of Su(var)205 (HP1a), egg, and pof products instead suggests a model where there is a simultaneous requirement for EGG and POF, which together create conditions to recruit HP1a to active gene bodies on chromosome 4, presumably utilizing H3K9 methylation by EGG. EGG and POF are reported to physically interact with each other in vivo [22], providing a mechanism for this process. How the complex is targeted to chromosome 4 active genes remains to be established. An interesting aspect of our study is that on chromosome 4, the association between HP1a, H3K9me2, and H3K9me3 is substan- tially different from what is observed in pericentric heterochro- matin (Figure 2). The loss of the strong correlation between H3K9me2 and H3K9me3 on chromosome 4 is likely due to differences in H3K9 HMTs. While little is known about G9a, both EGG and SU(VAR)3-9 have been examined in our study and by others [20–22,56,57]. Both EGG and SU(VAR)3-9 are found on chromosome 4, but the predominant H3K9 methylation signal depends on EGG [20–22,37]. Our analysis suggests that H3K9me2 and H3K9me3 enrichment on chromosome 4 reflects both HP1a-dependent and HP1a-independent mechanisms. H3K9me2 and H3K9me3 enrichment on chromosome 4 reflects an EGG-dependent mechanism to modify the histone over the body of the genes, and an EGG-independent mechanism to modify the histone associated with repeat sequences (Figure 8). Presumably the latter reaction is carried out by SU(VAR)3-9. We suggest that this activity of SU(VAR)3-9 was missed in the polytene chromosome studies of Su(var)3-9 mutants [13], as EGG appears to be responsible for ,80% of the H3K9me2/me3 signal on chromosome 4 in our analysis. Currently, it is unclear how the HMT activities on chromosome 4 are coordinated. In HeLa cells, several H3K9 HMTs interact with each other [58], thus providing potential mechanisms for coordination. However, how the enzymes on Drosophila chromosome 4 produce the H3K9me2 and H3K9me3 enrichment pattern as well as the active gene- specific increase in H3K9me3 remains to be discovered. The available data suggest the following model for the assembly of chromatin on chromosome 4 and regulation of the genes in this domain (Figure 9). Two mechanisms recruit HP1a to chromosome 4, one dependent on POF and EGG, the other independent of these components. POF is required for the recruitment of HP1a and H3K9 methylation in gene bodies of actively transcribed genes, and EGG appears to be required for significant recruitment or stabilization of POF. POF in turn interacts with the nascent transcript, positively affecting transcript output. Neither POF nor EGG is required for the recruitment of HP1a and the presence of H3K9me2/me3 at repeat clusters (and silent genes) on the chromosome 4. These findings suggest that the same general mechanisms that result in heterochromatic packaging of repeti- tious, TE-derived DNA in pericentric heterochromatin are at work here as well. Studies in plants and some fungi suggest that small RNAs play a role in targeting heterochromatin formation, and there is growing evidence for such a mechanism establishing heterochromatin patterns in the germline and early embryo of Drosophila [59,60]. However, direct targeting of one of the heterochromatin components by other means (such as direct DNA recognition) remains a possibility. The analysis above clearly shows that chromosome 4 is a mosaic of HP1a-associated domains, with each of the two modes of assembly detected here potentially impacting gene expression. Materials and Methods Datasets used are listed in Table S1 (cell lines) and Table S2 (mutants). Flybase version 5.12 was used for all analyses. Drosophila Chromosome 4 Chromatin Structure Figure 9. A model illustrating the two mechanisms proposed for HP1a assembly on chromosome 4. In active transcribed regions, POF and EGG are recruited, which leads to high levels of HP1a across the gene body (left). Details are unknown; POF is not reported to interact directly with HP1a, so other protein partners may be involved, in addition to HP1a binding to H3K9me2/3. The chromatin complex containing POF, HP1a and other partners has a general positive effect on RNA pol II – possibly by affecting transcription elongation - specifically on chromosome 4. In silent, repeat-rich regions, which lack POF enrichment, H3K9me marks deposited by a second histone methyltransferase (presumably SU(VAR)3-9) lead to a POF-independent assembly of HP1a–containing chromatin (right). Note that neither EGG, POF, nor SU(VAR)3-9 are known to interact directly with DNA; the binding described occurs in a chromatin context. doi:10.1371/journal.pgen.1002954.g009 Additional details on materials and methods can be found at www.modENCODE.org. Fly stocks and culture conditions Fly stocks were maintained on standard cornmeal media at 25uC with 70% humidity [61]. Mutant third instar larvae lacking HP1a were recovered from a cross of flies carrying the Su(var)20504 allele [62] over a GFP balancer to flies carrying the Su(var)20505 allele [62] over a GFP balancer by selecting for lack of GFP. Mutant third instar larvae lacking POF were recovered from a homozygous stock of the pof D119 allele [36]. Mutant third instar larvae lacking EGG were recovered from a heterozygous stock carrying the egg10.1-1a allele [21] over a GFP balancer by selecting for larvae lacking GFP. Cell lines S2-DRSC cells (stock #181) and ML-DmBG3-c2 cells (stock #68) were obtained from the Drosophila Genome Resource Center. Both cell lines are grown at 25uC with 70% humidity according to modENCODE protocols. S2-DRSC cells were grown to a density from 106 to 107 cells/ml in Schneider’s media supplemented with 10% FCS (fetal calf serum), and ML-DmBG3- c2 cells were grown to a density from 26106 to 1.26107 cells/ml in Schneider’s media supplemented with 10% FCS and 10 mg/ml insulin. Antibody characterization All antibodies used for ChIP experiments were characterized using immunoblotting or immunofluorescence to ensure the specificity of the antibody to recognize the histone modification or chromosomal protein in question. Validation protocols for histone antibodies were described in detail in a recent article and consisted of a test for cross-reactivity with non-histone Drosophila proteins as well as a test for modification specificity [63]. Other chromosomal proteins were tested by two methods, immuno- blotting or immunofluorescence, to check for cross-reactivity with non-target proteins. By western blot analysis, an antibody PLOS Genetics | www.plosgenetics.org 15 September 2012 | Volume 8 | Issue 9 | e1002954