Nutrient control of mammalian gene expression is a poorly understood process that is important to general cellular nutrition and the progression of numerous diseases. The proposed research will continue our work on transcriptional control of the human asparagine synthetase (AS) gene following amino acid deprivation. In addition, we have discovered that the AS gene is transcriptionally activated by glucose deprivation, a process mediated by the Unfolded Protein Response (UPR) signal transduction pathway. This observation is novel because most UPR activated genes are ER-bound proteins associated with protein processing. We have characterized a human AS genomic clone and shown that the AS proximal promoter has six protein binding sites, at least three of which (I, II, or III, and V, VI) are required for maximal nutrient control. None of these sequences corresponds to the reported mammalian UPR cis-element. Instead, the AS promoter appears to contain two unique cis-elements (sites V and VI) that mediate the regulated response to both amino acid and glucose limitation. These exciting results suggest that there are two independent mechanisms for UPR transcriptional control in mammalian cells, one of which overlaps with amino acid signaling. The global hypothesis is that there is a common set of unique cis-elements within the human AS gene that mediate increased transcription in response to multiple nutrient-sensing pathways. The proposed experiments contain both standard and innovative approaches to further characterize these nutrient-responsive cis-acting elements and to identify the corresponding transcription factors. Yeast one-hybrid screening, TJV cross-linking, and DNA affinity chromatography will be used to identify the proteins that bind to the AS promoter sites V and VI. The Pin point strategy involving transfection of trans-acting factor/FokI nuclease fusion constructs will document in vivo the specific transcription factors that bind to a particular AS promoter site. Mutagenesis, EMSA, Transcription Factor Decoy, and overexpression of likely trans-acting factors will further define these cis-elements and explore their role in nutrient-dependent regulation. The proposed experiments test important hypotheses and will generate valuable new information regarding the mechanisms by which mammalian cells respond to changes in amino acid and glucose availability.
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