Upstream Stimulatory Factors 1 and 2 activate the human hepatic lipase promoter via E-box dependent and independent mechanisms.
We studied the transcriptional regulation of the HL gene by USF1 and USF2 in HepG2 cells. The transcriptional activity of the HL(-685/+13) promoter construct was increased up to 25-fold by co-transfection with USF1 and USF2. Silencing of USF1 by RNA interference reduced promoter activity by 30-40%. Chromatin immunoprecipitation assays showed binding of endogenous USF1 and USF2 to the proximal HL promoter region. In gel shift assays, USF1 and USF2 bound to E-boxes at -307/-312 and -510/-516, and to the TATA-Inr region. Although the -514C-->T substitution abolished in vitro USF binding to the -510/-516 E-box, the increase in HL promoter activity by USF1 and USF2 was unaffected. Deletion and mutation analysis of the HL promoter region, and insertion of multiple E-box copies in front of a heterologous promoter, revealed that upregulation by USFs was mainly mediated through the -307/-312 E-box and the TATA-Inr region. We conclude that in HepG2 cells USF1 and USF2 regulate transcriptional activity of the HL gene through their binding to the E-box at -307/-312 and the TATA-Inr region.
van Deursen
Diederik
D
Department of Biochemistry, Cardiovascular Research School COEUR, Erasmus MC, NL-3000 CA Rotterdam, The Netherlands.
van Leeuwen
Marije
M
Vaulont
Sophie
S
Jansen
Hans
H
Verhoeven
Adrie J M
AJ
D000818:Animals; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D024721:E-Box Elements / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors / Q000502:physiology*
C504173:LIPC protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; D008049:Lipase