Impairment of transcription factor Gcr1p binding motif perturbs OPI3 transcription in Saccharomyces cerevisiae.

Abstract

In Saccharomyces cerevisiae, the transcription factor GCR1 plays a vital role in carbohydrate metabolism and in the current study we tried to elucidate its role in lipid metabolism. In silico analysis revealed the upstream activation sequence (UAS) in the promoter region of OPI3 possessed six conserved recognition sequences for Gcr1p and the ChIP assay confirmed the binding of Gcr1p on the OPI3 promoter region. The real-time quantitative polymerase chain reaction and promoter-reporter activity revealed a substantial reduction in OPI3 expression and was supported with decreased phosphatidylcholine (PC) level that is rescued with exogenous choline supplementation in gcr1∆ cells. Simultaneously, there was an increase in triacylglycerol level, accompanied with increased number and size of lipid droplets in gcr1∆ cells. The expression of pT1, pT2 truncations in opi3∆ cells revealed the -1 to -500 bp in the promoter region is essential for the activation of OPI3 transcription. The mutation specifically at UASCT box (-265) in the OPI3 promoter region displayed a reduction in the PC level and the additional mutation at UASINO (-165) further reduced the PC level. Collectively, our data suggest that the GCR1 transcription factor also regulates the OPI3 expression and has an impact on lipid homeostasis.