Chronic ethanol exposure produces functional adaptation of the central nervous system (CNS) such that alcoholics can tolerate and survive much higher levels of ethanol than naive individuals. Our central hypothesis is that expression of specific ethanol-responsive genes (EtRGs) could account for this CNS adaptive response. Our long term goal is that through identification of EtRGs and study of their function and mechanism(s) of regulation by ethanol, we can provide new options for genetic analysis and therapy of alcoholism. Studies of ours over the last four years have successfully identified and isolated EtRGs among which are the molecular chaperonins Hsc7O, GRP78 and GRP94. The products of these genes are thought to function in the intracellular trafficking of proteins. Previous work has shown that chronic ethanol (24-72 hours) increases the transcription of Hsc7O and GRP78. This proposal suggests that ethanol regulates the function of specific transcription factor(s) and thus produces a coordinate regulation of these three chaperonins.
Our specific aims can be summarized to include: 1) determine if ethanol regulates GRP94 transcription: 2) determine if the cis-acting promoter elements conferring ethanol-responsiveness are the same for GRP94, GRP78 and Hsc70; 3) identify or isolate the DNA-binding proteins involved in mediating ethanol induction of chaperoning transcription and determine the mechanism(s) of ethanol action. Nuclear runoff analysis will address whether ethanol regulates transcription of GRP94 and whether protein synthesis is required for ethanol induction of GRP94, GRP78 and/or Hsc70 transcription. A rat GRP94 genomic clone will be isolated and an ethanol-responsive cis-acting promoter region identified. Transient transfection analysis of deletion and point mutations will define ethanol-responsive cis-acting elements in the promoter regions of GRP94 and GRP78. We will compare these elements to our preliminary studies which implicate a consensus Sp1-binding site as required for ethanol induction of Hsc7O. We will also further confirm the possible role of Sp1 in ethanol-induced gene transcription by determining if basal transcription activity and the promoter context of Sp1 sites alters ethanol-responsiveness. Gel mobility shift assays (GMSA), in vitro and in vivo DNA footprinting will be used to determine if ethanol alters protein binding to the ethanol-responsive elements in the Hsc70, GRP78 and GRP94 promoters. Later parts of our studies will isolate transcription factors (other than Sp1) which may mediate ethanol regulation of GRP94, GP78 and/or Hsc70. Finally, fusion proteins containing the DNA-binding domain of the yeast transcriptional activator GAL4 and transcription activation domains of Sp1 will be studied to determine if they confer ethanol-responsiveness on a promoter containing GAL4 recognition sites. This will directly implicate Sp1 in at least some ethanol-responsive gene expression. In vitro transcription assays using Drosophila embryo extracts and Sp1 purified from control or ethanol-treated NG108-15 cells will be used to determine if ethanol activates Sp1 through a post-transcriptional mechanism.
