Transcriptional suppression of CTP:phosphoethanolamine cytidylyltransferase by 25-hydroxycholesterol is mediated by nuclear factor-Y and Yin Yang 1

Abstract

Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 μM) was determined to reside between −56 and −36 on the basis of analysis with several Pcyt2 promoter deletion–luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C−37CAAT−41 and YY1 (Yin Yang1) binds at C−42AT−40 in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C−14CA−12 in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter–reporter construct (−30/+61) by 25-HC was abolished when C−14CA−12 was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.