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J Biol Chem, Vol. 274, Issue 9, 5880-5887, February 26, 1999
From the Department of Molecular Physiology and Biophysics,
Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-0615
The cyclic AMP response element (CRE) of the rat
phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for
a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The
first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed p
CCAAT/Enhancer-binding Protein
Is an Accessory Factor for the
Glucocorticoid Response from the cAMP Response Element in the Rat
Phosphoenolpyruvate Carboxykinase Gene Promoter
CREC/EBP, binds C/EBP
and 
but not CREB, yet it
confers a nearly complete glucocorticoid response when transiently
transfected into H4IIE rat hepatoma cells. These results suggest that
one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in
which the CRE was replaced with a GAL4-binding sequence
(pCREGAL4), and various GAL4 DNA-binding domain (DBD)
fusion protein expression vectors. Although chimeric proteins
consisting of the GAL4 DBD fused to either CREB or C/EBP are able to
confer an increase in basal transcription, they do not facilitate the
glucocorticoid response. In contrast, a fusion protein consisting of
the GAL4 DBD and amino acids 1-118 of C/EBP provides a significant
glucocorticoid response. Additional GAL4 fusion studies were done to
map the minimal domain of C/EBP needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid
regions 1-84, 52-118, or 85-118 of C/EBP fused to the GAL4 DBD do
not mediate a glucocorticoid response. We conclude that the amino
terminus of C/EBP contains a multicomponent domain necessary to
confer accessory factor activity to the glucocorticoid response from
the CRE of the PEPCK gene promoter.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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