Doctoral Dissertations

Date of Award

5-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Wesley D. Wicks

Committee Members

David Brian, Elizabeth Howell, John Koontz

Abstract

Tyrosine hydroxylase (TH) is an important enzyme for biosynthesis of a number of neurotransmitters. It catalyzes the initial and rate limiting reaction in the catecholamine biosynthetic pathway. TH activity has been shown to be regulated by cAMP at the level of transcription and at the level of enzyme activity. The studies described in this dissertation focus on the mechanism by which cAMP regulates transcription of TH. Results obtained in this investigation showed that when a 32 bp oligonucleotide bearing the TH cAMP responsive element (ORE) sequence was inserted in front of a thymidine kinase (TK) promoter, a 15 fold induction in CAT activity by CAMP was observed with transfected JEG-3 cells. The TH ORE was shown to be able to mediate cAMP effect in both forward and reverse orientations. While insertion of one TH CRE has no effect on basal transcription, two copies of the TH CREs showed significant basal enhancement. However, the degree of CAMP responsiveness remains essentially the same. When the TH CRE-containing constructs were tested in a number of cell lines, it was found that cAMP responsiveness of this element was inactive in F9 cells, weakly active in L cells, and highly active in JEG-3 cells. The importance of the TH CRE core motif in cAMP responsiveness is underscored by the fact that a single mutation introduced in the core motif (T32+A) completely abolished the capability of the TH CRE to activate transcription. Contextual sequences were also shown to have significant influence on cAMP responsiveness. When the contextual sequences of the TH CRE were mutated to "consensus" contextual sequences based on six cAMP inducible genes, the artificial constructs showed greatly decreased CAMP responsiveness. Significant influence of basal expression by contextual sequences was demonstrated by two mutant constructs (C32mC and C32mN). These two mutants were generated by a novel method called palindrome-mediated mutagenesis discovered during the course of this investigation. In an effort to investigate which cAMP responsive binding protein(s) (CREB) mediates cAMP responsiveness of the TH gene, cotransfeetion experiments with three cloned CREBs were performed. Results showed that while CREB-1 was active on the somatostatin CRE, it has no specific influence on the TH CRE-bearing promoter with or without cotransfection of a protein kinase A (PKA) construct. CREB-2 was shown by using a chimeric construct (CREB-2~VP16) to be able to bind to the TH CRE in F9 cells, but it failed to activate transcription with or without cotransfection of PKA. ATF-a also showed no significant influence on the TH CRE in F9 cells. Consistent with these results, in vitro DNA binding analysis by others in our laboratory showed that considerable differences exist between these cloned CREBs and the TH CRE-binding protein(s) detected in nuclear extracts from rat brain, PC12 and JEG-3 cells. Taken together, results obtained from this investigation indicate that: (i) the TH CRE is a functional CRE sufficient to confer cAMP responsiveness on a heterologous promoter; (ii) contextual sequences are one of the key factors involved in modulating both basal and cAMP-stimulated transcription; (iii) one of the cloned CREBs (CREB-1) known to be active in CAMP response may not be involved in transactivation of the TH gene.

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