Mammalian cells respond to amino acid deprivation by increasing the transcription of a wide variety of genes by a pathway that will be referred to as the Amino Acid Response (AAR). However, the molecular mechanisms by which these events occur at the genomic level are not well understood. Our global hypothesis is that the human S25 ribosomal protein gene represents a model for investigating nutrient control, because it contains an amino acid starvation response element (AARE) that regulates its transcription. The goal is to characterize the AAR cis-acting element of the S25 gene and then use that information to identify the corresponding transcription factor(s). To identify amino acid-dependent changes in chromatin structure in vivo indicating possible sites of interaction with transcription factors, we will assay for DNase I hypersensitive sites near and within the S25 gene using human HepG2 hepatoma cells maintained in amino acid-complete MEM medium or MEM lacking histidine. To more precisely delineate the AARE site, we will perform functional analysis using a collection of reporter plasmids containing S25 gene deletion/substitution fragments. The AARE will be further characterized by high resolution detection of amino acid-dependent changes in protein-DNA interactions using dimethyl sulfate in vivo footprinting, followed by mutation of individual nucleotides. Transfection with an excess of the AARE sequence (Transcription Factor Decoy) will test whether adsorption and subsequent depletion of the corresponding trans- acting proteins suppresses amino acid-dependent regulation of S25 transcription. Electrophoresis Mobility Shift Analysis (EMSA) data, with wild-type and mutated oligonucleotides, will be correlated with the functional studies and will assess the amount of complex formation using nuclear extracts from amino acid-fed or amino acid-deprived cells. If the AARE sequence is novel, yeast one-hybrid cDNA library screening or purification of the trans-acting protein DNA affinity chromatography will be used to identify and clone the AARE binding protein. The Pin*Point strategy for directing a transcription factor/FokI nuclease fusion protein to specific cis-elements will document the S25 genomic sites for factor binding in vivo. The in vivo Pin Point assay complements and extends the in vitro EMSA data. 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 availability.
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