Identification of a novel sterol-independent regulatory element in the human low density lipoprotein receptor promoter.
The cytokine oncostatin M (OM) activates human low density lipoprotein receptor (LDLR) gene transcription through a sterol-independent mechanism. Previous studies conducted in our laboratory have narrowed the OM-responsive element to promoter region -52 to +13, which contains the repeat 3 and two TATA-like sequences. We now identify LDLR promoter region -17 to -1 as a sterol-independent regulatory element (SIRE) that is critically involved in OM-, transcription factor CCAAT/enhancer-binding protein (C/EBP)-, and second messenger cAMP-mediated activation of LDLR transcription. The SIRE sequence overlaps the previously described TATA-like element and consists of an active C/EBP-binding site (-17 to -9) and a functional cAMP-responsive element (CRE) (-8 to -1). We demonstrate that (a) mutations within either the C/EBP or CRE site have no impact on basal or cholesterol-mediated repression of LDLR transcription, but they completely abolish OM-mediated activation of LDLR transcription; (b) replacing the repeat 3 sequence that contains the Sp1-binding site with a yeast transcription factor GAL4-binding site in the LDLR promoter construct does not affect OM inducibility, thereby demonstrating that OM induction is mediated through the SIRE sequence in conjunction with a strong activator bound to the repeat 3 sequence; (c) electrophoretic mobility shift and supershift assays confirm the specific binding of transcription factors C/EBP and cAMP-responsive element-binding protein to the SIRE; (d) cotransfection of a human C/EBPbeta expression vector (pEF-NFIL6) with the LDLR promoter construct pLDLR234 increases LDLR promoter activity; and (e) OM and dibutyryl cAMP synergistically activate LDLR transcription through this regulatory element. This study identifies, for the first time, a cis-acting regulatory element in the LDLR promoter that is responsible for sterol-independent regulation of LDLR transcription.
Liu
J
J
Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, USA. liu@icon.palo-alto.med.va.gov
Ahlborn
T E
TE
Briggs
M R
MR
Kraemer
F B
FB
D001483:Base Sequence; D015342:DNA Probes; D006801:Humans; D011401:Promoter Regions, Genetic; D011973:Receptors, LDL / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D013261:Sterols / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D011973:Receptors, LDL; D013261:Sterols