The biological effects of the ski oncogene are pleiotropic because its complex molecular interactions allow it to impinge upon elaborate pathways that regulate cellular proliferation and differentiation. Retroviral-mediated overexpression of either v-ski or the proto-oncogene, c-ski, transforms avian embryo fibroblasts and converts them into skeletal myoblasts. v-Ski and c-Ski apparently produce these biological effects by binding specific DNA elements in combination with other proteins, and either activating or repressing transcription. The Ski-related protein SnoN dimerizes with Ski and shares its oncogenic and transcriptional regulatory activities. The overall goals of this work are to 1) identify genes regulated by Ski and Sno that are involved in their ability to induce oncogenic transformation and 2) characterize DNA and protein interactions that may underlie their transcriptional activities. To accomplish these goals we propose a combination of focused and genome-based approaches. The mRNA sequences that are induced and repressed in Ski transformed cells will be identified by PCR-based differential display using a high-density cloning and screening method. The roles of the identified genes in Ski-induced transformation will be studied in model in vitro systems. We will explore the role of Ski, and its phosphorylation, in the regulation of two genes whose expression is responsive to Ski (myogenin) or to sequence elements identified as Ski binding sites (PAI-2). A protein partner that may act as an effector of Ski's transcriptional regulatory function has been identified using the yeast two-hybrid protein interaction screen. This protein has functions in chromatin-mediated gene regulation; the effects of its interactions with Ski and Sno on gene regulation will be examined using model gene expression systems. A protein that co-binds a DNA element that mediates repression of gene expression by Ski/SnoN has been partially purified. Its identification and an analysis of its role in Ski transformation will also be pursued. Finally, a specially devised system for tight control of ski expression in a cell line from the ski knock-out mouse will be used to identify genes acutely regulated by Ski. This will involve the powerful oligonucleotide computer chip analysis system developed by Affymetrix to identify activated and repressed genes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043600-18
Application #
6632881
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Cole, John S
Project Start
1986-03-01
Project End
2004-02-29
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
18
Fiscal Year
2003
Total Cost
$311,580
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Zhang, Hong; Stavnezer, Ed (2009) Ski regulates muscle terminal differentiation by transcriptional activation of Myog in a complex with Six1 and Eya3. J Biol Chem 284:2867-79
Atanasoski, Suzana; Notterpek, Lucia; Lee, Hye-Youn et al. (2004) The protooncogene Ski controls Schwann cell proliferation and myelination. Neuron 43:499-511
Johnson, Michael; Morris, Shannon; Chen, Aiping et al. (2004) Selection of functional mutations in the U5-IR stem and loop regions of the Rous sarcoma virus genome. BMC Biol 2:8
Chen, Dahu; Xu, Weidong; Bales, Elise et al. (2003) SKI activates Wnt/beta-catenin signaling in human melanoma. Cancer Res 63:6626-34
Morris, Shannon; Johnson, Michael; Stavnezer, Ed et al. (2002) Replication of avian sarcoma virus in vivo requires an interaction between the viral RNA and the TpsiC loop of the tRNA(Trp) primer. J Virol 76:7571-7
Xu, W; Angelis, K; Danielpour, D et al. (2000) Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor. Proc Natl Acad Sci U S A 97:5924-9
Nicol, R; Zheng, G; Sutrave, P et al. (1999) Association of specific DNA binding and transcriptional repression with the transforming and myogenic activities of c-Ski. Cell Growth Differ 10:243-54
Cohen, S B; Zheng, G; Heyman, H C et al. (1999) Heterodimers of the SnoN and Ski oncoproteins form preferentially over homodimers and are more potent transforming agents. Nucleic Acids Res 27:1006-14
Nicol, R; Stavnezer, E (1998) Transcriptional repression by v-Ski and c-Ski mediated by a specific DNA binding site. J Biol Chem 273:3588-97
Cohen, S B; Nicol, R; Stavnezer, E (1998) A domain necessary for the transforming activity of SnoN is required for specific DNA binding, transcriptional repression and interaction with TAF(II)110. Oncogene 17:2505-13

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