Background Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. Methods and Results Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases. Conclusion Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer.
References
[1]
Varmeh-Ziaie S, Okan I, Wang Y, Magnusson KP, Warthoe P, et al. (1997) Wig-1, a new p53-induced gene encoding a zinc finger protein. Oncogene 15: 2699–2704.
[2]
Israeli D, Tessler E, Haupt Y, Elkeles A, Wilder S, et al. (1997) A novel p53-inducible gene, PAG608, encodes a nuclear zinc finger protein whose overexpression promotes apoptosis. EMBO J 16: 4384–4392.
[3]
Hellborg F, Qian W, Mendez-Vidal C, Asker C, Kost-Alimova M, et al. (2001) Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein. Oncogene 20: 5466–5474.
[4]
Mendez-Vidal C, Wilhelm MT, Hellborg F, Qian W, Wiman KG (2002) The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity. Nucleic Acids Res 30: 1991–1996.
[5]
Chen T, Brownawell AM, Macara IG (2004) Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5. Mol Cell Biol 24: 6608–6619.
[6]
Yang M, Wu S, Su X, May WS (2006) JAZ mediates G1 cell-cycle arrest and apoptosis by positively regulating p53 transcriptional activity. Blood 108: 4136–4145.
[7]
Prahl M, Vilborg A, Palmberg C, Jornvall H, Asker C, et al. (2008) The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA. FEBS Lett 582: 2173–2177.
[8]
Mendez Vidal C, Prahl M, Wiman KG (2006) The p53-induced Wig-1 protein binds double-stranded RNAs with structural characteristics of siRNAs and miRNAs. FEBS Lett 580: 4401–4408.
[9]
Vilborg A, Glahder JA, Wilhelm MT, Bersani C, Corcoran M, et al. (2009) The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element. Proc Natl Acad Sci U S A 106: 15756–15761.
[10]
Bae BI, Xu H, Igarashi S, Fujimuro M, Agrawal N, et al. (2005) p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease. Neuron 47: 29–41.
[11]
Ryan AB, Zeitlin SO, Scrable H (2006) Genetic interaction between expanded murine Hdh alleles and p53 reveal deleterious effects of p53 on Huntington's disease pathogenesis. Neurobiol Dis 24: 419–427.
[12]
Feng Z, Jin S, Zupnick A, Hoh J, de Stanchina E, et al. (2006) p53 tumor suppressor protein regulates the levels of huntingtin gene expression. Oncogene 25: 1–7.
[13]
Yang XW, Gong S (2005) An overview on the generation of BAC transgenic mice for neuroscience research. Curr Protoc Neurosci Chapter 5: Unit 5 20.
[14]
Gray M, Shirasaki DI, Cepeda C, Andre VM, Wilburn B, et al. (2008) Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. J Neurosci 28: 6182–6195.
[15]
Spampanato J, Gu X, Yang XW, Mody I (2008) Progressive synaptic pathology of motor cortical neurons in a BAC transgenic mouse model of Huntington's disease. Neuroscience 157: 606–620.
[16]
Wu H, Lima WF, Crooke ST (1999) Properties of cloned and expressed human RNase H1. J Biol Chem 274: 28270–28278.
[17]
Butler M, Hayes CS, Chappell A, Murray SF, Yaksh TL, et al. (2005) Spinal distribution and metabolism of 2′-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats. Neuroscience 131: 705–715.
[18]
Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 95: 14863–14868.
[19]
Kalscheuer VM, FitzPatrick D, Tommerup N, Bugge M, Niebuhr E, et al. (2007) Mutations in autism susceptibility candidate 2 (AUTS2) in patients with mental retardation. Hum Genet 121: 501–509.
[20]
Bedogni F, Hodge RD, Nelson BR, Frederick EA, Shiba N, et al. (2010) Autism susceptibility candidate 2 (Auts2) encodes a nuclear protein expressed in developing brain regions implicated in autism neuropathology. Gene Expr Patterns 10: 9–15.
[21]
Islam SM, Shinmyo Y, Okafuji T, Su Y, Naser IB, et al. (2009) Draxin, a repulsive guidance protein for spinal cord and forebrain commissures. Science 323: 388–393.
[22]
Tamada A, Kumada T, Zhu Y, Matsumoto T, Hatanaka Y, et al. (2008) Crucial roles of Robo proteins in midline crossing of cerebellofugal axons and lack of their up-regulation after midline crossing. Neural Dev 3: 29.
[23]
Thompson H, Andrews W, Parnavelas JG, Erskine L (2009) Robo2 is required for Slit-mediated intraretinal axon guidance. Dev Biol 335: 418–426.
[24]
Maestrini E, Pagnamenta AT, Lamb JA, Bacchelli E, Sykes NH, et al. (2010) High-density SNP association study and copy number variation analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility. Mol Psychiatry 15: 954–968.
[25]
Petek E, Schwarzbraun T, Noor A, Patel M, Nakabayashi K, et al. (2007) Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome. Mol Genet Genomics 277: 71–81.
