Fig. 4: A cohesin-mediated chromatin interaction between Ucp1-En4 and the Ucp1 promoter regulates Ucp1 transcription.

a Alignment of high-throughput chromosome conformation capture (Hi-C) data from inguinal adipose tissue at Ucp1 locus. Heatmap (upper panel) showing the interaction between Ucp1-En4 and Ucp1 promoter located within the same TAD. Lower panel showing 4C-seq and ChIP-seq (MED1 and CTCF) data. The orange rectangle represents Ucp1-En4, the red rectangle represents Ucp1 promoter, and the gray rectangles represent the boundaries of the interaction domain. CTCF-motif position and orientation are indicated by arrows. b qRT-PCR analysis of Rad21 mRNA expression in differentiated brown adipocytes transfected with control or Rad21 small interfering RNA (siRNA) (n = 5). c Chromatin interactions between Ucp1 promoter and other chromatin sites (−10, −6, and −3 kb) in differentiated brown adipocytes were assessed by quantitative chromosome conformation capture (3C) qPCR assays 48 h after transfection with control or Rad21 siRNA. Chromatin interactions were normalized to the Ercc3 locus. A −6 kb chromatin site was used as a negative control. The triangle represents the 3C-qPCR primer, and the arrow direction represents the 3C-qPCR primer direction (n = 5). d Sanger sequencing of 3C-qPCR product between Ucp1-En4 (−10 kb site) and the Ucp1 promoter. The red base indicates the DpnII digestion site formed. e qRT-PCR analysis of Ucp1 mRNA expression in differentiated brown adipocytes transfected with control or Rad21 siRNA (n = 5). Data are presented as mean ± SEM. P values were calculated by two-tailed unpaired Student’s t-tests.