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Papers In Press, published online ahead of print April 17, 2002
J. Biol. Chem, 10.1074/jbc.M201959200
Submitted on February 27, 2002
Revised on April 9, 2002
Accepted on April 17, 2002
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610-0245
Corresponding Author: mkilberg{at}ufl.edu
Transcription from the asparagine synthetase (A.S.) gene is increased in response to either amino acid (Amino Acid Response) or glucose (Endoplasmic Reticulum Stress Response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the A.S. promoter referred to as Nutrient Sensing Response Elements (NSRE) 1 and 2, both of which are necessary for gene activation. The NSRE-1 sequence was used to screen ATF/CREB family members by electrophoresis mobility shift assays (EMSA) and supershift by specific antibodies. The results indicated that ATF4 binds to the NSRE-1 sequence and that the amount of the ATF4 complex was increased when extracts from amino acid-deprived or glucose-deprived cells were tested. Using EMSA experiments and a probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1 sequence completely prevented formation of the ATF4-containing complexes, whereas mutation of the NSRE-2 sequence did not. Over-expression of ATF4 increased A.S. promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitro and in vivo evidence for a role of ATF4 in the transcriptional activation of the A.S. gene in response to nutrient deprivation.
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