Prerequisite Barcoding of Cell-Type-Rpecific Enhancers by ESC Transcription Factors in ESCs Licenses Their Robust Developmental Activation [RNA-seq]. Mus musculus

While cell-type-restricted enhancers are initially detected following cooperative binding of positionally-determined DNA binding transcription factors during determination/differentiation, it remains unknown whether there are preceding events in embryonic stem cells (ESCs) that are functionally important to activate cell-type-restricted enhancer networks. Here, using murine macrophages as a model, we report that, while largely devoid of characteristic enhancer marks (H3K4me1, H3K4me2, H3K27Ac, H3K27me3 and p300) in ESCs, macrophage enhancers are activated as transcription units mainly by the binding of a single, at most two, ESC transcription factors. This provides “premarking” of these enhancers, as is also observed for other cell types. In contrast, ESC-active enhancers are cooperatively bound by multiple ESC transcription factors, including Esrrb, Nanog, Oct4 and Sox2 (ENOS). Interestingly, the strength of this signature in ESCs is functionally important for subsequent robust cell-restricted enhancer activation during macrophage differentiation events, as independently demonstrated by analysis of multiple ENOS motif–deleted macrophage-restricted enhancers. The ENOS-determined location of hydroxymethylation of the enhancers in ESCs could 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 “barcoded” by binding of a single ESC transcription factor in ESCs, with the strength of their binding dictating enhancer activation in mature cells. Overall design: RNA-seq experiment was designed to understand transcription regulation in ESCs, the mutant ESC clones of Tlr1 locus generated by CRISPR/Cas9 and macrophage. The mutant clones (mutant #10, mutant #14) were deletion of 8bp Esrrb motif in Tlr1 enhancers, and no deletion in WT