Orphan CpG islands amplify poised enhancer regulatory activity and determine target gene responsiveness

CpG islands (CGIs) represent a widespread feature of vertebrate genomes, being associated with ~70% of all gene promoters. CGIs control transcription initiation by conferring nearby promoters with unique chromatin properties. In addition, there are thousands of distal or orphan CGIs (oCGIs) whose functional relevance is barely known. Here we show that oCGIs are an essential component of poised enhancers that augment their long-range regulatory activity and control the responsiveness of their target genes. Using a knock-in strategy in mouse embryonic stem cells, we introduced poised enhancers with or without oCGIs within topologically associating domains harboring genes with different types of promoters. Analysis of the resulting cell lines revealed that oCGIs act as tethering elements that promote the physical and functional communication between poised enhancers and distally located genes, particularly those with large CGI clusters in their promoters. Therefore, by acting as genetic determinants of gene–enhancer compatibility, CGIs can contribute to gene expression control under both physiological and potentially pathological conditions.T.P. is supported by a doctoral fellowship from the DAAD (Germany). V.S.-G. is supported by a doctoral fellowship from the University of Cantabria (Spain). Work in the Rada-Iglesias laboratory was supported by the EMBO Young Investigator Programme; CMMC intramural funding (Germany); the German Research Foundation (DFG) (Research Grant no. RA 2547/2-1); ‘Programa STAR-Santander Universidades, Campus Cantabria Internacional de la convocatoria CEI 2015 de Campus de Excelencia Internacional’ (Spain); the Spanish Ministry of Science, Innovation and Universities (Research Grant nos. PGC2018-095301-B-I00 and RED2018-102553-T REDEVNEURAL 3.0); and the European Research Council (ERC CoG ‘PoisedLogic’; grant no. 862022). The Landeira laboratory is funded by grants from the Spanish Ministry of Science and Innovation (grant nos. BFU2016-75233-P and PID2019-108108GB-I00) and the Andalusian Regional Government (grant no. PC-0246-2017)