Drosophila Chromosome 4 Chromatin Structure in the gene body of the mutants for chromosome 4 genes, but not for the control euchromatic genes. The TSS-proximal RNA pol II peak also changes in the mutants compared to wildtype, moving approximately 68 bp and 50 bp downstream, respectively. B. Scatter plot showing enrichment of RNA pol II at the promoter region (+/2500 bp of TSS, top panel) and gene body (bottom panel). In both HP1a and POF mutants, the RNA pol II enrichment decreases significantly only in the gene body. C. Pausing index (PI) changes specifically on chromosome 4 in HP1a and POF mutants. In both mutants, PI for chromosome 4 genes increases to a range similar to the other chromosomes. D. Examples of the shift of RNA pol II in four chromosome 4 gene regions in the mutants. In Pur-alpha, no probes were available from 581 kb–584 kb. E. Fold changes of expression level (FPKM) of chromosome 4 genes in the HP1a mutant compared to the wildtype. Data from third instar larvae. doi:10.1371/journal.pgen.1002954.g005 (600 bp downstream of the TSS to the end of the gene) [31]. Analyzing the changes in RNA pol II distribution separately for the promoter region and the gene body, we find that only the changes in the gene body are significant (Figure 5B; p,2.25e-6). The shift to TSS-biased enrichment is illustrated for several genes in Figure 5D. While the level of RNA pol II enrichment at the promoter does not change in the HP1a mutant, the location of the peak shifts by approximately 68 bp into the gene body, a position suggestive of a paused polymerase. In total, 54 of 73 genes larger than 600 bp on chromosome 4 show an increase in PI (calculated as in [31] for ChIP- chip data, Figure S11), which is significantly different from what we observe for the remainder of the genome (Figure 5C, p,1.2e-6). Thus, the shift in RNA pol II distribution is specific to chromosome 4 genes and does not occur at euchromatic genes in general. Next, we examined the effect of HP1a depletion on gene expression using RNA-seq data. On chromosome 4, there is a significant overall decrease in the expression level (Figure S12D; p,2.26e-6, paired Wilcoxon test; p = 0.205 on chromosome 3R, a control euchromatic region). Specifically, 67 of 84 genes (,80%) exhibit decreased expression upon HP1a depletion (Figure 5D) in contrast to ,60% genome-wide. Expression is significantly decreased for plexB, ci, CG31998, dpr7, Lin29, zfh2, onecut, mav, CG11360, Sox102F, unc-13, toy, CG32017, pho, and Caps (FDR = 0.01), while significantly increased expression is observed for CG1970, bt, ATPsyn-beta, and Rfabg (FDR = 0.01). Among the genes showing decreased expression in the HP1a mutant, a group of ,10 genes also loses H3K36me3 signal relative to wildtype. In addition, genes on chromosome 4 with decreased expression levels tend to show depletion of RNA pol II in Su(var)205 [HP1a] mutants (Figure S11C). The loss in expression after HP1a depletion indicates that most chromosome 4 genes are behaving as ‘‘heterochromatic genes’’ by this criterion [34]. Using an RNA-immunoprecipitation technique, Piacentini and colleagues have identified a set of euchromatic genes regulated by HP1a in S2 cells [35]. We compared the response of this gene set to HP1a depletion in HP1a mutant larvae to the response of chromosome 4 genes (Figure S12). While chromosome 4 genes show a strong reduction of RNA pol II over the gene bodies in the absence of HP1a (p,7.52e-7), the putative euchromatic HP1a- regulated genes show a slight increase in RNA pol II enrichment (p = 0.02). In addition, the euchromatic gene set did not show a reduction in gene expression in these larvae [Su(var)20504/ Su(var)20505]. Our exceptional findings for chromosome 4 genes demonstrate a unique role for HP1a in this domain, affecting RNA pol II distribution as well as overall gene expression. The data indicate that the specific, high enrichment of HP1a over actively transcribed gene bodies on chromosome 4 positively regulates gene expression, and reduces 59 accumulation of RNA polymerase at these genes, possibly by promoting elongation and/or by interfering with pausing. POF mutants also show an altered RNA pol II distribution on chromosome 4 We speculated that disrupting chromosome 4’s unique chro- matin structure by removing POF would also affect the RNA pol II distribution. Thus, we investigated the relationship between POF and polymerase pausing using homozygous pof D119 third instar larvae. pof D119 is a mutant that lacks the first and part of the second exon of the POF coding sequence [36]. 79% of POF enrichment seen in wildtype is absent in pof mutants (Figure S10B). The gene body POF signal is absent in the mutant, while the remaining signal is TSS-associated and most likely represents cross-reactivity. As observed for HP1a mutants, we see a shift from a broader distribution of RNA pol II in the wildtype to a TSS- biased enrichment in the pof mutant (Figure 5A and 5B), with a significant decrease of RNA pol II enrichment over the gene body (p,2.01e-7). Thus, most genes on chromosome 4 (63 of 73) show an increase in PI, similar to what we observed in mutants lacking HP1a (Figure S13). The PI changes on chromosome 4 (relative to wildtype) are significant (p,3.9e-5), but those for the remainder of the genome are not (Figure 5C). Also similar to HP1a mutants, this shift in RNA pol II distribution, specifically the reduction of RNA pol II over the gene body, is unique to chromosome 4. To develop a better understanding of the genes undergoing a shift in RNA pol II distribution in the HP1a and POF mutants, we examined their association with various chromosomal proteins in wildtype (Figure S14). We find no exceptional enrichment for any of the additional chromosomal proteins we examined. Correlating the change in PI observed for the chromosome 4 genes in the HP1a and POF mutants with protein enrichment levels yields only low correlation values, with the highest value being r = 0.39 for RPD3 (Figure S14B). Together, our data from the HP1a and POF mutants suggest that chromosome 4 genes specifically respond to the disruption of their unique chromatin environment. That environment is dominated by high levels of HP1a over actively transcribing gene bodies, along with enrichment of POF – conditions that surprisingly result in RNA pol II enrichment over the gene bodies and low pausing indices. HP1a recruitment to chromosome 4 occurs by two mechanisms, one POF-dependent, one POF-independent It is likely that loss of POF or HP1a has a profound impact on the overall chromatin composition of chromosome 4, as suggested by the altered polymerase dynamics. To test this hypothesis, we investigated the relationship between EGG, POF, HP1a, H3K9me2, and H3K9me3 in several mutants at the third instar larval stage. ChIP-chip analysis of pof D119 chromatin reveals that over 90% of the HP1a enrichment observed on chromosome 4 in wildtype is abolished, and HP1a is now enriched in scattered peaks rather than the very broad domains observed in wildtype (Figure 6B, upper panel); these peaks are absent in profiles from HP1a mutants. Pericentric heterochromatin of chromosomes X, 2, and 3 (not normally associated with POF) retains strong enrichment for HP1a (Figure 6B, lower panel). Furthermore, we find that the strong HP1a enrichment over active gene bodies seen on wildtype chromosome 4 is lacking in pof mutant larvae (Figure 6B and 6D). Interestingly, the HP1a peaks remaining in pof mutants are located near repeats, with a median distance to repeats of 38 bp, significantly shorter than the 132 bp expected under a random distribution derived from chromosome 4 (Figure PLOS Genetics | www.plosgenetics.org 9 September 2012 | Volume 8 | Issue 9 | e1002954