Drosophila Chromosome 4 Chromatin Structure Figure 7. Lack of HP1a does not lead to a loss of POF from chromosome 4. A. H3K9me2 and H3K9me3 levels decrease in HP1a mutants, while POF enrichment is not reduced. The smoothed M-value (Y-axis) is shown for pericentric heterochromatin (right) and chromosome 4 (left) comparing wildtype (dark color) and trans-heterozygous Su(var)20504/Su(var)20505 mutants. Error bars: SEM. B. Browser shot illustrating the retention of POF enrichment on chromosome 4 in HP1a mutants (top panel) and depletion of H3K9me2 and H3K9me3 both in chromosome 4 (top) and pericentric heterochromatin (bottom). The M-value scale (Y-axis) is identical for wildtype and mutant ranging from 0 to 3. C. Changes in H3K9me2, H3K9me3, and HP1a enrichment at TSSs of actively transcribed genes, over gene bodies of active genes, and in silent regions on chromosome 4 in HP1a mutant. Error bars: SEM. doi:10.1371/journal.pgen.1002954.g007 data support the conclusion that enrichment of HP1a, H3K9me2, and H3K9me3 in pericentric heterochromatin (including the basal portion of chromosome 4) is established by a different mechanism than the enrichment of these same marks over active gene bodies on chromosome 4. Inter-genic clusters of repeats on chromosome 4 are likely to be assembled into heterochromatin by the same mechanism as that operating in pericentric regions. Discussion The genome-wide enrichment profiles of 20 histone modifica- tions and 25 chromosomal proteins demonstrate the distinct nature of chromatin on Drosophila chromosome 4. As anticipated based on the behavior of transgene reporters [10,11,24], we found that chromosome 4 sequences are almost ubiquitously packaged with marks commonly associated with heterochromatin, H3K9me2, H3K9me3, HP1a, and HP2 (Figure 1). The TSSs of active genes are depleted for these marks (Figure 3). Surprisingly, ‘‘permissive’’ domains, which allow full expression of reporter genes, were found not to resemble euchromatin, but to show evidence of Polycomb regulation (associated with H3K27me3 and PC in some cell types) (Figure 1). The association with Polycomb marks is cell-type specific; thus, some genes on chromosome 4 appear to be able to switch between the two main silencing systems in what appears to be a developmentally regulated process. We do not know the state of the Polycomb regulated domains in the cells of the eye imaginal disc, where white reporter expression is required to result in a red eye phenotype. It is possible that in these cells the Polycomb regulated domains are associated with its activating antagonist, trithorax, and its partners. However, packaging in the PC state, which appears to exclude HP1a and H3K9me2/3 in this situation, is sufficient to allow DNase1 hypersensitive site (DH site) formation at the genes in these domains in BG3 cells, while such sites are not evident when the same genes are packaged with HP1a and H3K9me2/3 in S2 cells (modENCODE data tracks; www. modENCODE.org). Given that loss of DH site formation has been observed for the variegating reporter [38], a domain that permits DH site formation may be sufficient for reporter expression. Due to the unusual chromatin environment, chromosome 4 genes experience a unique regulatory system and display decreased polymerase pausing (Figure 4). Mutant analysis indicates that the RNA pol II distribution pattern is dependent on HP1a (Figure 5). In mutants lacking HP1a or POF, enrichment of RNA pol II decreases in the gene bodies, leading to an increase in PI due to the now strongly TSS-biased RNA pol II distribution (Figure 5). This shift in RNA pol II seen in pof mutants is potentially an indirect effect due to the inability to recruit HP1a to active gene bodies in the absence of POF. Alternatively, both HP1a and POF together might be required for the wildtype RNA pol II distribution – and the decrease of polymerase pausing - normally seen on chromosome 4. How HP1a, and possibly POF, influence polymerase distribution is still unknown. This influence might occur at various steps of RNA pol II regulation, either by interfering with the establishment, maintenance, or resolution of the paused polymerase, or by promoting elongation. For example, the Positive Transcription Elongation Factor b (P-TEFb) and PAF1C act by promoting elongation [39]. On the other hand, Min and colleagues found that in mouse embryonic stem cells, ‘‘bivalent’’ genes associated with PRC1 and PRC2 display low levels of polymerase pausing, possibly due to their chromatin structure [40]. POF’s influence could be mediated by its RNA recognition motif [16] and its ability to interact with RNA transcripts [19], leading to a positive effect on gene expression [19,36]. Our data, however, indicate that POF alone is insufficient to determine the RNA pol II distribution on chromosome 4 genes, and that HP1a is vital for their regulation. Thus, further work is needed to elucidate the exact mechanism of interaction between POF, HP1a, and the polymerase. Another protein to consider in the regulation of chromatin structure and RNA polymerase distribution on chromosome 4 genes is JIL-1, which is enriched on chromosome 4 (Figure 1). JIL- 1 is an H3S10 kinase; it limits heterochromatin extent, as in its absence, HP1a and H3K9me2 spread to new genomic regions [41–44]. Depletion of JIL-1 overall has little effect on gene expression [45], with the major effect being on the X chromo- some, with approximately 10% of the genes affected, based on our analysis. In contrast, ,5% of the chromosome 4 genes are affected, less than the percentage of X chromosome genes but slightly more than seen in the remainder of the genome. As in HP1a and POF mutants, the expression of the affected genes decreases. However, given the small number of genes affected by JIL-1 depletion, the impacts of HP1a/POF depletion are unlikely to be dependent on JIL-1. This interpretation is supported by the genetic interaction analysis of JIL-1 and HP1a, which indicates that their mutations counteract each other’s effects, and that the spread of H3K9me2 triggered by Jil-1 mutations is not dependent on HP1a [46]. While HP1a is best known for its role in heterochromatin formation and silencing, several reports have also linked HP1a to regulation of transcriptional activity of both heterochromatic and some euchromatic genes [47–49]. Heterochromatic genes light and rolled are reported to be dependent on a heterochromatic environment, and specifically on HP1a, for optimal expression [34,50], and we find that the majority of the chromosome 4 genes show a similar dependence (Figure 5D). The distribution of H3K9me2/me3 at several active heterochromatic genes shows depletion at the TSS [51], as reported here for chromosome 4 genes. However, it has recently been reported that two chromo- some 4 genes, CAPS and Dyrk3, lose DNase accessibility at the 59 DH site in the absence of HP1a [52]. Thus, while absence of HP1a and other silencing marks from the TSS is associated with gene expression in heterochromatic and chromosome 4 genes, the presence of HP1a in the domain as a whole appears to be required for DH site formation at these genes. In contrast, HP1a domains are prohibitive for DH site formation at the TSSs of eukaryotic reporter genes inserted into these regions [38]. In euchromatin, we have found HP1a associated with a number of TSSs, a finding that is supported by the detection of small amounts of HP1a in chromosome arms of polytene chromosomes [12]. Others have identified HP1a as a positive regulator of more than 100 genes, associating with the transcript and apparently facilitating elongation [35]. HP1a has been reported to interact PLOS Genetics | www.plosgenetics.org 13 September 2012 | Volume 8 | Issue 9 | e1002954