The SRE Motif in the Human PNPLA3 Promoter (-97 to -88 bp) Mediates Transactivational Effects of SREBP-1c.

Patatin-like phospholipase domain containing 3 (PNPLA3) is a non-secreted protein primarily expressed in liver and adipose tissue. Recently, numerous genetic studies have shown that PNPLA3 is a major susceptibility gene for nonalcoholic fatty liver disease (NAFLD). However, the mechanism involved in transcriptional regulation of the PNPLA3 gene remains unknown. We performed a detailed analysis of the human PNPLA3 gene promoter and identified two novel cis-acting elements (SRE and NFY binding motifs) located at -97/-88 and -26/-22 bp, respectively. Overexpression of SREBP-1c in HepG2 cells significantly increased PNPLA3 promoter activity. Mutation of either of the putative SRE or NFY binding motifs blocked the transactivation effects of SREBP-1c on the promoter. Overexpression of SREBP-1c and NFY together increased PNPLA3 promoter activity twice as much as that of SREBP-1c or NFY expression alone. This result suggests that SREBP-1c and NFY synergistically transactivate the human PNPLA3 gene. The ability of SREBP-1c and NFY to bind these cis-elements was confirmed using gel shift analysis. Putative SRE and NFY motifs also mediated synergistic insulin-induced transactivation of the PNPLA3 promoter in HepG2 cells. Additionally, the ability of SREBP-1c to bind to the PNPLA3 promoter was increased by insulin in a dose-dependent manner. Moreover, the treatment of HepG2 cells with the PI3K inhibitor LY294002 led to reduced insulin promoter-activating ability accompanied by a decrease in PNPLA3 and SREBP-1c protein expression. These results demonstrate that SREBP-1c is a direct activator of the human PNPLA3 gene and insulin transactivates the PNPLA3 gene via the PI3K-SREBP-1c/NFY pathway in HepG2 cells.