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内容記述 |
Ad4BP/SF-1 (Adrenal4 Binding Protein/Steroidogenic Factor-1 (NR5A1)), an orphan member of the nuclear receptor superfamily, was initially identified as a transcriptional activator of adrenal steroidogenic <i>P450</i> genes, and subsequent studies revealed that its expression is tightly regulated not only in the steroidogenic tissues such as gonad and adrenal gland, but also in the ventromedial hypothalamic nucleus (VMH), pituitary gonadotrope, and spleen. In addition to the findings above, <i>Ad4BP/SF-1</i> gene disrupted mice showed dramatic phenotypes such as adrenal and gonadal agenesis, markedly decreased gonadotropin expression, and abnormal formation of VMH. Based on these phenotypes, this factor is thought to be involved in establishment of the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes. Since Ad4BP/SF-1 is crucial for the development of reproductive organs as well as for the endocrine regulation, extensive studies have been performed to clarify the genetic cascades regulated by Ad4BP/SF-1, and a number of target genes have been identified in each tissue. However, despite its functional significance, the molecular mechanisms underlying tissue-specific expression of Ad4BP/SF-1 are largely unknown. In order to localize tissue-specific cis-regulatory regions of Ad4BP/SF-1 gene, several cosmid clones covering the <i>Ad4BP/SF-1</i> gene locus were initially analyzed. The genomic DNA fragments in the cosmid clones were ligated to 5.8-kb <i>Ad4BP/SF-1</i> gene promoter fragment, followed by the bacterial <i>lacZ</i> gene and SV40 poly (A) signal (Ad4BP-lacZ cassette), and the resultant constructs were subjected to transgenic mouse assays. Among these clones, one clone termed cIA3 showed an activity to induce lacZ expression in the fetal adrenal gland, fetal ventral diencephalon (future VMH), and Rathke’s pouch (pituitary primordium). The genomic region covered by cIA3 was analyzed further with sequentially truncated constructs, and finally he has identified the VMH-specific enhancer of Ad4BP/SF-1 gene within the 6th intron. Interestingly, phylogenetic footprinting analysis retrospectively revealed that the VMH-specific enhancer sequence was highly conserved not only in the mammalians but also in the birds and amphibians. The transgenic mouse lines harboring Ad4BP-lacZ cassette and the minimal enhancer fragment were then established, and expression profiles of lacZ were examined. From the results of these analyses, it was indicated that the enhancer has the potential to reproduce endogenous gene expression from the fetal ventromedial diencephalon to the adult VMH. In order to identify the functionally important sequences in the enhancer region, transgenic mouse analyses using the mutated or truncated enhancer fragments were performed. As the result, the enhancer was also characterized by the presence of one suppressive and two activating elements. Mutation of the former element resulted in ectopic <i>lacZ</i> reporter gene expression in an area dorsal to the intrinsic expression domain and in the ventricular zone, while mutations in the latter containing tandemly repeated ATTA motifs led to the disappearance of the reporter gene expression, suggesting the involvement of homeobox proteins in the gene regulation. Although the <i>trans</i>-acting factors capable to recognize those activating and suppressive elements have not been identified yet, electrophoretic mobility shift assays revealed that a same factor recognizes both the two activating elements.<br /> The pituitary gonadotrope-specific enhancer was also identified in the 6th intron of the gene by transgenic mouse assays. It was intriguing that the enhancer region was conserved only in mammalians but not in the amphibians, birds, nor in the fishes although Ad4BP/SF-1 is also expressed in the pituitary of these nonmammalian species. The transgenic mouse lines in which lacZ or EGFP is driven by the minimal enhancer fragment and <i>Ad4BP/SF-1</i> gene promoter were generated. LacZ staining analyses and immunohistochemical analyses revealed that the enhancer was capable to recapitulates endogenous gene expression in the fetal Rathke’s pouch later than embryonic day 13.5 to the adult pituitary gonadotrope. In addition, fluorescence-activating cell sorting and RT-PCR analyses revealed that the enhancer-driven reporter gene expression was strictly confined to the gonadotrope lineage, indicating that the enhancer function is tightly regulated only in the gonadotrope. Structurally, the enhancer included several elements completely conserved across the mammalian species, in addition to the GATA2-binding elements and EGR-1 binding element. Mutational analyses confirmed the significance of the completely conserved elements for the enhancer function, whereas mutations in the other elements showed no effects. One of the completely conserved elements was recognized by a <i>bicoid</i>-related homeoprotein, Pitx2 <i>in vitro</i>, and chromatin immunoprecipitation assays showed that the enhancer region was actually occupied by the transcriptional complex containing Pitx2 and RNA polymerase II <i>in vivo.</i> These results strongly suggested that Pitx2 regulates <i>Ad4BP/SF-1</i> gene transcription in the pituitary gonadotrope via direct interaction with the gonadotrope-specific enhancer.<br /> During tissue differentiation, it is conceivable that certain genes encoding transcription factors act as critical components forming a gene regulatory cascade. Ad4BP/SF-1 is a component of the cascade required for differentiation of the pituitary gonadotrope and VMH. Ad4BP/SF-1 is localized upstream of a set of tissue-specific genes that include steroidogenic <i>Cyp</i> genes, and at the same time it is localized downstream of other transcription factors regulating the gene transcription. Given that activation/inactivation of the components occurs in an upstream to downstream direction along the cascade during tissue differentiation, and that Ad4BP/SF-1 is an essential transcription factor for the tissue differentiation, identification of the components that function with Ad4BP/SF-1 and regulate <i>Ad4BP/SF-1</i>gene transcription is quite important to fully understand the entire gene cascade. Identification of the enhancers to drive expressions specific for the pituitary gonadotrope and VMH is a step leading to understanding the gene cascade. |
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