Inhibition of triglyceride metabolism-associated enhancers alters lipid deposition during adipocyte differentiation

Triglyceride (TG) metabolism is a complex and highly coordinated biological process regulated by a series of genes, and its dysregulation can lead to the occurrence of disorders in lipid metabolism. However, the transcriptional regulatory mechanisms of crucial genes in TG metabolism mediated by enhancer-promoter interactions remain elusive. Here, we identified candidate enhancers regulating the Agpat2, Dgat1, Dgat2, Pnpla2, and Lipe genes in 3T3-L1 adipocytes by integrating epigenomic data (H3K27ac, H3K4me1, and DHS-seq) with chromatin three-dimensional interaction data. Luciferase reporter assays revealed that 11 enhancers exhibited fluorescence activity. The repression of enhancers using the dCas9-KRAB system revealed the functional roles of enhancers of Dgat2 and Pnpla2 in regulating their expression and TG metabolism. Furthermore, transcriptome analyses revealed that inhibition of Dgat2-En4 downregulated pathways associated with lipid metabolism, lipid biosynthesis, and adipocyte differentiation. Additionally, overexpression and motif mutation experiments of transcription factor found that two TFs, PPARG and RXRA, regulate the activity of Agpat2-En1, Dgat2-En4, and Pnpla2-En5. Our study identified functional enhancers regulating TG metabolism and elucidated potential regulatory mechanisms of TG deposition from enhancer-promoter interactions, providing insights into understanding lipid deposition.