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Transcriptional Repression of the Neurofibromatosis-1 Tumor Suppressor by the T(8;21) Fusion Protein Genyan Yang Vanderbilt University

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Transcriptional Repression of the Neurofibromatosis-1 Tumor Suppressor by the T(8;21) Fusion Protein Genyan Yang Vanderbilt University Washington University School of Medicine Digital Commons@Becker Open Access Publications 2005 Transcriptional repression of the Neurofibromatosis-1 tumor suppressor by the t(8;21) fusion protein Genyan Yang Vanderbilt University Waleed Khalaf Herman B. Wells Center for Pediatric Research, Indianapolis Louis van de Locht University Medical Center St. Radboud, Nijmegen Joop H. Jansen University Medical Center St. Radboud, Nijmegen Meihua Gao Middle Tennessee State University See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Yang, Genyan; Khalaf, Waleed; van de Locht, Louis; Jansen, Joop H.; Gao, Meihua; Thompson, Mary Ann; van der Reijden, Bert A.; Gutmann, David H.; Delwel, Ruud; Clapp, D. Wade; and Hiebert, Scott .,W ,"Transcriptional repression of the Neurofibromatosis-1 tumor suppressor by the t(8;21) fusion protein." Molecular and Cellular Biology.25,14. 5869-5879. (2005). https://digitalcommons.wustl.edu/open_access_pubs/2251 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Authors Genyan Yang, Waleed Khalaf, Louis van de Locht, Joop H. Jansen, Meihua Gao, Mary Ann Thompson, Bert A. van der Reijden, David H. Gutmann, Ruud Delwel, D. Wade Clapp, and Scott .W Hiebert This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/2251 Transcriptional Repression of the Neurofibromatosis-1 Tumor Suppressor by the t(8;21) Fusion Protein Genyan Yang, Waleed Khalaf, Louis van de Locht, Joop H. Jansen, Meihua Gao, Mary Ann Thompson, Bert A. van der Downloaded from Reijden, David H. Gutmann, Ruud Delwel, D. Wade Clapp and Scott W. Hiebert Mol. Cell. Biol. 2005, 25(14):5869. DOI: 10.1128/MCB.25.14.5869-5879.2005. http://mcb.asm.org/ Updated information and services can be found at: http://mcb.asm.org/content/25/14/5869 These include: REFERENCES This article cites 66 articles, 36 of which can be accessed free at: http://mcb.asm.org/content/25/14/5869#ref-list-1 on January 6, 2014 by Washington University in St. Louis CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ MOLECULAR AND CELLULAR BIOLOGY, July 2005, p. 5869–5879 Vol. 25, No. 14 0270-7306/05/$08.00ϩ0 doi:10.1128/MCB.25.14.5869–5879.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Transcriptional Repression of the Neurofibromatosis-1 Tumor Suppressor by the t(8;21) Fusion Protein Genyan Yang,1 Waleed Khalaf,2 Louis van de Locht,3 Joop H. Jansen,3 Meihua Gao,4 Mary Ann Thompson,5 Bert A. van der Reijden,3 David H. Gutmann,6 Ruud Delwel,7 D. Wade Clapp,2 and Scott W. Hiebert1,8* Downloaded from Department of Biochemistry,1 Department of Pathology,5 and Vanderbilt-Ingram Cancer Center,8 Vanderbilt University School of Medicine, Nashville, Tennessee 37232; Departments of Microbiology, Immunology and Pediatrics, Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana2; Department of Hematology, University Medical Center St. Radboud, Nijmegen,3 and Department of Hematology, Erasmus University, Rotterdam,7 The Netherlands; Department of Mathematics, Middle Tennessee State University, Murfreesboro, Tennessee 371324; and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 631106 http://mcb.asm.org/ Received 9 November 2004/Returned for modification 19 January 2005/Accepted 14 April 2005 Von Recklinghausen’s disease is a relatively common familial genetic disorder characterized by inactivating mutations of the Neurofibromatosis-1 (NF1) gene that predisposes these patients to malignancies, including an increased risk for juvenile myelomonocytic leukemia. However, NF1 mutations are not common in acute myeloid leukemia (AML). Given that the RUNX1 transcription factor is the most common target for chromo- somal translocations in acute leukemia, we asked if NF1 might be regulated by RUNX1. In reporter assays, RUNX1 activated the NF1 promoter and cooperated with C/EBP␣ and ETS2 to activate the NF1 promoter over 80-fold. Moreover, the t(8;21) fusion protein RUNX1-MTG8 (R/M), which represses RUNX1-regulated genes, on January 6, 2014 by Washington University in St. Louis actively repressed the NF1 promoter. R/M associated with the NF1 promoter in vivo and repressed endogenous NF1 gene expression. In addition, similar to loss of NF1, R/M expression enhanced the sensitivity of primary myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor. Our results indicate that