CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy [MNase-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Marek Bartkuhn
PROVIDER: E-GEOD-51599 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.
Bohla Dorte D Herold Martin M Panzer Imke I Buxa Melanie K MK Ali Tamer T Demmers Jeroen J Krüger Marcus M Scharfe Maren M Jarek Michael M Bartkuhn Marek M Renkawitz Rainer R
PloS one 20140923 9
Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome ...[more]
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Project description:H3-ChIP-seq was performed in order to analyze changes in nucleosomal occupancy after depletion of CTCF/P190 and ISWI from Drosophila S2 cells Histone H3 ChIP-seq from Drosophila S2 cells after CTCF/CP190 or ISWI-specific RNAi treatment
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Project description:MNase-seq was performed in order to analyze changes in nucleosomal occupancy after depletion of CTCF/P190 and ISWI from Drosophila S2 cells
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Project description:During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we find that the interplay between these factors depends on their large-scale genomic context. The mostly unmethylated CpG islands have a reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated and their average density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the level of TET1 and 5hmCs goes down. In particular, this has a dramatic effect at variable CTCF sites enriched outside CpG islands, that are occupied by CTCF in ESCs but loose CTCF during differentiation. To rationalize this cell type dependent targeting of CTCF binding in competition with the histone octamer, we have developed a quantitative biophysical model, which allowed predicting differences in CTCF binding due to TET1/5hmC/5mC-dependent nucleosome repositioning. MNase-seq experiments for analysis of mononucleosomes were conducted as described previously (Teif et al. Nature Struct. Mol. Biol., 2012). In addition, a dinucleosome fraction also was gel purified and sequenced. Briefly, embryonic stem cells from 129P2/Ola mice were cultured in ESGRO complete medium (Millipore), harvested and resuspended in low salt buffer (10 mM Hepes, pH 8, 10 mM KCl, 0.5 mM DTT) at 4 M-BM-0C. After disruption of the cells with a douncer the nuclei were collected by centrifugation and washed once with the MNase Buffer (10 mM TrisM-bM-^@M-^SHCl, pH 7.5, 10 mM CaCl2), resuspended in the MNase Buffer and digested with 0.5 units MNase per microliter (Fermentas) and incubation for 6M-bM-^@M-^S11 minutes at 37 M-BM-0C. The MNase digestion was stopped by putting the samples on ice and adding EDTA to a concentration of 10 mM. After digestion with 0.1 M-BM-5g M-BM-5lM-bM-^@M-^S1 RNase A (Fermentas) and removal of protein by phenol and chloroform extraction, the DNA was ethanol precipitated and the resulting DNA pellet was dissolved in H2O. DNA fragments corresponding to mononucleosomes or dinucleosomes were separated on a 2 % agarose gel using an E-Gel electrophoresis system (Life Technologies). The libraries for sequencing were prepared according to the standard Illumina protocol. High-throughput paired-end sequencing of at least 50 bp read length was performed on the Illumina HiSeq 2000 platform at the DKFZ sequencing core facility in Heidelberg, Germany. We obtained about 150 million nucleosome positions per sequencing reaction. ChIP-seq experiments were conducted as described previously (Teif et al. 2012). For each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes, pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 M-BM-5g of either H3K9me3 (Abcam ab8898) or H3K4me3 (Abcam ab8580) antibody over night. After washes with sonication (10 mM TrisM-bM-^@M-^SHCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% NaM-bM-^@M-^Sdeoxycholate), high-salt-buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaM-bM-^@M-^Sdeoxycholate, 0.1% SDS), lithium buffer (20 mM TrisM-bM-^@M-^SHCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% NaM-bM-^@M-^Sdeoxycholate) and 10 mM TrisM-bM-^@M-^SHCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina HiSeq 2000 sequencing platform (single reads of 50 bp length).
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Project description:CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy [MNase-seq]
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