The repression of the lipolytic inhibitor G0s2 enhancers affects lipid metabolism.

The G0/G1 switch gene 2 (G0s2) is a selective inhibitor of adipose triglyceride lipase (ATGL) which is the rate-limiting enzyme for triglycerides (TGs) hydrolysis in adipocytes, and regulates the mobilization of TGs in adipocytes and hepatocytes. The expression and functional disorders of G0S2 are associated with various metabolic diseases and related pathological states, such as obesity and metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). However, the extent to which the transcriptional regulatory mechanisms mediated by the interaction between the G0s2 gene promoter and enhancer regions are involved remains unknown. Here, through the analysis of epigenomic data (H3K27ac, H3K4me1, and DHS-seq) and luciferase reporter assays, we identified three active enhancers of G0s2 in 3 T3-L1 adipocytes. Subsequently, using the dCas9-KRAB system for epigenetic inhibition of G0S2-En2, -En4, and -En5 revealed the functional role of these enhancers in regulating G0s2 expression and lipid droplet biosynthesis. Additionally, transcriptome analyses revealed that inhibition of G0S2-En5 downregulated pathways associated with lipid metabolism and lipid biosynthesis. Furthermore, overexpression of transcription factors (TFs) and motif mutation experiments identified that PPARG and RXRA regulate the activity of G0S2-En5. Taken together, we identified functional enhancers regulating G0s2 expression and elucidated the important role of the G0S2-En5 in lipid droplet biogenesis.