A Sox2 distal enhancer cluster that regulates embryonic stem cell pluripotency

SOX2 is part of the core regulatory network of transcription factors required for pluripotency maintenance and cellular reprogramming.  Sox2 is expressed at high levels in pluripotent cells and down-regulated upon differentiation to endoderm or mesoderm while being maintained in the neuroectodermal lineage.  Two gene proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display enhancer activity in reporter assays, but we show here that they are not sufficient for expression of Sox2 in embryonic stem (ES) cells.  Our data reveal that a distal enhancer cluster located >100 kb downstream of the Sox2 transcription start site (TSS) is required for Sox2 expression in mouse ES cells.

Chromatin looping brings the distal enhancer cluster into close physical proximity to the Sox2 promoter in ES cells but not in mouse embryonic fibroblasts (MEFs).  Furthermore, reporter analysis of putative enhancer regions surrounding Sox2 revealed three new classical enhancers active in ES cells but not MEFs: SRR18, SRR107 and SRR111.  Surprisingly deletion of any of the more proximal enhancers, SRR1, SRR2, or SRR18 did not affect Sox2 expression in ES cells.  Next, we deleted the 7.3 kilobase enhancer cluster containing SRR107 and SRR111 which revealed that these two enhancers are required for Sox2 expression in ES cells.  Deletion caused an 8-fold reduction in Sox2 mRNA levels and differentiation into trophoblast-like cells indicating that the proximal regulatory sequences alone are not sufficient for Sox2 expression in ES cells.  These findings identify a distal enhancer cluster essential for Sox2 transcription and maintenance of ES cell pluripotency and demonstrate the requirement for deletion studies in functional assessment of enhancers to determine their regulatory roles.

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Dr Peter Fraser
Brian Heap Room