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modENCODE_submission_4192 This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We will precisely identify sequence elements that direct DNA replication by using chromatin immunoprecipitation of known replication initiation complexes. These experiments will be conducted in multiple cell types and developmental tissues. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); Developmental Stage: Embryo 4-7h; Genotype: wild type; EXPERIMENTAL FACTORS: Strain Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); read length (read_length) ; Antibody dORC2 (target is Drosophila ORC2p); Developmental Stage Embryo 4-7h
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Additional file 2: Figure S1. Comparison of ChIP results in SuUR mutants and in wild type obtained with H3K27me3 antibodies from different vendors and with the antibodies against H3K27me2. A—scatter plot of ChIP-chip signals obtained with the Abcam #6002 antibodies in SuUR mutants (abscissa) and in wild type (ordinate) [13]. B—scatter plot showing H3K27me3 ChIP-seq signals obtained with Cell Signaling Technology #9733 (CST #9733) antibodies in the same genotypes. C—the same analysis performed with Millipore #07-452 antibodies against H3K27me2. Datapoints inside 193 SSRs are shown in red. In both cases (A and B) H3K27me3 antibodies produce the characteristic skew (arrows): SSRs systematically show stronger signal in wild type strain as compared to SuUR mutants. This tendency is absent in case of H3K27me2 (C).
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Insulators, chromatin domains and topologically associated domains (TADs). Interaction matrix representing a virtual Hi-C experiment (top). The grey scale above indicates interaction frequencies. Interactions occur predominantly within TADs (e.g. enhancer–promoter interactions), which are often grouped in subdomains. Interactions between TAD boundaries are thought to depend on the binding of CTCF (shown as a schematic ChIP-seq track in red) to its cognate DNA-binding motif (black arrows). CTCF sites not involved in binding to TAD boundaries are shown in pale red and grey motifs, respectively. Motifs involved in long-range chromatin interactions show an inverted repeat orientation (see Figure 2). As not all TAD boundaries are bound by CTCF it is likely that additional factors may be involved in their function (indicated by question mark). TADs are often co-incident with chromatin domains represented by a schematic ChIP-seq track for an active (H3K36me3; green) and a repressive (H3K27me3; blue) histone modification. Active TADs are gene-rich (black bars for active genes) in contrast to gene-poor repressed domains (grey bars). ... Insulator components with conserved features in vertebrates and Drosophila. ... Drosophila CP190 recruitment and strength of TAD boundaries/insulators correlate with combinatorial binding of architectural proteins. (a) The interaction matrix represents TADs. Boundaries between TADs are often marked by CP190 binding (schematic ChIP-seq track, blue). CP190 is recruited to chromatin by a wide variety of insulator binding factors (IBPs, as exemplified by CTCF, BEAF32 and Pita in schematic ChIP-seq tracks). Frequently, different insulator binding factors cluster together, suggesting a cooperative recruitment mode for targeting CP190 to chromatin. Combinatorial recruitment of CP190 to TAD boundaries may be functionally important since high occupancy of IBPs and other architectural proteins such as cohesin, condensin and TFIIIC predict the strength of insulator function as well as TAD borders [54••]. It should be noted that not all TAD boundaries are bound by known IBPs (?) and that many IBP binding sites are found within TADs. (b) The physical DNA string model summarizes the contact and binding data illustrated in (a).
