Identification of a distal enhancer of Ucp1 essential for thermogenesis and mitochondrial function in brown fat

Uncoupling protein 1 (UCP1) is a crucial protein located in the mitochondrial inner membrane that mediates nonshivering thermogenesis. However, the molecular mechanisms by which enhancer-promoter chromatin interactions control Ucp1 transcriptional regulation in brown adipose tissue (BAT) are unclear. Here, we employed circularized chromosome conformation capture coupled with next-generation sequencing (4C-seq) to generate high-resolution chromatin interaction profiles of Ucp1 in interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) and revealed marked changes in Ucp1 chromatin interaction between iBAT and eWAT. Next, we identified four iBAT-specific active enhancers of Ucp1, and three of them were activated by cold stimulation. Transcriptional repression of the Ucp1-En4 or Ucp1-En6 region significantly downregulated Ucp1 and impaired mitochondrial function in brown adipocytes. Furthermore, depletion of the cohesin subunit RAD21 decreased the interaction intensity between Ucp1-En4 and the Ucp1 promoter and downregulated Ucp1. EBF2 cooperated with the acetyltransferase CBP to regulate Ucp1-En4 activity and increase Ucp1 transcriptional activity. In vivo, lentivirus-mediated repression of Ucp1-En4 was injected into iBAT, resulting in impacted iBAT thermogenic capacity and impaired iBAT mitochondrial function under cold acclimation conditions. Studying the functional enhancers regulating Ucp1 expression in iBAT will provide important insights into the regulatory mechanisms of BAT activity.