MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

  • Bronson, Sarah (PI)
  • QUINN, PATRICK (PI)
  • Quinn, Patrick G. (PI)
  • BRONSON, SARAH K. (PI)

Project: Research project

Project Details

Description

The long-term objective of the research program described here is to
understand how cAMP regulates gene expression with particular emphasis on
the mechanism of action of the cAMP response element (CRE) binding protein
(CREBP) and the interplay of factors regulating expression of the
phosphoenolpyruvate carboxykinase (PEPCK) gene. The CRE plays an essential
role in both basal and cAMP-stimulated transcription of the PEPCK gene.
Regulated expression of PEPCK, which catalyzes the rate-limiting step in
gluconeogenesis, is essential for maintaining appropriate blood glucose
concentrations. Gene transcription is regulated by the interaction of
trans-acting protein factors with each other and RNA polymerase, which is
facilitated by binding of these factors to cis-acting DNA elements. Cyclic
AMP activates protein kinase A, resulting in phosphorylation of several
proteins, including the CRE binding protein. CREBP has recently been
purified, cloned and shown to be directly involved in regulation of gene
expression.

In order to understand how CREBP interacts with other factors to regulate
gene expression, it is necessary to identify the domains of CREBP that are
involved in transactivation. CREBP binds to the CRE as a dimer, and two
isoforms that differ by 14 amino acids and possess distinct transcriptional
capacities are present in cells due to alternative splicing of the CREBP
mRNA. Aim 1 is to determine the significance of the dimerization of CREBP
isoforms for transactivation and to establish whether the amount or
distribution of isoforms is regulated. This will be done by examining the
contribution of each peptide alone compared to the interactions of the same
and different isoforms in a dimer and by determining whether CREBP
synthesis or splicing is regulated by hormones. Aim 2 is to identify the
functional domains of CREBP involved in activation of basal and cAMP-
stimulated transcription. This will be done by creating specific mutations
in the CREBP cDNA and by fusing potential activation domains to the GAL4
DNA-binding domain. The transactivational capacity of mutated CREBP
proteins and of CREBP/GAL4 fusion factors will be assessed in transfection
and in vitro transcription assays. Aim 3 is to identify and characterize
the interactions of the CRE with heterologous promoter elements, especially
those involved in regulation of PEPCK. This will identify potential
targets of CREBP interactions. Domains of CREBP that are required for the
interplay of different transcription factors can then be identified by
examining the ability of different CREBP variants and fusion factors to
potentiate activation by these other factors.
StatusFinished
Effective start/end date5/1/916/30/07

Funding

  • NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES: $264,160.00

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