Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide maps of five major transcription factors in epiblast stem cells

ABSTRACT: ChIP-seq data of ZIC2, OTX2, SOX2, POU5F1 and POU3F1 in EpiSCs ChIP-seq using biotinylated transcription factors

ORGANISM(S): Mus musculus

SUBMITTER: Hisato Kondoh

PROVIDER: E-GEOD-74636 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

ChIP-seq analysis of genomic binding regions of five major transcription factors highlights a central role for ZIC2 in the mouse epiblast stem cell gene regulatory network.

Matsuda Kazunari K Mikami Tomoyuki T Oki Shinya S Iida Hideaki H Andrabi Munazah M Boss Jeremy M JM Yamaguchi Katsushi K Shigenobu Shuji S Kondoh Hisato H

Development (Cambridge, England) 20170428 11

To obtain insight into the transcription factor (TF)-dependent regulation of epiblast stem cells (EpiSCs), we performed ChIP-seq analysis of the genomic binding regions of five major TFs. Analysis of <i>in vivo</i> biotinylated ZIC2, OTX2, SOX2, POU5F1 and POU3F1 binding in EpiSCs identified several new features. (1) Megabase-scale genomic domains rich in ZIC2 peaks and genes alternate with those rich in POU3F1 but sparse in genes, reflecting the clustering of regulatory regions that act at shor ...[more]

PMID: 28455373

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Project description:Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of their target site recognition. Most previous studies have examined predicted off-target binding sites that differ from the perfect target site by one to four mismatches, which represent only a subset of genomic regions. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites. For two guide RNAs targeting the murine Snurf gene promoter, we observed very high binding specificity at the intended target site while off-target binding was observed at 2- to 6-fold lower intensities. We also identified significant gRNA-independent off-target binding. Interestingly, we found that these regions are highly enriched in the PAM site, a sequence required for target site recognition by CRISPR. To determine the relationship between Cas9 binding and endonuclease activity, we used targeted sequence capture as a high-throughput approach to survey a large number of the potential off-target sites identified by ChIP-seq or computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results demonstrate that even a simple configuration of a Cas9:gRNA nuclease can support very specific DNA cleavage activity and that most interactions between the CRISPR nuclease complex and genomic PAM sites do not lead to DNA cleavage. ChIP-seq using dCas9 to determine genome-wide binding of CRISPR/Cas9 noED: Cas9 doublemutant protein without an effector domain KRAB: Cas9 doublemutant protein fused to the KRAB repressor domain S1 gRNA: guide RNA targeting GCTCCCTACGCATGCGTCCC(AGG) in the mouse genome S2 gRNA: guide RNA targeting AATGGCTCAGGTTTGTCGCG(CGG) in the mouse genome VEGFA TS3 gRNA: guide RNA targeting GGTGAGTGAGTGTGTGCGTG(TGG) in the human genome

Dataset Information

Genome-wide maps of five major transcription factors in epiblast stem cells

Publications

ChIP-seq analysis of genomic binding regions of five major transcription factors highlights a central role for ZIC2 in the mouse epiblast stem cell gene regulatory network.

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