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A simple method for generating high-resolution maps of genome-wide protein binding.

ABSTRACT: Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution, and newer techniques require significant experimental alterations and complex bioinformatics. Previously, we have used a new crosslinking ChIP-seq protocol (X-ChIP-seq) to perform high-resolution mapping of RNA Polymerase II (Skene et al., 2014). Here, we build upon this work and compare X-ChIP-seq to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity, to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base-pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing.

SUBMITTER: Skene PJ

PROVIDER: S-EPMC4480131 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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A simple method for generating high-resolution maps of genome-wide protein binding.

Skene Peter J PJ Henikoff Steven S

eLife 20150616

Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution, and newer techniques require significant experimental alterations and complex bioinformatics. Previously, we have used a new crosslinking ChIP-seq protocol (X-ChIP-seq) to perform high-resolution mapping of RNA Polymerase II (Skene et al., 2014). Here, we build upon this work ...[more]

PMID: 26079792

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Project description:BackgroundGenome-wide DNA methylation at a single nucleotide resolution in different primary cells of the mammalian genome helps to determine the characteristics and functions of tissue-specific hypomethylated regions (TS-HMRs). We determined genome-wide cytosine methylation maps at 91X and 36X coverage of newborn female mouse primary dermal fibroblasts and keratinocytes and compared with mRNA-seq gene expression data.ResultsThese high coverage methylation maps were used to identify HMRs in both cell types. A total of 2.91% of the genome are in keratinocyte HMRs, and 2.15% of the genome are in fibroblast HMRs with 1.75% being common. Half of the TS-HMRs are extensions of common HMRs, and the remaining are unique TS-HMRs. Four levels of CG methylation are observed: 1) total unmethylation for CG dinucleotides in HMRs in CGIs that are active in all tissues; 2) 10% to 40% methylation for TS-HMRs; 3) 60% methylation for TS-HMRs in cells types where they are not in HMRs; and 4) 70% methylation for the nonfunctioning part of the genome. SINE elements are depleted inside the TS-HMRs, while highly enriched in the surrounding regions. Hypomethylation at the last exon shows gene repression, while demethylation toward the gene body positively correlates with gene expression. The overlapping HMRs have a more complex relationship with gene expression. The common HMRs and TS-HMRs are each enriched for distinct Transcription Factor Binding Sites (TFBS). C/EBPβ binds to methylated regions outside of HMRs while CTCF prefers to bind in HMRs, highlighting these two parts of the genome and their potential interactions.ConclusionsKeratinocytes and fibroblasts are of epithelial and mesenchymal origin. High-resolution methylation maps in these two cell types can be used as reference methylomes for analyzing epigenetic mechanisms in several diseases including cancer. Please see related article at the following link: http://www.epigeneticsandchromatin.com/content/7/1/34.

Dataset Information

A simple method for generating high-resolution maps of genome-wide protein binding.

Publications

A simple method for generating high-resolution maps of genome-wide protein binding.

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