Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family

Abstract

The melanocortin-4 receptor (MC4-R) plays a key role in the hypothalamic control of food intake, lending importance to the understanding of the mechanisms that regulate its expression. To identify factors controlling the expression of the human (h) MC4-R gene, a fragment containing 1253 bp of the 58-flanking region of the hMC4-R gene was isolated. A series of hMC4-R luciferase constructs were developed and used to transiently transfect HEK293 and GT1-7 cell lines, both expressing endogenous MC4-R mRNA. Deletion analysis of the 1253 bp fragment showed that the basal promoter activity is mainly restricted to the 179 bp upstream of the transcription start site in both cell types. Mutation of a putative Sp1-binding site located at position −76 bp resulted in a dramatic reduction of the luciferase activity in HEK293 and GT1-7 cells by 87 and 80% respectively. Both in vitro and in vivo studies (gel shift and chromatin immunoprecipitation analyses) revealed binding of both Sp1 and Sp3 to this site in HEK293 cells. Cotransfection with an Sp1 expression vector in Drosophila cells that do not express Sp1, in conjunction with treatment of HEK293 cells with mithramycin A, a specific inhibitor of Sp1, confirmed the role of Sp1. For the first time, we have demonstrated that the constitutive activity of the hMC4-R promoter is dependent upon Sp transcription factors.

DNA methylations of MC4R and HNF4α are associated with increased triglyceride levels in cord blood of preterm infants

“…The MC4R and HNF4α promoter regions contain putative binding sites for transcription factors, including C/EBP alpha and SP1, which are involved in the regulation of lipid metabolism (Supplemental Digital Content-Figure S1). [29,30] The expression levels of MC4R and HNF4α genes were significantly increased in cord blood cells of preterm infants compared with term infants ( P  = 0.04 and P  = 0.048, respectively; Fig. 1).…”

Mithramycin A sensitizes cancer cells to TRAIL-mediated apoptosis by down-regulation of XIAP gene promoter through Sp1 sites

“…For example, melanocortin-4 receptor (MC4-R) gene (25), myeloid Elf-1 like factor (MEF) gene, MUC6 mucin gene, DNA methyltransferase 3A (DNMT3A), and DNMT3B genes promoter involves Sp1 site proximal to transcriptional start site (26 -28). As shown in Fig.…”

Proximal Promoter of the Cytochrome P450 Oxidoreductase Gene: Identification of Microdeletions Involving the Untranslated Exon 1 and Critical Function of the SP1 Binding Sites

“…Deletion mutants were created by site-directed mutagenesis. Transient transfection was carried out using human embryonic kidney (HEK) 293 cells with endogenous SP families, because of their stable transfection efficiency and usefulness in in vitro functional studies for SP1 binding sites (13). HEK 293 cells were cultured in DMEM at 37 C, seeded in 12-well dishes, and transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) with 0.6 g of the reporter plasmids.…”