Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the fly D. melanogaster, and compare it to that from the yeast S. cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical −1, NFR (nucleosome-free region), +1 arrangement. However, Drosophila does not incorporate H2A.Z into the −1 nucleosome and does not bury its transcriptional start site in the +1 nucleosome. At thousands of genes, RNA polymerase II engages the +1 nucleosome and pauses. How the transcription initiation machinery contends with the +1 nucleosome appears to be fundamentally different between lower and higher eukaryotes.Knowledge of the precise location of nucleosomes in a genome is essential in order to understand the context in which chromosomal processes such as transcription and DNA replication operate. A common theme to emerge from recent genome-wide maps of nucleosome locations is a general deficiency of nucleosomes in promoter regions and an enrichment of certain histone modifications towards the 5′ end of genes [1][2][3][4][5][6][7] . A high resolution genomic map of nucleosome locations in the budding yeast S. cerevisiae has further revealed Correspondence and request for material should be addressed to B.F.P. (bfp2@psu.edu). * These authors contributed equally to this work.Author Information Sequence data deposition is through NCBI Trace Archives TI SRA000283, Sequencing Center = "CCGB", and microarray deposition through ArrayExpress, Accession numbers E-MEXP-1515 and -1519. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interest.Author Contributions T.M. prepared and purified the nucleosomes including Pol II-bound nucleosomes; C.J. analyzed the nucleosome mapping data and its relationship to other genomic features; I.P.I. performed computational analyses related to nucleosome positioning sequences; X.L. conducted ChIP-chip on Pol II; B.J.V. conducted ChIP-chip on nucleosome-Pol II interactions; S.J.Z. provided bioinformatics support; L.T. constructed libraries and sequenced nucleosomal DNA; J.Q. mapped sequencing reads to the yeast genome; RG provided H2A.Z antibodies; SCS directed the DNA sequencing phase; DSG directed embryo preparations and helped interpret the data; I.A. developed computational approaches to derive nucleosome maps from the read locations and developed the associated browser; B.F.P. directed the project, interpreted the data, and wrote the paper. S6). Those 112,750 nucleosomes detected three or more times were further analyzed, although patterns were identical when all nucleosomes were analyzed. The internal median error of the data was 4 bp (Fig. S7). H2A.Z nucleosomes were predominantly distributed at 175 bp intervals from the TSS (compared to 165 ...

The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning, and other early developmental processes; and the zinc-finger protein Zelda (ZLD) plays a key role in their transcriptional activation. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD before (mitotic cycle 8), during (mitotic cycle 13), and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8 embryos, most of which remain bound through mitotic cycle 14. As expected, early ZLD-bound regions include the promoters and enhancers of genes transcribed at this early stage. However, we also observed ZLD bound at cycle 8 to the promoters of roughly a thousand genes whose first transcription does not occur until the MZT and to virtually all of the thousands of known and presumed enhancers bound at cycle 14 by transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription but also plays a major role in activating the genome at the MZT.

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior–posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal–ventral patterning genes, whose expression we show to be quantitatively modulated by anterior–posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets.

Transcription factor binding in Drosophila

Distinct developmental fates in Drosophila melanogaster are specified by quantitative differences in transcription factor occupancy on a common set of bound regions.