[26]
Zhang XD, Wang Y, Zhang X, Han R, Wu JC, et al. (2009) p53 mediates mitochondria dysfunction-triggered autophagy activation and cell death in rat striatum. Autophagy 5: 339–350.
[27]
Ho SP, Hartig PR (1999) Antisense oligonucleotides for target validation in the CNS. Curr Opin Mol Ther 1: 336–343.
[28]
Geary RS, Yu RZ, Levin AA (2001) Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides. Curr Opin Investig Drugs 2: 562–573.
[29]
Sharif TR, Sharif M (1999) Overexpression of protein kinase C epsilon in astroglial brain tumor derived cell lines and primary tumor samples. Int J Oncol 15: 237–243.
[30]
Perletti GP, Concari P, Brusaferri S, Marras E, Piccinini F, et al. (1998) Protein kinase Cepsilon is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway. Oncogene 16: 3345–3348.
[31]
Cacace AM, Ueffing M, Philipp A, Han EK, Kolch W, et al. (1996) PKC epsilon functions as an oncogene by enhancing activation of the Raf kinase. Oncogene 13: 2517–2526.
[32]
Basu A, Persaud SD, Sivaprasad U (2008) Manipulation of PKC isozymes by RNA interference and inducible expression of PKC constructs. Methods Enzymol 446: 141–157.
[33]
Basu A, Johnson DE, Woolard MD (2002) Potentiation of tumor necrosis factor-alpha-induced cell death by rottlerin through a cytochrome-C-independent pathway. Exp Cell Res 278: 209–214.
[34]
Weterman MA, Ajubi N, van Dinter IM, Degen WG, van Muijen GN, et al. (1995) nmb, a novel gene, is expressed in low-metastatic human melanoma cell lines and xenografts. Int J Cancer 60: 73–81.
[35]
Metz RL, Yehia G, Fernandes H, Donnelly RJ, Rameshwar P (2005) Cloning and characterization of the 5′ flanking region of the HGFIN gene indicate a cooperative role among p53 and cytokine-mediated transcription factors: relevance to cell cycle regulation. Cell Cycle 4: 315–322.
[36]
Abdelmagid SM, Barbe MF, Arango-Hisijara I, Owen TA, Popoff SN, et al. (2007) Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function. J Cell Physiol 210: 26–37.
[37]
Tse KF, Jeffers M, Pollack VA, McCabe DA, Shadish ML, et al. (2006) CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma. Clin Cancer Res 12: 1373–1382.
[38]
Qian X, Mills E, Torgov M, LaRochelle WJ, Jeffers M (2008) Pharmacologically enhanced expression of GPNMB increases the sensitivity of melanoma cells to the CR011-vcMMAE antibody-drug conjugate. Mol Oncol 2: 81–93.
[39]
Rose AA, Siegel PM (2007) Osteoactivin/HGFIN: is it a tumor suppressor or mediator of metastasis in breast cancer? Breast Cancer Res 9: 403.
[40]
Rose AA, Annis MG, Dong Z, Pepin F, Hallett M, et al. (2010) ADAM10 releases a soluble form of the GPNMB/Osteoactivin extracellular domain with angiogenic properties. PLoS One 5: e12093.
[41]
Sultana R, Yu CE, Yu J, Munson J, Chen D, et al. (2002) Identification of a novel gene on chromosome 7q11.2 interrupted by a translocation breakpoint in a pair of autistic twins. Genomics 80: 129–134.
[42]
Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, et al. (1998) Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92: 205–215.
[43]
Seeger M, Tear G, Ferres-Marco D, Goodman CS (1993) Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron 10: 409–426.
[44]
Rothberg JM, Jacobs JR, Goodman CS, Artavanis-Tsakonas S (1990) slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev 4: 2169–2187.
[45]
Graeber MB, Muller U (1998) Recent developments in the molecular genetics of mitochondrial disorders. J Neurol Sci 153: 251–263.
[46]
Leonard JV, Schapira AH (2000) Mitochondrial respiratory chain disorders I: mitochondrial DNA defects. Lancet 355: 299–304.
[47]
Leonard JV, Schapira AH (2000) Mitochondrial respiratory chain disorders II: neurodegenerative disorders and nuclear gene defects. Lancet 355: 389–394.
[48]
Lagnado CA, Brown CY, Goodall GJ (1994) AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol Cell Biol 14: 7984–7995.
[49]
Zubiaga AM, Belasco JG, Greenberg ME (1995) The nonamer is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Biol 15: 2219–2230.
[50]
Bakheet T, Williams BR, Khabar KS (2003) ARED 2.0: an update of AU-rich element mRNA database. Nucleic Acids Res 31: 421–423.
[51]
Peng SS, Chen CY, Shyu AB (1996) Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements. Mol Cell Biol 16: 1490–1499.
[52]
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80.
[53]
Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, et al. (2007) NCBI GEO: mining tens of millions of expression profiles–database and tools update. Nucleic Acids Res 35: D760–765.
