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.
Highlights
Sox2 is one of the three core transcription factors (Oct4, Sox2, Nanog) responsible for maintaining embryonic stem cells (ESCs) pluripotency
Using a double-CRISPR excision strategy, we studied the in vivo function of this SE and demonstrated that it is responsible for over 90% of Sox2 gene expression in mouse ESCs
A different region upstream of the Sox2 locus is marked by H3K27ac in brain tissues (Fig. 1), suggesting that Sox2 is regulated by different mechanisms in ESCs and neural tissues involving different cis-regulatory elements
Summary
Sox is one of the three core transcription factors (Oct, Sox, Nanog) responsible for maintaining ESC pluripotency. These core pluripotency factors form autoregulatory loops and transcriptionally induce a cohort of other key pluripotency genes [1,2,3]. Elucidating the transcriptional regulation mechanisms of Sox gene is important for the understanding of both pluripotency and tumorigenesis. Enhancers play a critical role in regulating metazoan gene transcription [8,9,10,11]. Highly specific enhancer landscapes are responsible for cellular identity as they regulate distinct transcriptional programs in different cell types [12,13,14,15,16]. Compelling genomic evidence has predicted that SEs play a important role in the control of cell identity and diseases [17,18,19], but direct functional evidence is lacking
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