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Papers In Press, published online ahead of print October 24, 2001
Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610-0245
Corresponding Author: mkilberg{at}ufl.edu
Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (Amino Acid Response) or glucose (Unfolded Protein Response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the A.S. promoter, which are referred to as Nutrient Sensing Response Elements (NSRE) 1 and 2, both of which are absolutely necessary for gene activation. The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening. Based on those results, electrophoresis mobility shift assays (EMSA) for individual (C/EBP) family members were performed to test for supershift of complexes by specific antibodies. The results indicate that of all the family members C/EBPb binds to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex is increased when extracts from amino acid-deprived or glucose-deprived cells are tested. Using EMSA, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPb-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPb binding. Over-expression in HepG2 hepatoma cells of the activating isoform of C/EBPb increased A.S. promoter-driven transcription, whereas the inhibitory dominant negative isoform of C/EBPb blocked enhanced transcription following amino acid or glucose deprivation. Collectively, the results provide both in vitro and in vivo evidence for a role of C/EBPb in the transcriptional activation of the A.S. gene in response to nutrient deprivation.
J. Biol. Chem, 10.1074/jbc.M109533200
Submitted on October 2, 2001
Revised on October 22, 2001
Accepted on October 24, 2001
CCAAT/enhancer-binding protein beta (C/EBPb) is a mediator of the nutrient sensing response pathway that activates the human asparagine synthetase gene
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