The mammalian Fushi tarazu-F1 gene encodes steroidogenic factor 1 (SF- 1), an orphan nuclear receptor that plays important roles in early embryonic development and in maintaining the steroidogenic capacity of steroidogenic tissues in adulthood. The nuclear receptors are able to recruit coregulatory factors to enhance or inhibit transcription. SF-1 has been shown to function in conjunction with the homeodomain protein, Fushi tarazu (Ftz) which is involved in formation of a segment during development and also stimulates transcription synergistically in Drosophila. We hypothesize that this interaction ability of nuclear receptor with homeodomain protein in Drosophila embryogenesis suggests that SF-1 may have a similar function in development and regulating target gene expression in mammals. However, the mammalian homologue of Ftz protein has not been identified. Therefore, identification and characterization of the Ftz-like protein will provide new information concerning the molecular mechanisms of endocrine tissue development and function.
Aim 1 is to clone the mammalian homologue of the ftz-like gene. Screening of an embryonic stem (ES) cell lambda gt11 library will be performed using Drosophila gene ftz. In parallel, SF-1 will be used to detect candidate interacting protein in Farwestern blots and in expression library. In addition, Polymerase Chain Reaction (PCR) and Reverse Transcription-PCR will be used to amplify candidate mammalian ftz homologues.
Aim 2 is to characterize the Ftz-like protein and explore its interactions with SF-1. Development- and tissue-specific expression of gene will be determined by Northern blotting and in situ hybridization. In vivo and in vitro transfection, DNA binding and protein-protein interaction assays will be carried to define the functional role of the cofactor.
