding sites and the CTR midpoint is about 400 bp.</p

Cis-elements associated with CTRs in <i>Drosophila</i> S2 cell line.

<p>(A) Logos representation of motifs identified from DNA sequences underlying insulator protein binding sites associated with CTRs (CTR-associated) or in H3K27me3-enriched (Heterochromatic) regions. Motifs obtained with all binding sites are represented at the bottom. (B) Multi-A motifs are the discriminative motif identified by MEME for CTR-associated binding sites vs. heterochromatic binding sites. (C) Multi-A (AAAA/TTTT) content (normalized to genome average, red curve) and nucleosome density (blue curve) around CTR-associated insulator protein binding sites (solid line) and heterochromatic binding sites (dashed line). Data presents combined value for all the insulator proteins, dCTCF, Su(Hw), GAF, and BEAF-32. For CTR-associated binding sites, negative and positive distances denote euchromatic and heterochromatic side.</p

Histone modifications and gene expression levels on the euchromatic vs. heterochromatic side of the CTRs in <i>Drosophila</i> S2 cell line.

<p>(A) Enrichment levels of active (solid lines) and repressive (dashed lines) histone modifications around the H3K27me3 CTRs identified in S2 cells. Negative and positive distances indicate euchromatic and heterochromatic sides of the identified CTRs, respectively. (B) Expression levels of genes on the euchromatic or heterochromatic side of CTRs. Barplots represent Mean±SE for all genes (grey), genes within the 4 kb region on the euchromatic side (yellow) or the heterochromatic side (green) of CTRs. The expression levels for genes on euchromatic side of CTRs are significantly greater than those of the genes on the heterochromatic side (p<2.2E-16, Wilcoxon rank sum test). (C) An example of 7 CTRs (red bars) predicted by CTRICS. Bar height reflects T-score, top and bottom rows denotes the orientation of the CTRs. The panel below CTR shows H3K27me3 domains called by SICER. RNA-Seq signal, RNA Pol II binding, as well as active histone modification (H3K4me3) are depleted in heterochromatic regions which have high H3K27me3, while they are enriched in euchromatic regions.</p

Multi-A (AAAA/TTTT) content (normalized to genome average, red curve) and nucleosome density (blue curve) around individual subgroup of CTRs in <i>Drosophila</i> S2 cell line (For group F only those co-localized with GAF were included).

<p>The negative and positive distances denote the euchromatic and heterochromatic sides of CTR, respectively.</p

Proposed models for facultative heterochromatin boundary.

<p>Models represent distinct features of GAF-associated (A) vs. dCTCF-associated (B) CTRs. The red and blue dashed lines denote the position of CTR and chromatin barrier, respectively. The blue circles at the bottom of each model indicate the nucleosome turnover rate, the bigger the circles, the faster the nucleosomes turnover. For dCTCF-associated CTRs, the increased enrichment of H3.3 is coupled with decreased turnover rate.</p

Registration examples on the fish point set.

<p>From top to bottom are the four largest degradations: deformation (0.08), noise (0.05), occlusion (0.5), and outlier (2.0). The goal is to align the model point set (blue pluses) onto the scene point set (red circles).</p