Functional studies of cyclic AMP regulatory element binding proteins

Author

Qingqing Gong

Date of Award

12-1993

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

The CAMP regulatory element (ORE) for the Phosphoenolpyruvate Carboxykinase (PEPCK) gene has been localized between -61 and -113 in the promoter region. The gel retardation assay using a synthetic oligo bearing the CRE revealed that two DNA-protein complexes were formed with rat liver nuclear extract. Competition assays suggested that the upper complex is due to a CREB-like activity, while the lower complex results from the binding of NF-1 or an NF-1 like protein. Partial purification with DEAE cellulose chromatography showed that the CREB protein(s) binds tightly to DEAE, while the NF-1 like activity comes out in the flow through fractions. Considerable attempts made to further purify the CREB^PEPCK were unsuccessful due to inexplicable losses of CRE-specific binding activity. The rat brain tyrosine hydroxylase (TH) gene contains a functional CRE localized in the promoter region between -31 and -54. A DNA-protein complex with an Rf 0.3-0.4 was detected in a gel retardation assay with JEG-3 cell nuclear extract and a labeled 32 bp oligo (T32) containing the TH CRE. Binding specificity of the complex toward the CRE was demonstrated by the use of different DNA probes and DMS interference analysis. Similar results have been obtained using nuclear extracts from PC12 cells and rat brain. The molecular weight of the binding protein was approximately 45 Kd determined by a UV crosslinking assay. ATFa shares high sequence homology with another transcriptional factor, ATF2. Both can mediate Ela-stimulated transcription. To investigate possible functional similarities and differences between them, in vitro and in vivo experiments were carried out. The DNA binding specificities of the two are very similar. Both were not able to mediate cAMP stimulation under various conditions tested. Contrary to what has been suggested in the literature, we found that ATFa can stimulate transcription through a ORE in the TGF-β2 promoter in transfected CHO cells, a property shared by ATF2. The major functional difference discovered is that their transactivation functions are differentially regulated by pRb. While ATF2's activity is up-regulated by pRb, ATFa's activity is inhibited by the same protein. Further characterization showed that both interact directly with pRb in vitro.

Recommended Citation

Gong, Qingqing, "Functional studies of cyclic AMP regulatory element binding proteins. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10680