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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052064-07
Application #
6626961
Study Section
Nutrition Study Section (NTN)
Program Officer
May, Michael K
Project Start
1997-01-01
Project End
2005-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
7
Fiscal Year
2003
Total Cost
$327,680
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Bouman, L; Schlierf, A; Lutz, A K et al. (2011) Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress. Cell Death Differ 18:769-82
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
Kilberg, Michael S; Shan, Jixiu; Su, Nan (2009) ATF4-dependent transcription mediates signaling of amino acid limitation. Trends Endocrinol Metab 20:436-43
Palii, S S; Kays, C E; Deval, C et al. (2009) Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation. Amino Acids 37:79-88
Su, Nan; Thiaville, Michelle M; Awad, Keytam et al. (2009) Protein or amino acid deprivation differentially regulates the hepatic forkhead box protein A (FOXA) genes through an activating transcription factor-4-independent pathway. Hepatology 50:282-90
Abbatiello, Susan E; Pan, Yuan-Xiang; Zhou, Mi et al. (2008) Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients. J Proteomics 71:61-70
Thiaville, Michelle M; Pan, Yuan-Xiang; Gjymishka, Altin et al. (2008) MEK signaling is required for phosphorylation of eIF2alpha following amino acid limitation of HepG2 human hepatoma cells. J Biol Chem 283:10848-57
Su, Nan; Kilberg, Michael S (2008) C/EBP homology protein (CHOP) interacts with activating transcription factor 4 (ATF4) and negatively regulates the stress-dependent induction of the asparagine synthetase gene. J Biol Chem 283:35106-17

Showing the most recent 10 out of 41 publications