Functional and in vitro characterization of an insulin responsive regulatory region of the tyrosine aminotransferase gene promoter

Author

Richard S. Peterson

Date of Award

5-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

John W. Koontz

Committee Members

David A. Brian

Abstract

Previous studies from our laboratory have shown that in the KRC-7 rat hepatoma, insulin inhibits tyrosine aminotransferase (TAT) gene expression through a selective decrease in TAT transcription. To further characterize the insulin response similar studies were conducted with the Fao rat hepatoma, which possesses a differentiated phenotype. In agreement with a previous report, it was found that insulin treatment results in an increased rate of basal TAT transcription followed by an increase in hybridizable mRNA^TAT in Fao rat hepatoma cells. To further extend these results, the effect of insulin on dexamethasone-and 8Br-cAMP-mediated induction of TAT gene expression was examined. Although insulin was found to increase basal expression, it was also found to inhibit either dexamethasone- or 8Br-cAMP-mediated increases in mRNA^TAT. Insulin has been reported to inhibit either dexamethasone- or cAMP-mediated increases in mRNA^TAT in the KRC-7 rat hepatoma. These results suggest that both cell lines may share a common mechanism for the inhibition of enhancer-mediated induction. In an effort to identify cis-element(s) contained in the TAT 5'-flanking sequence which mediate the effect of insulin, a series of 5'-, 3'-, and internal deletion TAT promoter/chloramphenicol acetyltransferase reporter (TAT•CAT) chimeric constructs were transfected into Fao cells for transient expression. These investigations have led to the identification of an insulin-response element(s) (IREs) contained within the proximal promoter sequences located between -351 to -52 bp. In addition, transient transfection analysis indicates that a liver-specific enhancer activity, located at -3.6 Kb relative to the TAT cap site, is diminished in the Fao clone when compared to that observed in the KRC-7 clone. Gel shift experiments with fragments of the TAT promoter containing the putative IRE(s) with nuclear extracts from insulin-treated and control Fao cells indicate that specific protein-DNA complexes are formed. No observable differences in protein-DNA complex intensities and/or mobility's resulted with insulin treatment. This was followed with in vitro DNase I footprint analyses of TAT 5'- flanking sequences -351 to -2 bp with nuclear extract from insulin-treated or control cells. Although no insulin-dependent differences could be detected, the investigations have revealed multiple protected regions that suggest a close spacing of several DNA-binding activities. Transcription factor recognition-site assignments, inferred through footprint protection and computer-assisted sequence homologies, suggest important functional roles for liver-enriched DNA-binding proteins. Since no DNA-binding activities could be correlated with cellular responses to insulin, no insulin response element(s) could be defined using these methods. The results indicate alternative experimental approaches must be employed in an effort to further characterize the molecular mechanism by which insulin directs TAT gene expression in the Fao rat hepatoma.

Recommended Citation

Peterson, Richard S., "Functional and in vitro characterization of an insulin responsive regulatory region of the tyrosine aminotransferase gene promoter. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10745