CCAAT/enhancer-binding protein-beta is a mediator of the nutrient-sensing response pathway that activates the human...

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.