Differential expression of androgen receptor (AR) mRNA in rat liver during maturation and aging directly correlates with androgen responsiveness of the young-adult male liver, and androgen insensitivity during prepuberty and senescence. A 31 kDa androgen-repressible liver protein, designated as SMP-2, is expressed at relatively high abundance during the periods of hepatic androgen insensitivity compared to its very low level of expression in the androgen-responsive adult male. Nuclear run off studies show that age-dependent reactivation and androgen-mediated repression of SMP-2 are primarily regulated through altered rates of gene transcription. The proposed studies have been designed to examine the following: 1) Identification of putative transcription transactivators whose differential expression/activity during maturation and aging results in the differential temporal regulation of the androgen receptor (AR) gene in the liver. 2) Dissection of the DNA sequences of the androgen receptor gene which play critical roles in hepatic expression of the receptor. 3) Delineation of the cis elements responsible for androgenic repression of the SMP-2 gene. 4) The possible role of additional cis elements, distinct from androgen response elements, which may participate in the age-dependent regulation of the SMP-2 gene. 5) The role of androgen receptor and other non-receptor transcription regulatory proteins in the age-dependent regulation of SMP-2 gene expression. The DNA response elements of interest will be identified through gene transfer into cells containing androgen receptor. Additional cis elements responding to changes in maturation and aging will be detected via DNA transfection into cultured hepatocytes from young and old rats. Putative transcription transactivators regulating differential temporal expression of the AR gene in the liver, as well as some that are possibly involved in SMP-2 gene regulation during aging, will be identified using complementary analyses of specific protein-DNA binding and in vitro transcription. The DNA binding studies will utilize Southwestern blotting, mobility shift and DNase 1 footprinting analyses. Transcription competence of liver nuclear extracts from young and old rats will be compared in their ability to support SMP-2 promoter-directed in vitro transcription of appropriate templates. The results of these studies are expected to provide important new insights into gene regulation during aging.
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