Glycerol-3-phosphate acyltransferase 1 (GPAT1) acts as a rate limiting enzyme in triacylglycerol and phospholipid synthesis in mammals. GPAT1 regulates hepatic lipid accumulation associated with metabolic disorders. Here we have identified two transcriptional initiation sites and two promoters (promoter I and II) required for expression of the human GPAT1 (hGPAT1) gene. Promoter I regulates transcription of three alternative hGPAT1 mRNA variants, hGPAT1-V1, V2, and V3, while promoter II induces expression of a fourth variant, hGPAT1-V4. RT-PCR analysis and luciferase reporter assays revealed that promoter II acts in lipogenic tissues like the liver (and liver-derived HepG2 cells), whereas promoter! is differentially regulated and also acts in non-liver HeLa cells. Among liver-enriched transcription factors, HNF4 alpha and C/EBP alpha slightly activated hGPAT1 promoter I, while factors including HNF1 alpha altered promoter II activity. The lipogenic transcription factor SREBP1c greatly increased promoter II activity in HepG2 cells. The use of various truncated or mutated fragments of promoter II revealed that one sterol regulatory element-like motif and one inverted CCAAT box on promoter 11 contributed to the SREBP1c response. These cis-acting elements and trans-acting factors can be potential targets for manipulation of hepatic GPAT1 levels in humans. (C) 2012 Elsevier Inc. All rights reserved.