High-Resolution Mapping Reveals Links of HP1 with Active and Inactive Chromatin Components

Heterochromatin protein 1 (HP1) is commonly seen as a key factor of repressive heterochromatin, even though a few genes are known to require HP1-chromatin for their expression. To obtain insight into the targeting of HP1 and its interplay with other chromatin components, we have mapped HP1-binding sites on Chromosomes 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays and the DNA adenine methyltransferase identification (DamID) technique. The resulting high-resolution maps show that HP1 forms large domains in pericentric regions, but is targeted to single genes on chromosome arms. Intriguingly, HP1 shows a striking preference for exon-dense genes on chromosome arms. Furthermore, HP1 binds along entire transcription units, except for 5′ regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4 (H3K4me2), which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5′ ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. Additionally, we observed that HP1-chromatin and Polycomb-chromatin are nonoverlapping, but often closely juxtaposed, suggesting an interplay between both types of chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components

Global Chromatin Domain Organization of the Drosophila Genome

In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution

Preferential Binding of HP1 to Exon-Dense Genes

<p>(A–C) Pie charts showing the overlap of GATC fragments with promoters (black), 5′ UTRs (green), exons (yellow), introns (white), 3′ UTR (black), and intergenic regions (blue) for all fragments represented on the high-density oligonucleotide array (top) and only those fragments that are significantly bound by HP1 (bottom). This analysis was performed for (A) all fragments, (B) nonpericentric fragments, and (C) pericentric fragments. (D and E) Density plot showing the frequency distribution of exon densities (i.e., the fraction of sequence in transcription units that consists of exons) for genes with high HP1 levels (red) and genes with low HP1 levels (black), in (D) nonpericentric and (E) pericentric genes. Only genes with a length >5 kb were included in this analysis.</p

HP1 Binding to Individual Transposon Copies

<div><p>(A) Frequencies of copies of the different TE types that are target of HP1 (dark gray) in nonpericentric (top) and repeat-rich pericentric (bottom) regions. A TE copy was counted as an HP1 target if, in the unique flanking 1 kb on each side of the TE, at least one GATC fragment was significantly bound by HP1.</p><p>(B) HP1-Dam/Dam–binding ratios at unique sequences within 1 kb of a TE are plotted as a function of the FRI_20kb (see main text). Running mean with window size 20 is shown for HP1 binding as a function of the FRI_20kb (red line).</p></div

Most HP1-Bound Genes Are Actively Transcribed

<p>Density plot (smoothed histogram) showing the distribution of normalized expression levels of (A) nonpericentric and (B) pericentric genes that are strongly bound by HP1 (high HP1, average HP1 log₂-ratio along entire gene >2 [red]) or genes with low or no binding by HP1 (low HP1, average HP1 log₂-ratio along entire gene <2 [black]). Expression data were taken from Pickersgill et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030038#pgen-0030038-b050" target="_blank">50</a>].</p

HP1 Binding Is Linked to H3K4me2 and Histone H3.3 Patterns

<p>Alignment of HP1-bound genes to (A) their TSSs and (B) the 3′ end of their transcription units. TSS-aligned genes include upstream regions up until the next upstream gene; 3′ end aligned genes include downstream regions until the next downstream gene. Curves show running mean (window size 100) of HP1-binding ratios (log₂) for nonpericentric (green) and pericentric target genes (blue). (C) H3K4me2 levels of TSS-aligned genes in nonpericentric regions with high (red) or low (black) levels of HP1 as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030038#pgen-0030038-g002" target="_blank">Figure 2</a>. H3K4me2 levels were taken from Schubeler et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030038#pgen-0030038-b053" target="_blank">53</a>]. (D) Frequency distribution of H3K4me2 levels around the TSS (−500 to +1000 bp) for genes with high (black line) and low (gray lines) HP1 levels, either all genes (dotted gray line) or expression matched (solid gray line). (E and F) TSS alignment of H3.3 levels for genes with high (red) and low (black) HP1 levels in nonpericentric (E) and pericentric (F) regions. H3.3 data were taken from Mito et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030038#pgen-0030038-b051" target="_blank">51</a>]. In (C), (E), and (F) running mean window sizes correspond to 2% of the total number of datapoints.</p

HP1 and Polycomb Form Two Distinct, Nonoverlapping Chromatin Domains That Are Often in Close Proximity to Each Other

<p>Running mean (window size 20 GATC fragments) of HP1-Dam/Dam–binding ratios (black) and Pc-Dam/Dam–binding ratios (red) of (A) Chromosome 4; (B and C) pericentric regions of Chromosome 2; and (D) a repeat-rich region on the right arm of Chromosome 2 (cytological region 42AB). Positions of genes are indicated below each graph.</p

High-Resolution HP1-Binding Profiles

<p>(A and B) Maps of Chromosome 2, (C) Chromosome 4, (D) pericentric <i>light</i> gene, (E) pericentric <i>concertina</i> gene, and (F) cytological region 31. Inset in (A) shows a more detailed view of the centromere-proximal 0.4 Mb of 2L. Each stick represents the mean HP1–Dam/Dam binding ratio of a single GATC fragment, for one representative experiment. Fragments significantly bound by HP1 are marked in red, fragments not significantly bound by HP1 are shown in black. Gaps originate from nonunique sequences for which binding cannot reliably be determined. Positions of genes (open rectangles) and TEs (gray rectangles) are shown in D–F. Arrows in (D) and (E) indicate orientation of the genes.</p