Characterization of Protein Co-Localization and Histone Modifications at Enhancers Anchored in Chromatin Loops

Enhancers regulate genes over long genomic distances by physically looping to interact with promoters, a process highly dependent on specific protein recruitment and local histone modifications. We identified 1399, 1394, and 4063 enhancer-associated chromatin loops in GM12878, K562, and HepG2 cell lines, respectively, and found that dual-function proteins, characterized by both activating and repressing activities, preferentially localized at loop-anchored enhancers. Although protein co-localization patterns varied among cell types, CTCF, RAD21, SMC3, and ZNF143 consistently co-localized with one another at these enhancers. Furthermore, enhancer-associated loops localized primarily to the A compartment. Enhancers anchored in A-A loops showed greater enrichment of H3K4me1, H3K4me2, H3K9ac, and H3K27ac than those anchored in B-B loops. However, in K562 cells, enhancers anchored in B-B loops exhibited minor changes in active histone modification enrichment compared to their A-A counterparts, and even showed higher proportions of H3K4me2 and H3K9ac enrichment for enhancer–promoter loops. Nevertheless, across all three cell lines, A-A enhancer–promoter loops with both active anchors exhibited significantly higher target gene expression than other A-A loops and B-B loops. Our study suggests that enhancer-associated loop formation correlates with dual-function protein co-localization and active histone modification enrichment, which may collectively contribute to target gene transcription.