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Specific histone modifications are currently used to define
Specific histone modifications are currently used to define promotion info regions with different regulatory functions. In particular, monomethylation of lysine 4 of histone 3 (H3K4me1) characterizes enhancer regions, whereas its trimethylation (H3K4me3) defines promoters (Ernst et al., 2011; Heintzman et al., 2009). Acetylation of H3K27 defines transcriptionally active enhancers and large clusters of enhancers (super-enhancers [SEs]) involved in the definition of cell and tissue identity (Hnisz et al., 2013). In this study, we aimed to map transcriptional regulatory elements and define their usage during epithelial differentiation. By combining high-throughput identification of Pol-II-transcribed (capped) RNAs defined by Cap Analysis of Gene Expression (DeepCAGE) (Carninci et al., 2006) with genome-wide profiling of histone modifications determined by chromatin immunoprecipitation (ChIP-seq), we mapped active enhancer and SE elements in prospectively isolated TA progenitors and terminally differentiated keratinocytes. For KSCs, which lack markers for prospective isolation, we exploited the integration characteristics of the Moloney murine leukemia retrovirus (MLV), which integrates in active promoters and enhancers (Biasco et al., 2011; Cattoglio et al., 2010; De Ravin et al., 2014) as a consequence of the direct binding of the viral integrase to the bromodomain and extraterminal (BET) proteins BRD2, BRD3, and BRD4 that tether the pre-integration complex to acetylated chromatin regions (De Rijck et al., 2013; Gupta et al., 2013; Sharma et al., 2013). By using MLV vector integration clusters as surrogate genetic markers of active regulatory elements, we mapped a collection of putative enhancers and SEs active in bona fide KSCs, retrospectively defined by their capacity to maintain long-term keratinocyte cultures.
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Acknowledgments
This work was supported by grants from the European Research Council (ERC-2010-AdG, GT-SKIN) and the Italian Ministry of Education, University and Research (EPIGEN Epigenomics Flagship Project).