Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

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

C/EBPβ Induces Chromatin Opening at a Cell-Type-Specific Enhancer▿

We have used the chicken mim-1 gene as a model to study the mechanisms by which transcription factors gain initial access to their target sites in compacted chromatin. The expression of mim-1 is restricted to the myelomonocytic lineage of the hematopoietic system where it is regulated synergistically by the Myb and CCAAT/enhancer binding protein (C/EBP) factors. Myb and C/EBPβ cooperate at two distinct cis elements of mim-1, the promoter and a cell-type-specific enhancer, both of which are associated with DNase I hypersensitive sites in myelomonocytic cells but not in mim-1-nonexpressing cells. Previous work has shown that ectopic expression of Myb and C/EBPβ activates the endogenous mim-1 gene in a nonhematopoietic cell type (fibroblasts), where the gene is normally completely silent. Here, we investigated the molecular details of this finding and show that the activation of mim-1 occurs by two independent mechanisms. In the absence of Myb, C/EBPβ triggers the initial steps of chromatin opening at the mim-1 enhancer without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with chromatin opening at the promoter. Our work identifies a novel function for C/EBPβ in the initial steps of a localized chromatin opening at a specific, physiologically relevant target region

Targeting the Transcriptional Machinery with Unique Artificial Transcriptional Activators

The link between a growing number of human diseases and misregulation of gene expression has spurred intense interest in artificial transcriptional activators that could be used to restore controlled expression of affected genes. To expand the repertoire of activation domains available for the construction of artificial transcriptional regulators, a selection strategy was used to identify two unique activation domain motifs. These activation domains bear little sequence homology to endogenous counterparts and bind to unique sites within the transcriptional machinery. A comparison with two well-characterized activation domains, VP2 and P201, demonstrated for the first time that functional potency is not solely dictated by binding affinity. Finally, the selection strategy described is readily applicable to the identification of small molecule activation domains

“Jump Start and Gain” Model for Dosage Compensation in Drosophila Based on Direct Sequencing of Nascent Transcripts

Dosage compensation in Drosophila is mediated by the MSL complex, which increases male X-linked gene expression approximately two-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 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 coordinated regulation of a diverse set of genes

Nature and function of insulator protein binding sites in the Drosophila genome

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

Enrichment of HP1a on drosophila chromosome 4 genes creates an alternate chromatin structure critical for regulation in this heterochromatic domain

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 T-m 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