Zhou Harry Y, Katsman Yulia, Dhaliwal Navroop K, Davidson Scott, Macpherson Neil N, Sakthidevi Moorthy, Collura Felicia, Mitchell Jennifer A
Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada;
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
Genes Dev. 2014 Dec 15;28(24):2699-711. doi: 10.1101/gad.248526.114.
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.
在胚胎干细胞（ES细胞）中，Sox2转录因子必须被强劲转录以维持多能性。两个基因近端增强子，即Sox2调控区域1（SRR1）和SRR2，在报告基因检测中显示出活性，但删除SRR1对多能性没有影响。基于预测Sox2基因130kb范围内转录增强子元件的计算模型，我们鉴定并通过功能验证了Sox2转录所需的序列。我们的报告基因检测揭示了三个新的增强子——SRR18、SRR107和SRR111，它们在ES细胞中通过形成染色质环与Sox2启动子形成染色质复合体。利用CRISPR/Cas9系统和F1 ES细胞（小家鼠（129）×栗色小鼠），我们对每个增强子区域进行了杂合缺失，结果表明，只有位于Sox2下游>100kb处包含SRR107和SRR111的远端簇，才是ES细胞中Sox2顺式调控所必需的。此外，这个远端Sox2控制区域（SCR）的纯合缺失导致Sox2 mRNA和蛋白质水平显著降低、ES细胞集落形态丧失、全基因组基因表达变化，以及在自发分化为胚状体时神经外胚层形成受损。总之，这些数据确定了一个对ES细胞中Sox2转录至关重要的远端控制区域。
