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H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

ABSTRACT: The bithorax complex (BX-C) in the fruit fly, Drosophila melanogaster, is a cluster of homeotic genes that determines the identities the body segments. Expression of these genes is governed by cis-regulatory domains, one for each parasegment, which are arranged on the chromosome in the order of the parasegments they affect. Stable repression of these domains depends on the functions of the Polycomb Group, including its ability to methylate lysine 27 of histone H3. To learn whether PcG proteins generate parasegment-specific chromatin signatures, we have used transgenes to mark and isolate nuclei from single parasegments. These nuclei were profiled for histone modifications and chromatin proteins. The H3K27me3 profiles across the BX-C in successive parasegments show a striking “stairstep” pattern that reveals sharp boundaries of the BX-C regulatory domains. The borders of H3K27me3 modification domains are sharp, and align precisely with binding sites for the CCCTC-binding protein (CTCF). H3K27ac is broadly enriched across active domains, in a pattern complementary to K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely localized H3K27 modification as a central determinant of segment identity. Nuclei from single parasegments of the Drosophila embryo were isolated and ChIP-seq performed for H3K27me3, CTCF, H3K27ac, H3K4me3, Ph, Pol2, Pc, and Suz12. For each sample, two biological replicates were performed.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Ruslan Sadreyev

PROVIDER: E-GEOD-55257 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

Bowman Sarah K SK Deaton Aimee M AM Domingues Heber H Wang Peggy I PI Sadreyev Ruslan I RI Kingston Robert E RE Bender Welcome W

eLife 20140731

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The ...[more]

PMID: 25082344

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Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain; (2) to profile key epigenetic marks H3K4me3, H3K27me3 and DNA methylation in wild type and Smchd1 null NSCs; (3) to identify the chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs. Methods: Chromatin immunoprecipitation for Smchd1, H3K4me3, H3K27me3 and Ctcf was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked NSCs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Smchd1, H3K4me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit or Ovation Ultralow system (NuGen) according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. To examine the level of DNA methylation, genomic DNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) and methylated DNA was isolated via binding to the methyl-CpG binding domain of human MBD2 protein coupled beads using the MethylMiner methylated DNA enrichment kit (Life Technologies) according to the manufacturer’s instructions. Isolated DNA was used to generate sequencing libraries as for the ChIP-seq experiment with Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer's instruction and sequenced on the Illumina HiSeq 2000 platform for 49 bp single-end reads. Sequencing analysis was performed as described for the ChIP-seq experiments. Chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain was determined by Smchd1 ChIP-seq. Enrichment of H3K4me3 and H3K27me3 in wild type and Smchd1 null NSCs were assessed by H3K4me3 and H3K27me3 ChIP-seq, respectively. DNA methylation in wild type and Smchd1 null NSCs was assessed by MBD-seq. Chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs was determined by by Ctcf ChIP-seq.

Dataset Information

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

Publications

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

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