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PLoS One. 2014 Dec 8;9(12):e114485. doi: 10.1371/journal.pone.0114485. eCollection 2014.

CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells.

Author information

1
Ludwig Institute for Cancer Research, San Diego, California, United States of America.
2
Ludwig Institute for Cancer Research, San Diego, California, United States of America; The Biomedical Sciences Graduate Program, University of California San Diego, School of Medicine, San Diego, California, United States of America.
3
Ludwig Institute for Cancer Research, San Diego, California, United States of America; Medical Scientist Training Program, University of California San Diego, School of Medicine, San Diego, California, United States of America.
4
Ludwig Institute for Cancer Research, San Diego, California, United States of America; Department of Cellular and Molecular Medicine, Institute of Genome Medicine, Moores Cancer Center, University of California San Diego, School of Medicine, San Diego, California, United States of America.

Abstract

The pluripotency of embryonic stem cells (ESCs) is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although previous studies have identified transcriptional regulators of this core network, the cis-regulatory DNA sequences required for the transcription of these key pluripotency factors remain to be defined. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-enhancer (SE) located 100kb downstream of Sox2 in mouse ESCs. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy we deleted the entire 13-kb SE and characterized transcriptional defects in the resulting monoallelic and biallelic deletion clones with RNA-seq. We showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. Our results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs.

PMID:
25486255
PMCID:
PMC4259346
DOI:
10.1371/journal.pone.0114485
[Indexed for MEDLINE]
Free PMC Article

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