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DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.

FACT (facilitates chromatin transcription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation of spt16 results in a global loss of nucleosomes as well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037 Spt16-suppressed noncoding transcripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion. [ABSTRACT FROM AUTHOR]

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships. [ABSTRACT FROM AUTHOR]

An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR. [ABSTRACT FROM AUTHOR]

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation. [ABSTRACT FROM AUTHOR]