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Distribution of lncRNA types in the different euchromatin regions. Figure S2. Occupied regions for each chromatin signature. Table S1. The length of lncRNAs. Table S2. RNA-seq datasets. Table S3. Statistics of exon numbers in lncRNA and mRNA genes from different sources. Table S4. Raw Ct values of RT-qPCR experiments for un-transcribed regions and the selected lncRNAs. Table S5. ChIP-seq datasets. Table S6. The primer list of the selected lncRNAs for RT-qPCR experiments. (PDF 356 kb)
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Full title: Complex patterns of genome accessibility discriminate sites of PcG repression, H4K16 acetylation and replication initiation Histone modifications have been proposed to regulate gene expression in part by modulating DNA accessibility and higher-order chromatin structure. However, there is limited direct evidence to support structural differences between euchromatic and heterochromatic fibers in the nucleus. To ask how histone modifications relate to chromatin compaction, we measured DNA accessibility throughout the genome by combining M.SssI methylase footprinting with methylated DNA immunoprecipitation (MeDIP-footprint). In the Drosophila genome, we find that accessibility to DNA methylase is variable in a manner that relates to the differential distribution of active and repressive histone modifications. Active promoters are highly permissive to M.SssI activity, yet inactive chromosomal domains decorated with H3 lysine 27 trimethylation are least accessible providing in vivo evidence for Polycomb-mediated chromatin compaction. Conversely, DNA accessibility is increased at active chromosomal regions marked with H4 lysine 16 acetylation and at the dosage-compensated male X chromosome suggesting that Drosophila transcriptional dosage compensation is facilitated by more permissive chromatin structure. Interestingly early replicating chromosomal regions and sites of replication initiation show also higher accessibility linking temporal and spatial control of genome duplication to the structural organization of chromatin. In conclusion, using a novel protocol we generated a comprehensive view of DNA accessibility and uncover different levels of chromatin organization, which are delineated by distinct patterns of posttranslational histone modifications and replication. Keywords: cell type comparison, ChIP-chip, MeDIP-footprint, RNA-seq, ChIP-seq MeDIP-footprint and ChIP-chip: ChIP-chip was performed for H3K4me3, H3K36me2, H3K36me3, H3K27me3, and H3K9me2 in Kc cells. We measured DNA accessibility throughout the genome by combining M.SssI methylase footprinting with methylated DNA immunoprecipitation (MeDIP-footprint) in Kc and S2 cells. RNA-seq: cDNA from RNA from Drosophila Kc cells was sequenced using Illumina deep sequencing. Reads were mapped and the abundance of all transcripts was determined. ChIP-seq: PSC ChIP from Drosophila Kc cells was sequenced using Illumina deep sequencing in three lanes. Reads were mapped and the binding profile of PSC was determined.
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Distribution of lncRNA types in the different euchromatin regions. Figure S2. Occupied regions for each chromatin signature. Table S1. The length of lncRNAs. Table S2. RNA-seq datasets. Table S3. Statistics of exon numbers in lncRNA and mRNA genes from different sources. Table S4. Raw Ct values of RT-qPCR experiments for un-transcribed regions and the selected lncRNAs. Table S5. ChIP-seq datasets. Table S6. The primer list of the selected lncRNAs for RT-qPCR experiments. (PDF 356 kb)
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MSL3-Independent Chromatin Entry Sites Are a Subset of the Wild-Type Binding Pattern for MSL Complex and Coincide with the Strongest Enrichment Peaks Detected by ChIP-seq Two representative chromatin entry sites, CES11D1 (A) and CES15A8 (B), are shown. ChIP-chip profiles were generated from y w; MSL3-TAP; msl3 embryonic chromatin (WT) using IgG to IP the TAP epitope, or msl3 mutant embryos (CES) using anti-MSL2 antibodies. DNA resulting from ChIP was hybridized to custom NimbleGen tiling arrays (Alekseyenko et al., 2006). The y axis shows the log2 ratio of IP/Input signal. The ChIP-seq tag profile (Solexa) was obtained from an MSL3-TAP transformed male cell line, Clone 8, using IgG to IP the TAP epitope. The ChIP-seq profile displays broad distribution along WT MSL targets and high peaks that correspond to entry sites. The y axis shows the tag density. Gray lines within ChIP-chip and ChIP-seq panels indicate the regions identified as bound clusters (See Experimental Procedures for details). Genes are color-coded based on their transcriptional status (transcribed, red; nontranscribed, black; genes that are differentially transcribed in S2 and Clone 8 cells, salmon; and genes without transcriptional data, gray). Genes on the top row are transcribed left to right, and genes on the bottom row are transcribed from right to left. Numbers along the x axis refer to chromosomal position (bp) (Dm1 release coordinates). Polytene map cytological locations are indicated below.
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Heat map visualization of modENCODE ChIP-seq (left) and RNA-seq (right) data. ... Drosophila
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Additional file 1: Figure S1. dCTCF and CP190 peaks co-localize with previously identified dCTCF and CP190 binding sites. Peaks for dCTCF and CP190 were identified using MACS2 based on publicly available (GSE41354) ChIP-seq profiles published in Ong et al. [42] (PMID 24055367). Average binding of dCTCF as well as CP190 before and after expression of FLAG-dSUMO is shown across the known dCTCF (left) and CP190 (right) binding sites.
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Additional file 1: Figure S1. dCTCF and CP190 peaks co-localize with previously identified dCTCF and CP190 binding sites. Peaks for dCTCF and CP190 were identified using MACS2 based on publicly available (GSE41354) ChIP-seq profiles published in Ong et al. [42] (PMID 24055367). Average binding of dCTCF as well as CP190 before and after expression of FLAG-dSUMO is shown across the known dCTCF (left) and CP190 (right) binding sites.
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