Abstract

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications can functionally define active enhancers remains unclear. Here we combine chromatin immunoprecipitation with a massively parallel reporter assay to identify functional enhancers in human embryonic stem cells (hESCs) genome-wide in a quantitative unbiased manner. While active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac and H3K4me1 function as enhancers, with activity changing markedly with culture conditions. Our analysis also reveals a novel enhancer set associated with housekeeping genes. Moreover, while transposable elements associate with putative enhancers only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalogue of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.\n\nHighlightsO_LIA catalog of functional enhancers in hESCs including a novel housekeeping class\nC_LIO_LIActive enhancers feature specific transcription factors and transposable elements\nC_LIO_LIMajor shifts in enhancer activity occur during induction of naive pluripotency\nC_LIO_LISuper-enhancers consist of small units with enhancer function\nC_LI