Decreases in s-adenosylmethionine (SAMe), the major methyl donor, or phosphatidylcholine (PC), a methylated phospholipid, are associated with lipid accumulation in mammalian metabolic disease. However, the molecular mechanisms that link these metabolites to pathways stimulating lipogenesis are not fully understood. Using C. elegans and mammalian models, we previously found that activity of a lipogenic transcription factor, SBP-1/SREBP-1, was increased when SAMe and PC levels fell. For example, knockdown of the SAMe synthase
sams-1 decreases both SAMe and PC and results in upregulation of SBP-1 target genes such
fat-7.
fat-7 expression is returned to wild type levels when PC biosynthesis is rescued by diet. In order to identify additional effectors, we performed a small scale RNAi screen for factors that could activate SBP-1 in a PC dependent manner by comparing
fat-7::GFP levels in normal and low PC background (
sams-1(lof)). We reasoned that factors affected by low PC in this pathway might be involved in intracellular transport (COP II or COP I components), act in synthesis of complex lipids (phospholipids, ceremides, sphingolipids), regulate phospholipid transport (flippases, lipid transfer proteins) or function in phospholipid-based signaling pathways (phospholipases). In order to insure complete coverage in our screen, we combined clones from the Ahringer and Orfeome collections and made RNAi targeting vectors for unrepresented genes.We identified multiple RNAi clones that increased
fat-7::GFP in a
sams-1-dependent fashion, suggesting they could be linked to low-PC activation of SBP-1. As secondary screens, we asked which candidates had similar effects on endogenous
fat-7, and finally, determined those that caused increases in nuclear localization of a translational fusion of GFP::SBP-1. Through this process we identified two genes,
lpin-1 and
arf-1.2, as potential effectors of low-PC effects on SBP-1.
lpin-1 is a phosphatidic acid phosphatase important for triglyceride synthesis and lipid droplet function.
arf-1.2 is a small GTPase that regulates COP I Golgi to ER traffic and is also important for lipid droplet formation. We extended these studies to mammalian cells and found that siRNA of LPIN1 or ARF1 is sufficient to activate SREBP-1 and that LPIN1 is required for SREBP-1 activation when PC biosynthesis is inhibited. Finally, we find that both inhibition of PC biosynthesis and LPIN1 knockdown decrease levels of active ARF1. This suggests a pathway in which LPIN1 acts between signals from PC levels and regulation of ARF1 activity in the Golgi to affect activation of SBP-1/SREBP-1.