Classical enhancers couple cis -regulatory logic with transcriptional condensates and 3D genome architecture

Abstract Deciphering the regulatory logic of enhancers remains a central question in understanding cell- and tissue-specific gene expression in multicellular organisms. This is particularly pertinent at multipartite enhancer clusters such as super-enhancers, where multiple enhancers contribute to the expression of a single gene. Gene expression has been studied largely through sequence-dependent recruitment of transcription factors (TF) and co-activators, whereas 3D chromatin structure has been attributed to architectural proteins such as cohesion and CTCF1–3. However, the contribution of DNA sequence encoded in enhancers to shaping higher-order genome organization remains poorly understood. Here we show that classical enhancers, embedded within multipartite super-enhancer structures, act as determinant regulatory elements that initiate the gene regulatory cascade by linking DNA sequence recognition to 3D chromatin architecture. Classical enhancers are more evolutionarily conserved and display stronger regulatory activity than facilitator elements, which lack intrinsic enhancer activity but potentiate classical enhancer function. We show that classical enhancers are selectively bound by specific TFs with strong intrinsically disordered regions, such as NFE2L2 in liver cancer cells, capable of driving transcriptional condensate formation through phase separation. NFE2L2 depletion reduced enhancer activity and induced widespread chromatin reorganization, characterized by increased cohesin and CTCF occupancy at super-enhancer boundaries and beyond. This “cohesin clogging” impaired DNA loop extrusion, led to formation of smaller topologically associated domains, and weakened enhancer–promoter contacts. These findings highlight that sequence-specific TFs have multifaceted roles beyond transcriptional control, establishing a direct mechanistic link between enhancer sequence, TF binding, condensate formation, and 3D genome organization, with the regulatory logic being encoded in the DNA sequence itself. Competing Interest Statement The authors have declared no competing interest. Data availability Sequencing data have been deposited at ENA as PRJEB100961 and are publicly available as of the date of publication. UCSC genome browser tracks are available at: https://genome.ucsc.edu/s/villet/Tiusanen_Patel_et_al_2025. Previously published sequencing datasets and annotation links utilized in this study are provided in Supplementary Table 5. Previously published data are available under the following accession code: GEO: GSE180158, GSM2428726, GSE180158, GSE254242. ENCODE: ENCFF920MZO, ENCFF655BEL, ENCFF000PIE, ENCFF000PHU, ENCFF000XTR, ENCFF000XTQ, ENCFF000XUL, ENCFF000XUK, ENCFF128UUS, ENCFF015SPJ, ENCFF492CBJ, ENCFF807WOU, ENCFF416JVM, ENCFF382VQI, ENCFF364UNM. Processed lenti-MPRA probe data for HepG2 cells was downloaded from ENCODE: ENCFF774DYO.