Pluripotency Factors Functionally Premark Cell-Type-Restricted Enhancers in ESCs

While enhancers for embryonic stem cell (ESC)-expressed genes and lineage-determining factors are characterized by conventional marks of enhancer activation in ESCs(1,2,3), it remains unclear whether enhancers destined to regulate cell-type-restricted transcription units might also have some currently overlooked, distinct signatures in ESCs. Here, we report that cell-type-restricted enhancers, are unexpectedly premarked and activated as transcription units by the binding of a single, or two, ESC transcription factors, although not exhibiting traditional enhancer epigenetic marks in ESCs, thus uncovering the initial temporal origins of cell-type-restricted enhancers. This premarking is required for future cell-type-restricted enhancer activity in the differentiated cells, with the strength of the ESCs signature being functionally important for subsequent robustness of cell-type-restricted enhancer activation. This model has been experimentally validated in macrophage-restricted enhancers and neural precursor cells (NPCs)-restricted enhancers using ESCs-derived macrophages or NPCs, edited to contain specific ESC transcription factor motif deletions. The ESC transcription factor-determined DNA hydroxyl-methylation of the enhancers in ESCs may serve as a potential molecular memory for subsequent enhancer activation in the mature macrophage. These findings suggest that the massive repertoire of cell-type-restricted enhancers are essentially hierarchically and obligatorily “premarked” by binding of a defining ESC transcription factor in ESCs, dictating robustness of enhancer activation in mature cells.