Characterization of promoter elements mediating ethanol regulation of hsc70 gene transcription.
Chronic exposure to ethanol increases transcription of the molecular chaperone Hsc70 in NG108-15 neuroblastoma X glioma cells. This and other ethanol-induced changes in gene expression may contribute to central nervous system tolerance and dependence in alcoholics. Here, we characterized sequences in the hsc70 promoter that are required for ethanol-induced transcriptional regulation. Deletion analysis of the hsc70 promoter showed that the 74-base pair region proximal to the transcription start site was sufficient for ethanol responsiveness. Point mutation or deletion of a consensus Spl-binding site at -67/-61 base pairs greatly reduced the induction by ethanol. Hsc70 promoter constructs with diminished ethanol responsiveness in NG108-15 cells similarly had decreased transcriptional activation by exogenous Sp1 in Drosophila SL2 cells. Some artificial promoter constructs containing multiple Sp1 sites were highly responsive to ethanol, but others were not, suggesting that the organization of the proximal promoter region was an additional factor that affected the ethanol response. Gel mobility shift analysis confirmed that an Sp1-like protein bound to the -67/-61 consensus Sp1 site. However ethanol exposure did not alter Sp1 DNA-binding activity. Together, our findings show that ethanol induction of Hsc70 requires a functional Sp1-binding site. Additional proximal promoter elements may also play a role in determining whether an Sp1-containing promoter will respond to ethanol.
Wilke
N
N
The Ernest Gallo Clinic and Research Center, Program in Biomedical Sciences and Department of Neurology, University of California, San Francisco, CA 94110, USA.
Sganga
M W
MW
Gayer
G G
GG
Hsieh
K P
KP
Miles
M F
MF
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D016384:Consensus Sequence; D004330:Drosophila / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005809:Genes, Regulator / Q000187:drug effects*; D006168:Guinea Pigs; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics; D017354:Point Mutation / Q000502:physiology; D011401:Promoter Regions, Genetic; D051381:Rats; D016329:Sp1 Transcription Factor / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494372:Hspa8 protein, mouse; C494373:Hspa8 protein, rat; D018832:Molecular Chaperones; D016329:Sp1 Transcription Factor; D000431:Ethanol