Transcription factors CCAAT/enhancer-binding protein beta and nuclear factor-Y bind to discrete regulatory elements in the very low density lipoprotein receptor promoter

Expression of the very low density lipoprotein receptor (VLD-R) is barely d etectable in liver, but occurs in adipose tissue, skeletal muscle, heart, a nd placenta, where it is postulated to supply triglyceride to tissues that utilize fatty acids, To investigate its tissue-specific expression, cell li nes were transfected with luciferase reporter gene constructs driven by the 5'-flanking region of the VLDL-R gene. Transcriptional activity of a 4.2-k b promoter fragment was 5-fold higher in BeWo placental cells than in Huh-7 hepatoma cells, consistent with relative endogenous expression of the VLDL -R, By deletion analysis, DNase I protection assays and site-directed mutag enesis, two regulatory elements were essential for maximal promoter activit y in BeWo cells: footprint site D (-856 to -830) and an inverted CCAAT box (-703 to -707), Mutation of either element reduced promoter activity by 60% in BeWo cells, but had little effect in Huh-7 cells, suggesting that these elements direct cell-type specific transcription. Electrophoretic mobility -shift assays with BeWo nuclear extracts revealed that the inverted CCAAT b ox binds transcription factor NF-Y, and site D binds CCAAT/enhancer-binding protein b (C/EBP beta) and minor amounts of C/EBP alpha and C/EBP delta.jl r Overexpression of a dominant negative NF-YA vector confirmed involvement of NF-Y in the regulation of the VLDL- receptor gene through the CCAAT box. However overexpression of C/EBP could not stimulate transcription from the VLDL-receptor promoter nor from site D fused to a heterologous promoter, s uggesting that the simultaneous binding of an accessory factor(s) may be ne cessary for C/EBP transactivation via the D site.-Kreuter, R., A. K. Soutar , and D. P.Wade. Transcription factors CC;JAT/enhancer-binding protein beta and nuclear factor-Y bind to discrete regulatory elements in the very low density lipoprotein receptor promoter.