necessary enzymes for generating the H3K9 methylation in these domains. POF deposition is independent of HP1a Polytene chromosome analysis had suggested that HP1a and POF enrichment on chromosome 4 are interdependent [22,36]. In order to verify this at a higher resolution, we carried out POF ChIP-chip analysis in HP1a mutants. As expected, ,94% of the HP1a enrichment in wildtype is absent in the trans-heterozygous mutant third instar larvae (Su(var)20504/Su(var)20505), both in pericentric heterochromatin and on chromosome 4. However, the POF distribution and its enrichment levels on chromosome 4 are unaffected in this mutant strain (p.0.05; Figure 7 and Table S7). This finding implies that POF recruitment to chromosome 4 is largely independent of HP1a. Alternatively, HP1a could be involved in an initial recruitment in the early embryo (when heterochromatin is formed), but be unessential for maintenance of POF association. H3K9me2/3 distributions on chromosome 4 depend both on HP1a-dependent and independent mechanisms The significantly altered H3K9 methylation on chromosome 4 in the pof mutants suggests the possibility of a similar effect in mutants lacking HP1a, leading us to investigate H3K9 methyla- tion levels on chromosome 4 and in pericentric heterochromatin in HP1a mutants. We find that both H3K9me2 and H3K9me3 are significantly decreased in pericentric heterochromatin (p,0.001, Figure 7A). Significant depletion of H3K9me2 (to 11.1% of wildtype) and H3K9me3 (to 33.3% of wildtype) is seen on chromosome 4 as well (Figure 7A and Table S7). With the exception of the first (centromere-proximal) 70 kb of assembled chromosome 4 sequence (discussed below), the regions of H3K9me3 enrichment that remain in the HP1a mutant are correlated with POF binding sites (69.1% retained in POF-positive regions while 30.9% retained in POF-negative regions, Table S7). In contrast, H3K9me2 is lost at similar rates in POF-positive and – negative portions of chromosome 4. Overall, it appears that HP1a is required in pericentric heterochromatin and chromosome 4 for wildtype levels of H3K9 methylation, but that a low level of both methyl marks is able to persist in the absence of HP1a. We suggest the possibility that the HP1a-dependent H3K9 methylation is mediated by the HP1a-interacting H3K9 HMT SU(VAR)3-9 [13], while the residual H3K9 methylation observed in the mutant is mediated by a different HMT, such as EGG or G9a. EGG is required for recruitment and/or maintenance of POF and HP1a at the majority of binding sites on chromosome 4 The altered H3K9 methylation in mutants lacking POF or HP1a led us to consider the involvement of the H3K9 HMTs in generating the distinct chromatin structure of chromosome 4. EGG is the Drosophila SETDB1 class H3K9 histone methyl- transferase, and it has been reported to be a major H3K9 methylation-producing methyltransferase on chromosome 4 based on immunohistochemistry and position effect variegation exper- iments [20–22,37]. Examining chromatin from homozygous egg10.1-1a third instar larvae ([21]; mutants, derived from a heterozygous stock carrying a GFP balancer), we find a number of significant changes in enrichment profiles compared to wildtype, primarily on chromosome 4 (Figure 8A and 8B). Overall levels of POF were significantly depleted (decreased by 63%, Figure 8A), with only 18% of binding sites remaining on chromosome 4 (Table S7). Similarly, the HP1a-enriched regions were reduced by 83.2% Drosophila Chromosome 4 Chromatin Structure and the level of enrichment of HP1a was decreased by 79% (Table S7, Figure 8A). These findings are consistent with the depletion of POF and HP1a seen on egg mutant polytene chromosomes [22]. Interestingly, some strong HP1a binding sites remain, which suggests that recruitment of HP1a to these sites is independent of EGG (Figure 8B). 63.9% of the HP1a peaks remaining in egg mutants coincide with HP1a peaks retained in the pof mutant (Table S7). HP1a peaks retained in egg mutants (and pof mutants) are within TE-rich regions (medium distance to a TE is 19 bp compared to the 135 bp of random expectation, p,0.001, Figure S15B). Thus, the enrichment profiles from egg mutants suggest that EGG is required for the majority of the recruitment and/or maintenance of POF and HP1a at actively transcribed genes, but not at some repeats. Loss of EGG protein alters the distribution of H3K9me2 and H3K9me3 on chromosome 4 We also investigated the effects of reduced EGG levels on H3K9 methylation. In egg10.1-1a mutants, we observed a significant reduction of H3K9me2 and H3K9me3 on chromosome 4 (decreased by 61% and 84%, respectively in Figure 8A, Table S7). While the overall H3K9me2/me3 level on transcribed genes of chromosome 4 dropped significantly (Figure 8B), there were several residual enriched areas, where H3K9me2/me3 was maintained despite the absence of EGG (22.7%/19.4% enriched regions remaining respectively, Table S7; Figure 8B, top panel; and Figure 8D). The remaining H3K9me2 and H3K9me3 enrichment is similar to that observed in the pof mutant (Figure 6). This finding implies that these residual H3K9me2/ me3 enriched domains are produced by an H3K9 HMT other than EGG. However, whether this activity is restricted to the mutant condition or is present in the wildtype as well is currently unclear and will require further experiments. It is interesting to note that the residual H3K9me2 enriched regions coincide with regions of residual HP1a binding in this mutant. As HP1a is known to bind to H3K9me2/H3K9me3, this finding suggests that the residual H3K9me2/me3 is capable of recruiting HP1a in the absence of EGG and POF. Conversely, the presence of HP1a could recruit an HMT such as SU(VAR)3-9, a known HP1a binding protein, to the region, resulting in H3K9 methylation. The ,70 kb closest to the centromere in the assembled sequence of chromosome 4 is a pericentric- heterochromatin-like domain where HP1a, H3K9me2, and H3K9me3 deposition are independent of POF and EGG In several of our analyses the most centromere-proximal portion of the assembled chromosome 4 sequences shows a response to the depletion of the various proteins that is clearly distinct from that of the remainder of the chromosome. For example, in chromatin from third instar larvae lacking POF (pof D119), HP1a, H3K9me2 and H3K9me3 are maintained at a level and density similar to wildtype in the ,70 kb of assembled sequence adjacent to the centromere (Figure S16). A similar effect is seen in EGG mutant larvae - HP1a and H3K9 methylation are reduced along most of chromosome 4, but maintained in this same ,70 kb region (Figure 8C). Overall, this domain behaves similarly to the pericentric heterochromatin regions of chromosomes X, 2, and 3, where mutations in pof and egg do not affect the enrichment of HP1a and H3K9 methylation. This finding suggests that the proximal ,70 kb of chromosome 4 can be considered to be pericentric heterochromatin, with chromatin characteristics dis- tinct from those of the remainder of distal chromosome 4. Our PLOS Genetics | www.plosgenetics.org 11 September 2012 | Volume 8 | Issue 9 | e1002954