the NF1 tumor suppressor gene is a direct transcriptional target of RUNX1 and the t(8;21) fusion protein, suggesting that suppression of NF1 expression contributes to the molecular pathogenesis of AML. Von Recklinghausen’s disease, or neurofibromatosis type I, cient to promote tumorigenesis (16). These effects are due in is a common inherited tumor predisposition syndrome with an part to the role of the NF1 gene product, neurofibromin, as a overall incidence of approximately 1 in 3,000 worldwide. Af- negative regulator of RAS and other small GTP binding pro- fected individuals are prone to the development of benign teins, acting to accelerate GTP hydrolysis (5, 11, 25). Loss of tumors, such as neurofibromas, and malignant cancers, includ- NF1 results in increased RAS-mediated signaling in response ing glioma, malignant peripheral nerve sheath tumors, and to multiple stimuli, including granulocyte-macrophage colony- leukemia (28, 59). The disease is characterized by inactivating stimulating factor (GM-CSF), leading to excessive prolifera- mutations of the Neurofibromatosis-1 (NF1) gene (11, 17, 61), tion and increased cell survival of lymphoid and myeloid pro- which indicates that NF1 is a tumor suppressor gene. Patients genitor cells (26, 35, 67). with mutant NF1 are at increased risk for clonal myeloprolif- RUNX1 (runt-related 1, also known as AML 1 or AML1) is erative diseases, including juvenile monomyelocytic leukemia a DNA binding transcription factor that acts as a molecular (JMML) (59, 60). In contrast, NF1 mutation has rarely been switch to activate or repress transcription. Although RUNX1 detected in acute myeloid leukemia (AML) (29, 42, 55, 56). can associate with coactivators, it appears to activate transcrip- Relatively small reductions in the expression of NF1 pro- tion through composite sites in which the RUNX1 DNA bind- duce phenotypic changes in cell proliferation. For example, ϩ Ϫ ing site is adjacent to sites for factors that RUNX1 can phys- Nf1 / astrocytes exhibit increased cell proliferation and mo- ically associate with, such as C/EBP␣, PU.1, and ETS1, to form tility (3). Similarly, loss of only one NF1 allele is sufficient to an active transcriptional complex (22, 31, 54, 57, 64, 66). For partially complement defects in coat color and mast cells in example, when expressed alone, RUNX1, C/EBP␣, and PU.1 mice containing mutations in the c-Kit receptor tyrosine kinase (W41 mice) that attenuate Ras-dependent signaling (26). In are poor activators of the M-CSF promoter. However, when ␣ addition, repression of NF1 by human immunodeficiency virus coexpressed, RUNX1 synergizes with C/EBP or PU.1 to po- Tax predisposes transgenic mice expressing Tax to neurofibro- tently activate transcription (54). Conversely, when expressed mas, indicating that loss of NF1 expression by transcriptional in some cell types, RUNX1 is capable of repressing transcrip- regulation, rather than NF1 mutational inactivation, is suffi- tion through the recruitment of mSin3 and Groucho corepres- sors and histone deacetylases (HDACs) (13, 38, 46). The function of RUNX1 is perhaps disrupted more than any * Corresponding author. Mailing address: Department of Biochem- other transcription factor in acute leukemia. Chromosomal istry, Vanderbilt University School of Medicine, PRB 512, 23rd and Pierce, Nashville, TN 37232. Phone: (615) 936-3582. Fax: (615) 936- translocations that affect RUNX1 include t(12;21), which is 1790. E-mail: [email protected]. present in 20 to 25% of pediatric acute B-cell lymphoblastic 5869 5870 YANG ET AL. MOL.CELL.BIOL. leukemias, and t(8;21), which is the most frequent chromo- Recombinant retroviral plasmids were transfected into a GPϩE 86 packaging somal translocation associated with AML, accounting for 10 to cell line as previously described (20). Stable populations of packaging cells expressing the retrovirus were selected by serially separating green fluorescent 15% of the cases (58). In addition, inv(16), which may be the ϩ protein-positive (GFP ) cells by fluorescence-activated cell sorter (FACS) until second most frequent chromosomal abnormality in AML (58), more than 95% of the cells were GFPϩ. The transduction protocol has been fuses the RUNX1-interacting factor, core binding factor ␤ previously described (19). Briefly, embryonic day 13.5 fetal liver cells were pre- (CBF␤), to a smooth-muscle myosin heavy-chain gene (40). stimulated for 48 h in liquid cultures of IMDM (Invitrogen Corporation, Grand The inv(16) fusion protein dominantly inactivates RUNX1- Island, NY) containing 20% fetal bovine serum (BioWhittaker,
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