title: A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.
abstract: The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Harry Y
authors.initials: HY
authors.lastname: Zhou
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Yulia
authors.initials: Y
authors.lastname: Katsman
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Navroop K
authors.initials: NK
authors.lastname: Dhaliwal
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Scott
authors.initials: S
authors.lastname: Davidson
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Neil N
authors.initials: NN
authors.lastname: Macpherson
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Moorthy
authors.initials: M
authors.lastname: Sakthidevi
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
authors.forename: Felicia
authors.initials: F
authors.lastname: Collura
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada; Center for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario M5S 3G5, Canada ja.mitchell@utoronto.ca.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D005810:Multigene Family / Q000235:genetics; D054258:Neural Plate / Q000166:cytology; D011401:Promoter Regions, Genetic / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics
chemical_list: D002843:Chromatin; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse
keywords: Sox2; chromatin; embryonic stem cell; enhancer elements; gene expression regulation; transcription