AP-1 elements in the promoter and second intron mediate endoplasmic reticulum stress-induced expression of the GPAT3 gene.

An excessive accumulation of hepatic lipids is a characteristic feature of metabolic dysfunction-associated steatotic liver disease (MASLD) and its severe form, metabolic dysfunction-associated steatohepatitis (MASH). Acyl-CoA:glycerol-sn-3-phosphate acyltransferase 3 (GPAT3) and other members of the GPAT family are enzymes which play an important role in glycerolipid synthesis. Previous articles have reported that GPAT3 mRNA and ER stress marker genes are upregulated in patients with MASH. Here, we study the regulatory mechanism of GPAT3 gene expression in human hepatoma cells suffering from ER stress. Transcriptome profiling showed that among the genes implicated in the formation of glycerolipids, GPAT3 is one of the most strongly activated genes in response to tunicamycin, an inducer of ER stress. CRISPR/Cas9-mediated disruption of activating transcription factor 4 (ATF4) resulted in reduced GPAT3 expression under ER stress. Luciferase reporter assays of GPAT3 gene fragments encompassing ATF4 ChIP-seq peaks and mutational analysis revealed that activator protein-1 (AP-1) sites located in GPAT3 promoter and intron 2 mediate the activation of transcription in response to ER stress and ATF4. CRISPR/Cas9-mediated deletion of the region containing AP-1 sites from GPAT3 intron 2 caused a reduction of GPAT3 expression and triglyceride content in both unstressed cells and under ER stress. Thus, the results indicate that the induction of GPAT3 expression in response to ER stress is mediated by ATF4 via AP-1 elements in the promoter and second intron.