Abstract
The human asparagine synthetase (AS) gene responds to depletion of mammalian cells for either amino acids or carbohydrates. Five specific cis-elements have been implicated: three GC boxes (GC-I, GC-II and GC-III) and two nutrient-sensing response elements (NSRE-1, -2). This study shows that all three GC boxes are required to maintain basal transcription and to obtain maximal induction of the AS gene by amino acid limitation. However, there is not complete redundancy among the three GC boxes, and there is a hierarchy of importance with regard to transcription (GC-III > GC-II > GC-I). In vitro, two GC boxes formed protein-DNA complexes (GC-II and GC-III) with Sp1 and Sp3. Although transcription of the AS gene is elevated by nutrient limitation, the absolute amount of these protein-DNA complexes and the total pools of Sp1 and Sp3 did not increase. A small, but detectable portion of Sp1 was modified by phosphorylation following amino acid deprivation. In vivo, expression of Sp1 and Sp3 in Drosophila SL2 cells increased AS promoter activity. Sp1 expression increased basal transcription but did not cause a further increase when SL2 cells were amino acid-deprived. Sp3 expression enhanced both the basal and the starvation-induced transcription.
Highlights
Metabolite control of gene transcription in mammalian cells is an important factor in regulating protein expression in response to nutrient changes in the environment
Within the initial 173 nt upstream of the transcription start site (Fig. 1), there are three GC-rich sequences (GC-I, GC-II, GC-III) and two cis-elements (NSRE-1 and NSRE-2) that make up the nutrient sensing response unit
A GC Box Is Necessary for Maximal Transcription of the asparagine synthetase (AS) Gene—To determine the importance of each of the GC boxes I-III in the transcriptional activity of the human AS promoter, each site was mutated in the context of the ASϪ173/ϩ51/growth hormone (GH) reporter construct (Table I)
Summary
Metabolite control of gene transcription in mammalian cells is an important factor in regulating protein expression in response to nutrient changes in the environment. The first analysis of amino acid-dependent control of the human AS gene was triggered by the observation that a mutation in AS led to a block in G1 phase of the cell cycle [13] Those studies led Guerrini et al [14] to identify an amino acid responsive sequence in the proximal promoter, which corresponds to the NSRE-1 site shown by Barbosa-Tessman et al [4, 5] to mediate both amino acid and glucose effects. This report shows that each of the three GC boxes within the human AS proximal promoter are required to maintain the highest basal transcription rates and that they act as modulators to allow maximal activation via the NSRE sites following nutrient deprivation. Sp3 was capable of enhancing both the basal and the starvation-induced activation of the AS promoter
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