During the life-span of the rat, the liver of both male and female animals goes through three phases of androgen responsiveness, i.e., prepubertal ( <40 days) androgen-insensitivity, an androgen responsive phase of young-adulthood and androgen-insensitivity of senescence ( <750 days). These age-dependent changes in androgen sensitivity are correlated with the hepatic expression of the androgen receptor (AR) mRNA. Because of such a marked alteration in the expression of AR gene during the three periods of life, the rat liver offers a unique model for exploring the molecular mechanism of the temporal regulation of this gene. Such an age-dependent regulation of this receptor gene may be mediated through a programmed alteration of DNA-protein interactions that are critical for transcription. This proposal is designed to undertake a systematic analysis of DNA-protein interactions that are involved in the regulation of AR gene expression in the liver. In order to achieve this goal, the upstream regulatory region of the AR gene will be cloned and characterized. Fragments of the cloned gene will be used to identify specific protein-binding regions by DNase I in vitro foot-printing analysis. Age-specific alterations of DNA-binding nuclear factors will be subsequently characterized by band-shift analysis of labeled oligonucleotide duplexes corresponding to the foot-print regions of the AR gene. Cellular existence of DNA-protein interactions will be substantiated by in vivo foot-printing of primary heptocytes and DNase I hypersensitivity analysis of isolated liver nuclei. Once the age- specific DNA-protein interactions are characterized, their functional relevance will be substantiated by transfection assay of the wild type and appropriate mutant of the AR gene promoters ligated to heterologous reporter genes. The biological role of these interactions will be further authenticated by examining the specific requirements of the cis/trans interactions for in vitro transcription of promoter-reporter constructs. Results of these studies are expected to provide new knowledge concerning the molecular mechanism of age-dependent changes in AR gene expression. Furthermore, receptor dysregulation is one of the major causes of the age-associated loss of systemic coordination. These studies will therefore, make an important contribution toward an overall understanding of the molecular basis of aging.
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