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  • Title: Role of basic helix-loop-helix (bHLH) and CREB transcription factors in the regulation of Sertoli cell androgen-binding protein expression.
    Author: Saxlund MA, Sadler-Riggleman I, Skinner MK.
    Journal: Mol Reprod Dev; 2004 Jul; 68(3):269-78. PubMed ID: 15112319.
    Abstract:
    Differentiation of Sertoli cells is marked by the presence of novel gene products such as transferrin and androgen-binding protein (ABP). Transcriptional regulation of Sertoli cell differentiation is, in part, controlled through the binding of specific transcription factors to response elements within these genes promoters. Transferrin gene expression has been shown to be regulated by the binding and interactions of basic helix-loop-helix (bHLH) and cAMP response element binding protein (CREB) to an E-box and cyclic AMP response element (CRE), respectively. Interaction between the bHLH and CREB is facilitated through subsequent binding of CREB-binding protein (CBP)/p300. The hypothesis tested in the current study is that ABP expression is regulated by a similar mechanism. The ABP promoter activation was analyzed through the use of transfection assays, site-directed mutagenesis, and electromobility shift assays (EMSA). Transient transfections of rat Sertoli cells used a reporter construct containing the proximal 619 bp of the ABP promoter. Observations suggest that cAMP and follicle stimulating hormone (FSH) upregulate the expression of ABP. Mutational studies of the three E-boxes and the CRE of the 619-bp ABP promoter indicate that all of these elements are critical for stimulation of promoter activity. EMSA revealed a weak interaction between an E-box-2 and the CRE that are overlapping in the promoter. An artificial promoter that contains only an E-box and CRE was created to further test this hypothesis. The artificial promoter was stimulated by both FSH and cAMP. Experiments with mutants of the artificial promoter demonstrate that both response elements contribute to the optimal activation of the promoter construct. The overexpression of the bHLH inhibitor Id (i.e., inhibitor of differentiation) that binds bHLH proteins and eliminates DNA binding was found to suppress hormone activation of the ABP promoter. Combined observations of the ABP promoter and artificial promoter provide insight into a common mechanism for gene regulation in differentiated Sertoli cells involving a role for both the bHLH and CREB family of transcription factors.
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