A flexible repertoire of transcription factor binding sites and diversity threshold determines enhancer activity in embryonic stem cells

Summary Transcriptional enhancers are critical for development, phenotype evolution and often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. We found genomic regions with conserved binding of multiple transcription factors (TFs) in mouse and human embryonic stem cells (ESCs) are enriched in a diverse repertoire of transcription factor binding sites (TFBS) including known and novel ESC regulators. Remarkably, using a diverse set of TFBS from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site directed mutagenesis of conserved TFBS in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for >10 different TFBS for robust activity. Specific TFBS, including the OCT4:SOX2 co-motif, are dispensable, despite co-binding the OCT4, SOX2 and NANOG master regulators of pluripotency. These findings reveal a TFBS diversity threshold overrides the need for optimized regulatory grammar and individual TFBS that bind specific master regulators. Highlights ➢ Comparative epigenomics determines the enhancer sequence code and synthetic enhancer design. ➢ A diversity of >10 TFBS are required and sufficient for robust enhancer activity ➢ >40 TFBS contribute to enhancer activity implicating new TFs in pluripotency maintenance ➢ Increased TFBS diversity, above a threshold, overrides the need for regulatory grammar