We previously demonstrated that activation of the insulin-receptor promoter occurs between Xho I and Hindlll restriction enzyme sites located 877 and 578 bp upstream, respectively, from the translational initiation site. Deletion mutants 5' of this promoter region were constructed with the reporter gene, CAT, and transiently transfected into HepG2 and MCF-7 cells. This study demonstrated that most of the promoter activity could be localized to a 40-bp region between –618 and –578. When attached to a heterologous SV40 early promoter, this 40-bp insulin-receptor regulatory region, in either orientation, stimulated the SV40 early promoter by approximately two- to threefold after transfection into HepG2 cells. EMSA demonstrated that the purified transcription factor, Sp1, binds to this transcription activator. DNA binding of protein obtained from crude HepG2 nuclear extracts demonstrated electrophoretically retarded bands that competed for the consensus Sp1 element; these bands were not observed in a similar analysis with crude MCF-7 nuclear extracts. However, in both HepG2 and MCF-7 cells, a protein was identified that specifically binds to this important insulin-receptor promoter region, but does not bind to the Sp1 consensus element. We conclude that activation of insulin-receptor gene transcription occurs in a 40 bp region 578 bp upstream from the translational initiation site, and that Sp1 and another nuclear factor other than Sp1 may be important in regulating transcription In HepG2 cells.
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Original Articles|
January 01 1993
Nuclear Protein—Binding Analysis of a GC-Rich Insulin-Receptor Promoter Regulatory Region
James R Levy;
James R Levy
Department of Research, McGuire Veterans Affairs Medical Center
Richmond, Virginia
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Victoria Hug
Victoria Hug
Department of Research, McGuire Veterans Affairs Medical Center
Richmond, Virginia
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Address correspondence and reprint requests to James R. Levy, MD, McGuire Veterans Affairs Medical Center, Medical Service 111-P, 1201 Broad Rock Boulevard, Richmond, VA 23249.
Diabetes 1993;42(1):66–73
Article history
Received:
January 27 1992
Revision Received:
September 15 1992
Accepted:
September 15 1992
PubMed:
8380564
Citation
James R Levy, Victoria Hug; Nuclear Protein—Binding Analysis of a GC-Rich Insulin-Receptor Promoter Regulatory Region. Diabetes 1 January 1993; 42 (1): 66–73. https://doi.org/10.2337/diab.42.1.66
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