Author Summary How DNA is packaged into chromatin has profound implications for gene regulation. While certain chromatin conformations are accessible to RNA polymerase and allow expression, other chromatin structures prevent transcrip- tion. In many genomes, genes that need to be expressed and repetitive sequences that need to be silenced are interspersed at close intervals. We use Drosophila melano- gaster chromosome 4 as one example of such a complex domain and ask how the genes on this chromosome are packaged and regulated. While the transcription start sites of active genes on chromosome 4 exhibit the expected pattern of chromatin marks, we see an unusual combina- tion of marks over expressed gene bodies, including enrichment of HP1a and H3K9me3. Deposition of HP1a over the gene bodies is dependent on POF (painting of fourth), while its association with intergenic repeat clusters is accomplished by a different mechanism. In this environment, promoter proximal RNA polymerase pausing is largely absent, despite the fact that genome-wide, approximately 10%–15% of all active genes display pausing. A redistribution of polymerase on chromosome 4 genes, including depletion in the gene body, is observed on HP1a depletion. These findings demonstrate how gene regulation mechanisms can be modulated in specific domains of the genome and illustrate the necessity of examining regulatory pathways within chromatin sub- domains, rather than relying on genome-wide averages or on a limited set of reporter genes. interspersed, with these repetitious elements found both within and between genes. In Drosophila melanogaster, the small chromosome 4 has an organization that is reminiscent of mammalian genomes. Its 1.2 Mb distal arm hosts approximately 80 genes and has a repeat density of ,30% [4]. (While the entire chromosome 4 is 4.2 Mb [5], we will refer here to the 1.2 Mb distal arm of chromosome 4 as ‘‘chromosome 4’’, as the other 3 Mb are composed of highly repetitive sequence for which no genome assembly is available.) Despite a high gene density, similar to that in euchromatic domains in Drosophila, chromosome 4 exhibits hallmarks of heterochromatin. It replicates late [6] and lacks recombination in the laboratory setting, although there is evidence of recombination events over evolutionary time [7,8]. In most cases reporter transgenes inserted into chromosome 4 are silenced by position effect, resulting in a variegating phenotype [9–11]; the entire chromosome is highly enriched for heterochromatic marks such as heterochromatin protein 1a (HP1a) and H3K9 di- and trimethyla- tion [12,13]. Recent work has demonstrated that chromosome 4 displays distinct chromatin profiles compared to both pericentric heterochromatin and euchromatic regions [14,15]. How genes are regulated in TE-rich domains with dense heterochromatic marks remains elusive, and Drosophila chromo- some 4 provides a system to study the mechanisms involved. Chromosome 4 also provides a potential example of domain-wide regulation with the presence of the painting of fourth (POF) protein, which exclusively binds to the distal 1.2 Mb portion of chromosome 4 [16]. POF binds to gene bodies, and expression of chromosome 4 genes is decreased in its absence [17]. In addition, compensation of gene expression in aneuploid chromosome 4 animals is mediated by POF, supporting its role in gene regulation on this chromosome [18]. Recent work has shown that POF binds nascent RNAs of chromosome 4 genes and that its association with chromosome 4 is dependent on active transcription [19]. Drosophila Chromosome 4 Chromatin Structure Intriguingly, HP1a is enriched in gene bodies with POF, and polytene chromosome analysis has suggested that HP1a and POF are interdependent for deposition on chromosome 4 [17]. Also present on chromosome 4 are two H3K9 histone methyltransfer- ases (HMTs), EGG – a SETDB1 class enzyme – and SU(VAR)3-9 [13,20–22]. SU(VAR)3-9 has been suggested to be enzymatically inactive on chromosome 4 (its main function appears to be in pericentric heterochromatin), while EGG appears responsible for maintaining the bulk of the H3K9me2 and H3K9me3 in this domain [20–22]. Co-immunoprecipitation experiments indicate that POF can interact with EGG [22], suggesting that regulation of gene expression on chromosome 4 involves EGG, POF, and HP1a. To further explore how genes function in a heterochromatin- like milieu in general, and on Drosophila chromosome 4 specifically, we examined the enrichment profiles of 20 histone modifications and 25 chromosomal proteins, drawing on new as well as previously published datasets profiled by the model organism Encyclopedia of DNA Elements (modENCODE) Drosophila group [23]. In addition, we mapped H3K9me2/3, H3K36me3, HP1a, POF, and RNA polymerase II (RNA pol II) by chromatin immunoprecipitation-microarray (ChIP-chip) tech- nology in mutant larvae lacking HP1a, POF, or EGG. Our results indicate that chromosome 4 genes are governed by a unique regulatory system characterized by a lack of RNA polymerase pausing, which may be a consequence of the presence of HP1a. We find that efficient POF recruitment is dependent on EGG, but not HP1a. Our results argue that HP1a is recruited to chromosome 4 by two mechanisms: the majority of HP1a (associated with genes) is dependent on POF, while a smaller fraction (associated with TE-rich regions) is POF-independent. We suggest a model where EGG, POF, and HP1a bind to active genes on chromosome 4 and together positively regulate their expres- sion. Results Mapping of additional chromatin components confirms that chromosome 4 is a distinct heterochromatic domain, rich in transcribed genes Earlier studies of chromosome 4 using cytological approaches established the enrichment of HP1a and noted a banded pattern, suggesting interspersed domains of low HP1a density that might favor gene expression. However, while low-resolution mapping with an hsp70-white reporter transgene indicated a few permissive domains (allowing full expression, red eye), the bulk of the insertions, including 12 within genes, resulted in a variegating phenotype, indicating heterochromatin packaging [9–11]. More recently, we used high-resolution genome-wide enrich- ment profiles of 16 histone marks and two proteins to identify and map predominant combinatorial chromatin states within hetero- chromatin [15]. Here, we expand this analysis to include four additional histone marks and 18 additional chromosomal proteins, whose enrichment in the original five predominant combinatorial chromatin states of heterochromatin is shown in Figure 1A. [Throughout this article, we will define pericentric heterochro- matin by enrichment in H3K9me2 as described in [15].] Several of the new proteins are enriched in heterochromatin states preferentially found on chromosome 4 (Figure 1A, states B–E). For example, chromosome 4 contains higher levels of POF, JIL-1, MOD(MDG4), HIS2AV (Figure 1A, states B–D), and some Polycomb-associated proteins (Figure 1A, state E) compared to pericentric heterochromatin in BG3 cells (Figure 1B). (For S2 cell data see Figure S1.) Compared to the pericentric heterochromatin PLOS Genetics | www.plosgenetics.org 2 September 2012 | Volume 8 | Issue 9 | e1002954