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High-resolution nucleosome mapping of targeted regions using BAC-based enrichment.

ABSTRACT: We report a target enrichment method to map nucleosomes of large genomes at unprecedented coverage and resolution by deeply sequencing locus-specific mononucleosomal DNA enriched via hybridization with bacterial artificial chromosomes. We achieved ? 10 000-fold enrichment of specific loci, which enabled sequencing nucleosomes at up to ? 500-fold higher coverage than has been reported in a mammalian genome. We demonstrate the advantages of generating high-sequencing coverage for mapping the center of discrete nucleosomes, and we show the use of the method by mapping nucleosomes during T cell differentiation using nuclei from effector T-cells differentiated from clonal, isogenic, naïve, primary murine CD4 and CD8 T lymphocytes. The analysis reveals that discrete nucleosomes exhibit cell type-specific occupancy and positioning depending on differentiation status and transcription. This method is widely applicable to mapping many features of chromatin and discerning its landscape in large genomes at unprecedented resolution.

SUBMITTER: Yigit E

PROVIDER: S-EPMC3627574 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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High-resolution nucleosome mapping of targeted regions using BAC-based enrichment.

Yigit Erbay E Zhang Quanwei Q Xi Liqun L Grilley Dan D Widom Jonathan J Wang Ji-Ping JP Rao Anjana A Pipkin Matthew E ME

Nucleic acids research 20130214 7

We report a target enrichment method to map nucleosomes of large genomes at unprecedented coverage and resolution by deeply sequencing locus-specific mononucleosomal DNA enriched via hybridization with bacterial artificial chromosomes. We achieved ≈ 10 000-fold enrichment of specific loci, which enabled sequencing nucleosomes at up to ≈ 500-fold higher coverage than has been reported in a mammalian genome. We demonstrate the advantages of generating high-sequencing coverage for mapping the cente ...[more]

PMID: 23413004

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Project description:Genome-wide mapping of nucleosomes has revealed a great deal about the relationships between chromatin structure and control of gene expression, and has led to mechanistic hypotheses regarding the rules by which chromatin structure is established. High-throughput sequencing has recently become the technology of choice for chromatin mapping studies, yet analysis of these experiments is still in its infancy. Here, we introduce a pipeline for analyzing deep sequencing maps of chromatin structure and apply it to data from S. cerevisiae. We analyze digestion series where nucleosomes are isolated from under- and over-digested chromatin. We find that certain classes of nucleosomes are unusually susceptible or resistant to overdigestion, with promoter nucleosomes easily digested and mid-coding region nucleosomes being quite stable. We find evidence for highly sensitive nucleosomes located within “nucleosome-free regions,” suggesting that these regions are not always completely naked but instead are likely associated with easily-digested nucleosomes. Finally, since RNA polymerase is the dominant energy-consuming machine that operates on the chromatin template, we analyze changes in chromatin structure when RNA polymerase is inactivated via a temperature-sensitive mutation. We find evidence that RNA polymerase plays a role in nucleosome eviction at promoters, and is also responsible for retrograde shifts in nucleosomes during transcription. Loss of RNA polymerase results in a relaxation of chromatin structure to more closely match in vitro nucleosome positioning preferences. Together, these results provide analytical tools and experimental guidance for nucleosome mapping experiments, and help disentangle the interlinked processes of transcription and chromatin packaging. Keywords: Genomic Solexa/Illumina sequencing of mononucleosomes. Titration series: gel-purified mononucleosomal DNA from three different titration levels – underdigested, typical digestion, and overdigested. RP021 series: MNase-seq at 0, 20, and 120 minutes after shifting these cells from 25 C to 37 C.

Dataset Information

High-resolution nucleosome mapping of targeted regions using BAC-based enrichment.

Publications

High-resolution nucleosome mapping of targeted regions using BAC-based enrichment.

Similar Datasets

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