SMAD3 and EGR1 physically and functionally interact in promoter-specific fashion

Gonadotropin-releasing hormone (GNRH1) stimulates luteinizing hormone beta subunit (LHB/Lhb) transcription. The transforming growth factor beta superfamily ligand activin A partially inhibits this effect on the human LHB promoter while potentiating GNRH1-induction of the murine Lhb gene. Here, we investigated the mechanisms underlying the species-specific modulation of the GNRH1 response by activin signalling. GNRH1 stimulates LHB/Lhb transcription via induction of early-growth response 1 (EGR1), which binds to the proximal promoter of both species. Activin A decreased GNRH1-induced recruitment of EGR1 to the human, but not murine, promoter. We hypothesized that the activin A signalling protein, SMAD3, might play a role in this system. Indeed, we observed both physical and functional interactions between SMAD3 and EGR1. The two proteins interacted via the SMAD3 MH2 domain and the EGR1 DNA-binding domain. Analogous to the species-specific activin A effect on the GNRH1 response, SMAD3 over-expression partially inhibited EGR1-induction of the human promoter, while potentiating EGR1-induced murine Lhb promoter activity. The proximal murine Lhb promoter contains three minimal SMAD-binding elements (SBEs) that are absent from human LHB. Introduction of the SBEs into the human promoter converted SMAD3 from an inhibitor to a stimulator of EGR1-induced transcription. The converse was observed when the SBEs in the murine promoter were replaced by the corresponding human sequences. Together, our results suggest a model in which activin A inhibits GNRH1-induction of human LHB transcription via an interaction between SMAD3 and EGR1 that inhibits the latter's recruitment to the proximal promoter. In contrast, in mouse, the presence of SBEs in the promoter allows SMAD3 and EGR1 to function synergistically to regulate Lhb transcription. The basis for their functional cooperativity is not completely clear, but may involve enhancement of EGR1's physical interaction with other important co-factors, including paired-like homeodomain transcription factor 1 (PITX1).