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Characterization of the 5'-regulatory regions of the rat and human apelin genes and regulation of breast apelin by USF

Department of Surgery, University of Texas Medical Branch, Galveston, Texas, USA

¹Correspondence: Department of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555, USA. E-mail: ggreeley{at}utmb.edu

ABSTRACT

Apelin, a peptide widely expressed in the body, is the endogenous ligand for the APJ receptor. To investigate how the apelin gene is regulated transcriptionally, we cloned and characterized 3000 and 4000 bp 5'-upstream fragments of the rat and human apelin genes. Putative CAAT-like box, but not TATA-box sites were identified. The rat (–207/–1 bp) and human (–100/+74 bp) core promoter sequences contain putative binding sites for upstream stimulatory factor (USF)-1/-2. Mutagenesis and overexpression assays showed that USF up-regulates basal and inducible apelin transcription. EMSA and supershift experiments indicated binding of USF-1/-2 to the rat (–114/–109 bp) and human (–84/–79 bp) apelin promoters. ChIP experiments show that USF is recruited to the putative USF binding site in the human apelin promoter in cultured breast cells. In concert with increased breast apelin expression during pregnancy and lactation in rats, EMSAs demonstrate an elevated binding of pregnant and lactating rat breast nuclear proteins to a consensus USF oligonucleotide. In vivo ChIP assays verified increased USF binding to the apelin promoter in breast of lactating rats. Together, our findings show that USF exerts a stimulatory role in regulation of breast apelin expression during pregnancy and lactation.—Wang, G., Qi, X., Wei, W., Englander, E. W., and Greeley, G. H., Jr. Characterization of the 5'-regulatory regions of the rat and human apelin genes and regulation of breast apelin by USF.

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