Massive zygotic transcription begins in many organisms during the midblastula transition when the cell cycle of the dividing egg slows down. A few genes are transcribed before this stage but how this differential activation is accomplished is still an open question. We have performed ChIP-seq experiments on tightly staged Drosophila embryos and show that
massive recruitment of RNA polymerase II (Pol II) with widespread pausing occurs de novo during the midblastula transition. However, ∼100 genes are strongly occupied by Pol II before this timepoint and most of them do not show Pol II pausing, consistent with a requirement for rapid transcription during the fast nuclear cycles. This global change in Pol II pausing correlates with distinct core promoter elements and associates a TATA-enriched promoter with the rapid early transcription. This suggests that promoters are differentially used during the zygotic genome activation, presumably because they have distinct dynamic properties.... A spreadsheet summarizing our classification and analysis of pre-MBT and MBT gene groups. The first sheet gives an explanation for all column headings. The second sheet lists all data for all our annotated genes, including our ten custom transcripts. It includes the classifications into pre-MBT and MBT gene groups, the Pol II ChIP-seq enrichment values at the transcription start site (TSS) and transcription unit (TU) for all replicates, phastCon conservation scores, and the presence or absence of all core promoter motifs analyzed in this study, as well as the presence of the TATA element identified by de novo motif analysis.... ChIP-seq... Drosophila melanogaster
ChIP-seq... Drosophila simulans... We subsampled reads from R2 ChIP-seq to 100x coverage using BBnorm (v37.54) with the parameters "threads=24 prefilter=t target=100", and created de novo contigs from the subsampled ChIPseq reads (ChIPtigs) with Spades v3.11.0 (-t 24 -careful –sc;).... Drosophila melanogaster... Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are amongst the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species Drosophila simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.... Drosophila... We created a custom Drosophila-specific consensus repeat library modified from RepBase v20150807 to include all complex satellite DNAs from Drosophila melanogaster.
Contributors:Kok, Kurtulus, Ay, Ahmet, Li, Li M., Arnosti, David N.
Expression profiling analysis: Transcriptome data from four biological replicates were generated using 8x15K Customized Drosophila Genome Oligo Microarrays (Agilent). Slide image data was quantified using Agilent's Feature Extraction software.... ChIP-Seq experiments were visualized as custom tracks using Integrative Genomics Viewer (Broad Institute). Total uniquely mapped tags were normalized to 10 million reads to generate tracks using HOMER.... Drosophila melanogaster... Metazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, we studied the conserved Hairy/Enhancer of Split family repressor Hairy, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. Our findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. We speculate that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits.