Characterization of the murine and human Glycerol-3-phosphate acyltransferase 2 gene promoters

GARCIA FABIANI, MARIA BELEN; PELLON MAISON, MAGALI; MONTANARO, MAURO ALDO; QUIROGA, IVANA YOSELI; COLEMAN, ROSALIND; GONZALEZ BARO, MARIA DEL ROSARIO

Waterville Valley, NH

Congreso; Gordon Research Conference ?Molecular and Cellular Biology of Lipids; 2013

Gordon Research Conference

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in de novo glycerolipid synthesis. In mammals, four genes encoding GPATs have been cloned, and their products are present in different tissue and cellular locations and functions. GPAT1, 3 and 4 are expressed in lipogenic organs such as liver and adipose tissue. Unlike the other 3 isoforms, GPAT2, located in the outer mitochondrial membrane, is highly expressed in rat and mouse testis and certain cancer cells, tissues that do not specialize in lipid synthesis and storage. Gpat2 transcription in testis peaks at the beginning of rat and mouse spermatogenesis indicating transcriptional regulation, possibly related to hormones. We have cloned the murine Gpat2 promoter region (~6.7, 3.4 and 1.3kpb upstream from the transcription initiation site) into a luciferase-reporter vector and transfected them into CHO-K1 cells. Only the 3.4 kbp construct significantly activated transcription, so we used it for further experiments. Among several hormones, both cis-9- and all-trans-retinoic acids increased the promoter´s activity, consistent with the important role of retinoids in testis function. To identify the minimal promoter region and important elements for expression and regulation we generated a series of 5-prime deletions which were cloned into a luciferase-reporter vector. Results from promoter-reporter assays clearly identified the regulatory region between -2273 and -3282 bp as the most active. The constructs containing this region were also responsive to retinoid activation. We also determined that the region -1972 to -2273 contained essential response elements for Gpat2 transcription, since transcriptional activity was 10-fold higher in the 2.2kbp construct than in the 1.9kbp one. In silico data indicated epigenetic regulation for human GPAT2 gene. Human and mouse cell lines that do not express GPAT2 were treated with a DNA methylation inhibitor and GPAT2 expression was increased only in the human cell lines, evidencing DNA methylation as a possible regulatory GPAT2 expression mechanism in humans. Funding: NIH R03TW0008698 (USA); ANPCyT PICT 2246, CONICET PIP 0527 (Argentina)