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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK059315-03
Application #
6635350
Study Section
Nutrition Study Section (NTN)
Program Officer
May, Michael K
Project Start
2001-07-01
Project End
2006-02-28
Budget Start
2003-05-01
Budget End
2004-02-29
Support Year
3
Fiscal Year
2003
Total Cost
$239,443
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Shan, Jixiu; Lopez, Maria-Cecilia; Baker, Henry V et al. (2010) Expression profiling after activation of the amino acid deprivation response in HepG2 human hepatoma cells. Physiol Genomics :
Gjymishka, Altin; Su, Nan; Kilberg, Michael S (2009) Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway. Biochem J 417:695-703
Shan, Jixiu; Ord, Daima; Ord, Tõnis et al. (2009) Elevated ATF4 expression, in the absence of other signals, is sufficient for transcriptional induction via CCAAT enhancer-binding protein-activating transcription factor response elements. J Biol Chem 284:21241-8
Gjymishka, Altin; Palii, Stela S; Shan, Jixiu et al. (2008) Despite increased ATF4 binding at the C/EBP-ATF composite site following activation of the unfolded protein response, system A transporter 2 (SNAT2) transcription activity is repressed in HepG2 cells. J Biol Chem 283:27736-47
Gutierrez, Jemy A; Pan, Yuan-Xiang; Koroniak, Lukasz et al. (2006) An inhibitor of human asparagine synthetase suppresses proliferation of an L-asparaginase-resistant leukemia cell line. Chem Biol 13:1339-47
Kilberg, M S; Pan, Y-X; Chen, H et al. (2005) Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. Annu Rev Nutr 25:59-85
Pan, Yuan-Xiang; Chen, Hong; Kilberg, Michael S (2005) Interaction of RNA-binding proteins HuR and AUF1 with the human ATF3 mRNA 3'-untranslated region regulates its amino acid limitation-induced stabilization. J Biol Chem 280:34609-16
Chen, Chin; Dudenhausen, Elizabeth E; Pan, Yuan-Xiang et al. (2004) Human CCAAT/enhancer-binding protein beta gene expression is activated by endoplasmic reticulum stress through an unfolded protein response element downstream of the protein coding sequence. J Biol Chem 279:27948-56
Palii, Stela S; Chen, Hong; Kilberg, Michael S (2004) Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic element. J Biol Chem 279:3463-71
Leung-Pineda, Van; Pan, YuanXiang; Chen, Hong et al. (2004) Induction of p21 and p27 expression by amino acid deprivation of HepG2 human hepatoma cells involves mRNA stabilization. Biochem J 379:79-88

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