Drosophila Chromosome 4 Chromatin Structure Figure 8. Lack of EGG leads to large-scale changes in POF, HP1a, and H3K9 methylation specifically on chromosome 4. A. Depletion of EGG alters H3K9me2/3, HP1a, and POF enrichment. Scaled enrichment is shown on chromosome 4 (left) and in pericentric heterochromatin (right) comparing wildtype (dark color) and egg 10.1-1a homozygous mutant (light color). Error bars: SEM. B. Browser shots showing the reduction of POF, HP1a, and H3K9me2/3 on chromosome 4 (top panel) and the relatively small change in pericentric heterochromatin observed in egg mutants. The M- value scale (Y-axis) is identical for wildtype and mutant ranging from 0 to 3. C. The 70 kb proximal region on chromosome 4 shows minimal changes in POF, HP1a, and H3K9me2/3 levels in egg mutants, distinct from the alterations in the remainder of the chromosome illustrated in B. The M-value scale (Y-axis) is identical for wildtype and mutant ranging from 0 to 3. D. Changes in H3K9me2/me3, HP1a, and POF enrichment (Y-axis: smoothed M- values) are examined separately for TSS of actively transcribed genes, their gene bodies, and silent regions on chromosome 4. Error bars: SEM. Data from third instar larvae. doi:10.1371/journal.pgen.1002954.g008 with dKDM4, an H3K36 demethylase, [53], whose yeast homologs promote transcript elongation [54]. Thus, there are precedents for an ‘‘activating’’ role for HP1a, and an interaction with dKDM4 provides an attractive model for how HP1a might influence RNA pol II processivity and pausing. However, what remains to be determined is why polymerase pausing would be affected specifically on chromosome 4 rather than also affecting genes in pericentric heterochromatin. We note that while the overall pericentromeric domains are strongly enriched for HP1a, one does not see the increase over the gene body observed for the chromosome 4, and hence these genes do not exhibit the same contrast between TSS and gene body observed for chromosome 4 genes (Figure 3). POF may play a role in enhancing HP1a presence at active genes on chromosome 4. The chromatin structure analysis we present from mutants lacking POF, HP1a, and EGG is mostly in agreement with previously published results based on polytene chromosome analysis. On chromosome 4, lack of POF induces loss of HP1a [36], H3K9me2 [22], and H3K9me3 (our study). However, our higher resolution analysis reveals a pool of HP1a, H3K9me2, and H3K9me3 associated with repeated sequences on chromosome 4 that is independent of POF. Also in contrast to prior findings [36], our results indicate that POF is maintained on chromosome 4 independent of HP1a, as mutants lacking HP1a still show a normal POF enrichment pattern. Note that HP1a depletion was accomplished here by a heteroallelic cross; thus HP1a was present during the initial assembly of heterochromatin. It has been postulated that POF is recruited to chromosome 4 from a site close to the centromere of the chromosome, based on translocation studies [55]. However, the affinity of POF for transcribed genes leads to an enrichment pattern that changes from cell type to cell type, arguing against a simple recruitment and spreading model (comparison of modENCODE data from Bg3 and S2 cells). Our analysis of mutants (resulting in depletion) PLOS Genetics | www.plosgenetics.org 14 September 2012 | Volume 8 | Issue 9 | e1002954