Search

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function., United States. Dept. of Energy (Contract DE-AC02-05CH11231), RC2 HG005639, U01 HG004279, R01 GM082798, R37 GM45744, R01 GM071923, U54 HG004592, National Science Foundation (U.S.) (NSF 0905968)

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal ‘arms’, and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function., National Science Foundation (U.S.) (1122374)

Summary Dosage compensation in Drosophila is mediated by the MSL complex, which increases male X-linked gene expression approximately 2-fold. The MSL complex preferentially binds the bodies of active genes on the male X, depositing H4K16ac with a 3′ bias. Two models have been proposed for the influence of the MSL complex on transcription: one based on promoter recruitment of RNA polymerase II (Pol II), and a second featuring enhanced transcriptional elongation. Here, we utilize nascent RNA sequencing to document dosage compensation during transcriptional elongation. We also compare X and autosomes from published data on paused and elongating polymerase in order to assess the role of Pol II recruitment. Our results support a model for differentially regulated elongation, starting with release from 5′ pausing and increasing through X-linked gene bodies. Our results highlight facilitated transcriptional elongation as a key mechanism for the coordinated regulation of a diverse set of genes.

Conrad et al . (Reports, 10 August 2012, p. 742) reported a doubling of RNA polymerase II (Pol II) occupancy at X-linked promoters to support 5′ recruitment as the key mechanism for dosage compensation in Drosophila . However, they employed an erroneous data-processing step, overestimating Pol II differences. Reanalysis of the data fails to support the authors’ model for dosage compensation.

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome., Author Summary The genomes of complex organisms encompass hundreds of millions of base pairs of DNA, and regulatory molecules must distinguish specific targets within this vast landscape. In general, regulatory factors find target genes through sequence-specific interactions with the underlying DNA. However, sequence-specific factors typically bind only a fraction of the candidate genomic regions containing their specific target sequence motif. Here we identify potential roles for chromatin environment and flanking sequence composition in helping regulatory factors find their appropriate binding sites, using targeting of the Drosophila dosage compensation complex as a model. The initial stage of dosage compensation involves binding of the Male Specific Lethal (MSL) complex to a sequence motif called the MSL recognition element [1]. Using data from a large chromatin mapping effort (the modENCODE project), we successfully identify an active chromatin environment as predictive of selective MRE binding by the MSL complex. Our study provides a framework for using genome-wide datasets to analyze and predict functional protein–DNA binding site selection.

Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA., 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 transcription. 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 melanogaster 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 combination 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.