Unknown,Transcriptomics,Genomics,Proteomics

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High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm

ABSTRACT: Mammalian embryonic stem (ES) cells and sperm exhibit unusual chromatin packaging that plays important roles in cellular function. Here, we extend a recently developed technique, based on deep paired-end sequencing of lightly digested chromatin, to assess footprints of nucleosomes and other DNA-binding proteins genome-wide in murine ES cells and sperm. In ES cells, we recover well-characterized features of chromatin such as promoter nucleosome depletion, and further identify widespread footprints of sequence-specific DNA-binding proteins such as CTCF, which we validate in knockdown studies. We document global differences in nuclease accessibility between ES cells and sperm, finding that the majority of histone retention in sperm preferentially occurs in large gene-poor genomic regions, with only a small subset of nucleosomes being retained over promoters of developmental regulators. Finally, we describe evidence that CTCF remains associated with the genome in mature sperm, where it could play a role in organizing the sperm genome. We use Micrococcal Nuclease (MNase) to map chromatin structure in mouse ES cells and sperm. Specifically, we generate paired-end deep-sequencing libraries that are able to distinguish DNA digestion products by size, thus allowing us to simultaneously map nucleosomes as well as other DNA-binding proteins such as transcription factors.

ORGANISM(S): Mus musculus

SUBMITTER: Benjamin Carone

PROVIDER: E-GEOD-58101 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm.

Carone Benjamin R BR Hung Jui-Hung JH Hainer Sarah J SJ Chou Min-Te MT Carone Dawn M DM Weng Zhiping Z Fazzio Thomas G TG Rando Oliver J OJ

Developmental cell 20140703 1

Mammalian embryonic stem cells (ESCs) and sperm exhibit unusual chromatin packaging that plays important roles in cellular function. Here, we extend a recently developed technique, based on deep paired-end sequencing of lightly digested chromatin, to assess footprints of nucleosomes and other DNA-binding proteins genome-wide in murine ESCs and sperm. In ESCs, we recover well-characterized features of chromatin such as promoter nucleosome depletion and further identify widespread footprints of se ...[more]

PMID: 24998598

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Project description:As nucleosomes are widely replaced by protamine in mature human sperm, epigenetic contributions to embryo development appear limited. However, our genome-wide approaches find nucleosomes at low levels genome-wide, but also significantly enriched at imprinted gene clusters, miRNA clusters, HOX gene clusters, and the promoters of other developmental transcription and signaling factors. Developmental promoters were often DNA hypomethylated, and bore histone modifications localized to discrete locations: H3K4me2 is enriched at certain developmental promoters, whereas large blocks of H3K4me3 localize to a subset of developmental promoters, regions in HOX loci, certain non-coding RNAs, and generally to paternally-expressed imprinted loci. In contrast, H3K4me3 is generally absent at paternally-repressed imprinted loci. Interestingly, repressive H3K27me3 is enriched at many developmental promoters that lack early expression in embryos, with significant overlap with bivalent (H3K4me3/H3K27me3) promoters in ES cells. Taken together, epigenetic marking in sperm is extensive, and correlated with developmental regulators. *Use of ChIP-seq to identify regions enriched for histone, and several histone variants in human sperm. *Histone Localization: Chromatin was prepared from 40 million sperm as described previously (1) in the absence of crosslinking reagent, treated with sequential and increasing MNase (10U-160U), and centrifuged to sediment protamine-associated DNA, releasing mononucleosomes. The pooled mononucleosomes were gel purified (~140-155 bp) for sequencing and array analysis. Mononucleosomes for ChIP-seq were sequenced from a single donor (3 replicas combined) or a donor pool (4 donors each ~6 million reads) and data was normalized to 20 million input control reads (pooled donor sperm genomic DNA). *Chromatin IP and Preparation for Genomics Methods: ChIP methods were as described previously (2) but were performed without a cross linking agent and slight modifications to the salt levels, 250 mM NaCl, 200 mM LiCl, and replaced the TE wash with 150 mM PBS wash. A subset of the modifications H3K4me3, H3K27me3 and H2A.z were done from a donor pool and only analyzed by Solexa. Each IP was repeated three time and the three replicas were pooled. For sequencing, DNA lengths corresponding to mononucleosomes with adapters (220-280 bp) were gel purified after the addition of the Illumina adaptors. The sequencing data was normalized to histone donor pool (D1 was subsampled to 5million and combined with the reads from D2-D4). 1. Zalenskaya, I.A., Bradbury, E.M. & Zalensky, A.O. Chromatin structure of telomere domain in human sperm. Biochem Biophys Res Commun 279, 213-8 (2000). 2. Gordon, M. et al. Genome-Wide Dynamics of SAPHIRE, an Essential Complex for Gene Activation and Chromatin Boundaries. Mol Cell Biol 27, 4058-69 (2007). Samples corresponding to each GSE15690_Seq* processed data supplementary file: GSE15690_Seq_H2AZ.txt: GSM392695 GSE15690_Seq_H3K27Me3.txt: GSM392699, GSM392700 GSE15690_Seq_H3K4Me3.txt: GGSM392696-GSM392698 GSE15690_Seq_HistoneMnase_D1.txt: GSM392701-GSM392707, GSM392714-GSM392716 GSE15690_Seq_HistoneMnase_PooledDonor.txt: GSM392708-GSM392713, GSM392717-GSM392720

Dataset Information

High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm

Publications

High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm.

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