Endocrine Abstracts

The transcription factor, Steroidogenic factor-1 (SF-1/NR5A1) is the only specific marker of the VMH, to date. Total SF-1 deficiency results in defective VMH neuronal differentiation and architecture, and mice heterozygous for SF-1 (SF-1^+/−), or conditionally depleted of SF-1 in the VMH show reduced levels of the neurotrophin Bdnf and an increased propensity for weight gain. Recent studies have suggested several putative SF-1 target genes in the VMH that are implicated with transcription, metabolism and feeding. In silico and in vitro analyses were performed to investigate whether tow of these candidates, PACAP and H2AZ, are SF-1 target. Analyses of the 5′-untranslated region of the murine PACAP and H2AZ promoters revealed multiple potential SF-1 binding sites. Using specific oligonucleotides encoding this region, electrophoretic mobility shift assays (EMSA) were performed using endogenous SF-1 from gonadotroph αT3-1 cell nuclear extracts. A single putative SF-1 response element in the PACAP promoter bound at least 4 protein species, of which one was identified as SF-1. To establish whether SF-1 could directly regulate PACAP transcription, we performed a series of reporter gene assays using deletion constructs of the murine PACAP promoter transiently transfected in to rat phaechromocytoma PC-12 cells, with increasing concentrations of SF-1. Promoter constructs between −481 and −1474 bp were significantly stimulated by SF-1 (by up to 22-fold over basal, P<0.05). Two consensus SF-1 sites were found in the H2AZ promoter, both of which bound SF-1 as determined by EMSA analysis. The presence of an anti-phosphoSF-1 antibody resulted in a supershift of the SF-1 complex, confirming its identity. Additional protein complexes resembling members of the USF family of transcription factors were identified by cold competition using consensus USF binding sites. In summary, the promoters of PACAP and H2AZ contain putative SF-1 response elements that preferentially bind SF-1, and SF-1 can regulate the expression of PACAP in vitro. The physiological relevance of this regulation remains to be elucidated, but may be involved in the regulation of feeding behaviour by SF-1 in the VMH.