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A central question in biology is how enhancers are made accessible. The Drosophila embryo is a good model system to study this question as the gene regulatory networks regulating early developmental events have been well characterized. Zelda (Zld) is a uniformly distributed transcription factor (TF) integral to these networks, acting prior to and in collaboration with the patterning TFs to regulate target enhancers. Here we test the hypothesis that Zld directs TF binding, examplified by Dorsal (Dl) which patterns the dorsoventral axis, across the genome by displacing nucleosomes at enhancers. By performing ChIP-seq and MNase-seq experiments on early embryos with or without Zld, we demonstrated that early enhancers are characterized by an intrinsically high nucleosome barrier, which is overcome by Zld, and that without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that enhancers are initially specified across the genome by the binding of Zld, which locally decreases nucleosome occupancy, thereby assisting TFs in accessing their binding motifs and promoting transcriptional activity. Zld, Dl, Pol II ChIP-seq and MNase-seq profiles comparing 2-3h wild-type (wt) and zld- embryos, and MNase-seq profiles comparing 2-4h wt and gd7 embryos, all in 2 replicates
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Genome-wide binding profile of PcG proteins Pho and Ph, H3K27me3 and Engrailed in Drosophila third instar larval brains and disks, in duplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed. Drosophila third instar larval brains and disks were dissected and fixed with 2% HCHO before sonication. After sonication, chromatin immunoprecipitation using anti-Pho, -Ph, -H3K27me3, and -Engrailed antibodies was carried out.
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Genome-wide binding profile of PcG proteins- Ph, H3K27me3 and Cg in Drosophila third instar larval brains and disks, in duplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed. Drosophila third instar larval brains and discs were dissected and fixed with 2% HCHO before sonication. After sonication, chromatin immunoprecipitation was carried out.
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Chromatin immunoprecipitation using antibodies against pMad on 2 to 2.5 hr and 3 to 3.5 hr Drosophila embryos, detected by SOLiD sequencing
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Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but the DNA elements to which they can bind are not always sufficient to specify their binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones which package DNA into nucleosomes. Here, we examine using the ChIP-seq assay, the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. We detect HSF binding to 464 sites, the vast majority of which contain HSF Sequence-binding Elements (HSEs) in Drosophila S2 cells, but these HSF-bound sites represent only a small fraction of HSEs present in the genome. We find a strong correlation of bound HSEs to active chromatin marks present prior to HSF binding, indicating an HSE’s residence in open chromatin is a primary determinant of whether HSF can bind following heat shock. A single mock immunoprecipitation (IP) using the pre-innoculated animal serum was used as a background dataset for this study. For each condition (NHS and 20minute HS), we performed two independent HSF-ChIP-seq experiments. In addition, we performed two independent HSF-ChIP-seq experiments for each condition (NHS and 20minute HS) in cells that were highly depleted of HSF by RNAi.
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We report here the genome-wide localization of the histone acetyltransferase MYST5 (cg1984) in Drosophila. ChIP-seq analysis was performed with two anti-MYST5 antibodies in S2 cells. We found MYST5 to bind to the promoters of actively transcribed genes. MYST5 furthermore showed extensive colocalization with boundary/ insulator factors, including Chriz/ Chromator, CP190, dCTCF and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains. Examination of genome-wide MYST5 localization in S2 cells
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Here we examine changes in the distribution of Drosophila insulator proteins during the ecdysone response. We performed ChIP-seq analysis in Kc cells at 0, 3, and 48 hours of ecdysone treatment with antibodies against CP190, Su(Hw), dCTCF, and BEAF-32B. Examination of 4 different insulator proteins at 3 time points of ecdysone treatment.
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We map the genome-wide binding profiles for dTFIIIC220, as well as the Drosophila cohesin and condensin complexes in the late-stage embryonic cell line Kc167 Examination of several proteins in the Kc167 cell line
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We analyzed ORC2 ChIP-Seq from hand dissected salivary glands of wandering third instar larvae from OrR or SuUR Drosophila. Goals were to ascertain the difference in binding profile between salivary glands expressing and not expressing the Supressor of UnderReplication protein. One replicate is included for each of OrR (WT) or SuUR salivary glands.
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Chromatin immunoprecipitation using antibodies against Brk on 2 to 2.5 hr and 3 to 3.5 hr Drosophila embryos, detected by SOLiD sequencing
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