The enzyme acetyl-CoA carboxylase-alpha (ACC-α) is rate limiting for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA) is dominantly active in lipogenic tissues. This promoter is in principal repressed but activated under favorable nutritional conditions. Previous analyses already coarsely delineated the repressive elements on the distal promoter but did not resolve the molecular nature of the repressor. Knowledge about the molecular functioning of this repressor is fundamental to understanding the nutrition mediated regulation of PIA activity. We analyzed here the molecular mechanism calibrating PIA activity.
We finely mapped the repressor binding sites in reporter gene assays and demonstrate together with Electrophoretic Mobility Shift Assays that nuclear factor-Y (NF-Y) and CCAAT/enhancer binding protein-β (C/EBPβ) each separately repress PIA activity by binding to their cognate low affinity sites, located on distal elements of the promoter. Simultaneous binding of both factors results in strongest repression. Paradoxically, over expression of NFY factors, but also - and even more so - of C/EBPβ significantly activated the promoter when bound to high affinity sites on the proximal promoter. However, co-transfection experiments revealed that NF-Y may eventually diminish the strong stimulatory effect of C/EBPβ at the proximal PIA in a dose dependent fashion. We validated by chromatin immunoprecipitation, that NF-Y and C/EBP factors may physically interact.
The proximal promoter segment of PIA appears to be principally in an active state, since even minute concentrations of both, NF-Y and C/EBPβ factors can saturate the high affinity activator sites. Higher factor concentrations will saturate the low affinity repressive sites on the distal promoter resulting in reduced and calibrated promoter activity. Based on measurements of the mRNA concentrations of those factors in different tissues we propose that the interplay of both factors may set tissue-specific limits for PIA activity.
Keywords: ACC-alpha, Bos taurus, CCAAT-enhancer binding protein, Fat synthesis, Gene regulation, Nuclear factor Y