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Originally published In Press as doi:10.1074/jbc.M109533200 on October 24, 2001

J. Biol. Chem., Vol. 276, Issue 51, 48100-48107, December 21, 2001
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CCAAT/Enhancer-binding Protein-beta Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene*

Fai Siu, Chin Chen, Can Zhong, and Michael S. KilbergDagger

From the Department of Biochemistry and Molecular Biology, Centers for Mammalian Genetics and Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610-0245

Transcription from the human asparagine synthetase (AS) 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 AS promoter, which are referred to as nutrient-sensing response element (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, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-beta (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies. The results indicated that of all the family members, C/EBPbeta bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested. Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPbeta -containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPbeta binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPbeta increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPbeta 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/EBPbeta in the transcriptional activation of the AS gene in response to nutrient deprivation.

* This work was supported by NIDDK Grant DK-52064 (to M. S. K.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, University of Florida College of Medicine, P. O. Box 100245, Gainesville, FL 32610-0245. Tel.: 352-392-2711; Fax: 352-392-6511; E-mail: mkilberg@ufl.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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