Abstract
Previously, we identified the low density lipoprotein receptor (LDLR) promoter region -17 to -1 as a novel sterol-independent regulatory element (SIRE) that mediates the stimulating effect of oncostatin M (OM). The goal of this study was to identify the OM-induced transcription activator that binds to the SIRE sequence. By conducting a electrophoretic mobility shift assay (EMSA) followed by UV crosslinking and SDS-PAGE, we show that a protein with a molecular mass of 85 kDa was present in the OM-induced SIRE DNA-protein complex. Western blotting and supershift assays reveal that the 85 kDa factor is early growth response gene 1 (Egr1). The interaction of Egr1 with the SIRE sequence was further confirmed in vivo by chromatin immunoprecipitation assays. The functional role of Egr1 in LDLR transcription was assessed by cotransfection of an Egr1 expression vector with an LDLR promoter reporter construct. We show that overexpression of Egr1 significantly increases LDLR promoter activity when cotransfected with CCAAT/enhancer binding protein beta (c/EBPbeta) or with cAMP-responsive element binding protein (CREB) expression vectors. Our studies clearly demonstrate that Egr1 is the OM-induced transcription factor that binds to the SIRE sequence of the LDLR promoter and also suggest that Egr1 may have a functional role in OM-induced upregulation of LDLR transcription through interaction with other SIRE binding proteins such as c/EBPbeta or CREB.
Highlights
We identified the low density lipoprotein receptor (LDLR) promoter region ؊17 to ؊1 as a novel sterol-independent regulatory element (SIRE) that mediates the stimulating effect of oncostatin M (OM)
We have previously demonstrated that cAMP-responsive element binding protein (CREB)/activating transcription factor (ATF), c/EBP, and c-jun bind to the SIRE motif of the LDLR promoter in a constitutive manner, whereas an unknown transcription factor present in the C2 complex only interacts with this promoter region upon OM stimulation [20]
We report for the first time that early growth response gene 1 (Egr1) interacts with the LDLR promoter through the SIRE sequence that does not contain a canonical or a homologous Egr1 binding site
Summary
We identified the low density lipoprotein receptor (LDLR) promoter region ؊17 to ؊1 as a novel sterol-independent regulatory element (SIRE) that mediates the stimulating effect of oncostatin M (OM). We identified a cis regulatory element in the human LDLR promoter that is responsible for cytokine oncostatin M (OM), CCAAT/enhancer binding protein (c/EBP), and cAMP-stimulated transcription of LDLR [20]. This regulatory sequence designated as the sterolindependent regulatory element (SIRE) lies down stream of the SRE-1 and Sp1 sites. Transcription of the low density lipoprotein receptor (LDLR) is largely controlled by a cholesterol-mediated feedback mechanism through interaction of a sterol regulatory element-1 (SRE-1) and SRE binding proteins (SREBPs) [1,2,3,4,5,6,7,8,9].
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