The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.

The S.A.B. laboratory research is supported by the European Research Council (ERC), the Worldwide Cancer Research Foundation, the Foundation La Marató de TV3, the Spanish Ministry of Economy and Development, the Foundation Vencer el Cancer (‘‘Beat Cancer’’), the Government of Cataluña (SGR and Mario Salvia’ grants), the Foundation Fundación Botín, and the Institute for Research in Biomedicine (IRB-Barcelona). L.R. is a La Caixa Foundation Ph.D. fellow. G.S. was supported by an AXA postdoctoral fellowship. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). L.D.C. was supported by grants from the Spanish Ministerio de Educación y Ciencia (SAF2013-48926-P) and the European Commission’s 7th Framework Program 4DCellFate grant number 277899. We are grateful to the Common Fund’s Epigenomic Program from the NIH (USA) for providing the bisulphite whole genome sequencing data of human EpSCs