1 Computational and RNA Biology, Department of Biology, Faculty of Science, Københavns Universitet2 Institut for Biokemi og Molekylær Biologi3 National Cancer Institute4 Human Resources, Faculty Services, Faculty of Life Sciences, Københavns Universitet5 University of Southern Denmark6 Human Resources, Faculty Services, Faculty of Life Sciences, Københavns Universitet7 Computational and RNA Biology, Department of Biology, Faculty of Science, Københavns Universitet
Transcription factors have recently been shown to colocalize in hotspot regions of the genome, which are further clustered into super-enhancers. However, the detailed molecular organization of transcription factors at hotspot regions is poorly defined. Here, we have used digital genomic footprinting to precisely define factor localization at a genome-wide level during the early phase of 3T3-L1 adipocyte differentiation, which allows us to obtain detailed molecular insight into how transcription factors target hotspots. We demonstrate the formation of ATF-C/EBP heterodimers at a composite motif on chromatin, and we suggest that this may be a general mechanism for integrating external signals on chromatin. Furthermore, we find evidence of extensive recruitment of transcription factors to hotspots through alternative mechanisms not involving their known motifs and demonstrate that these alternative binding events are functionally important for hotspot formation and activity. Taken together, these findings provide a framework for understanding transcription factor cooperativity in hotspots.