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1083-351X286212011May27The Journal of biological chemistryJ Biol ChemGenomic selection identifies vertebrate transcription factor Fezf2 binding sites and target genes.186411864918641-910.1074/jbc.M111.236471Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.ChenLishanLDepartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94158, USA.ZhengJiashunJYangNanNLiHaoHGuoSuSengR01 NS042626NSNINDS NIH HHSUnited StatesNS042626NSNINDS NIH HHSUnited StatesJournal ArticleResearch Support, N.I.H., Extramural20110406
United StatesJ Biol Chem2985121R0021-92580Nerve Tissue Proteins0Transcription Factors0Zebrafish Proteins9007-49-2DNAIMAnimalsDNAgeneticsmetabolismHEK293 CellsHumansMicrocephalygeneticsmetabolismMutationNerve Tissue ProteinsgeneticsmetabolismProsencephalonembryologyResponse ElementsphysiologySELEX Aptamer TechniqueTranscription FactorsgeneticsmetabolismTranscription, GeneticZebrafishembryologygeneticsZebrafish Proteinsgeneticsmetabolism20114860201148602011729602012527ppublish21471212PMC309968010.1074/jbc.M111.236471S0021-9258(20)51121-6Iyer V. R., Horak C. E., Scafe C. S., Botstein D., Snyder M., Brown P. O. (2001) Nature 409, 533–53811206552Johnson D. S., Mortazavi A., Myers R. M., Wold B. (2007) Science 316, 1497–150217540862Blackwell T. K., Weintraub H. (1990) Science 250, 1104–11102174572Ellington A. D., Szostak J. W. (1990) Nature 346, 818–8221697402Tuerk C., Gold L. (1990) Science 249, 505–5102200121Djordjevic M. (2007) Biomol. Eng. 24, 179–18917428731Jolma A., Kivioja T., Toivonen J., Cheng L., Wei G., Enge M., Taipale M., Vaquerizas J. M., Yan J., Sillanpää M. J., Bonke M., Palin K., Talukder S., Hughes T. R., Luscombe N. M., Ukkonen E., Taipale J. (2010) Genome Res. 20, 861–873PMC287758220378718Shtatland T., Gill S. C., Javornik B. E., Johansson H. E., Singer B. S., Uhlenbeck O. C., Zichi D. A., Gold L. (2000) Nucleic Acids Res. 28, E93.PMC11316211058143Shostak Y., Van Gilst M. R., Antebi A., Yamamoto K. R. (2004) Genes Dev. 18, 2529–2544PMC52954015489294Matsuo-Takasaki M., Lim J. H., Beanan M. J., Sato S. M., Sargent T. D. (2000) Mech. Dev. 93, 201–20410781957Hashimoto H., Yabe T., Hirata T., Shimizu T., Bae Y., Yamanaka Y., Hirano T., Hibi M. (2000) Mech. Dev. 97, 191–19511025224Guo S., Wilson S. W., Cooke S., Chitnis A. B., Driever W., Rosenthal A. (1999) Dev. Biol. 208, 473–48710191060Levkowitz G., Zeller J., Sirotkin H. I., French D., Schilbach S., Hashimoto H., Hibi M., Talbot W. S., Rosenthal A. (2003) Nat. Neurosci. 6, 28–3312469125Jeong J. Y., Einhorn Z., Mathur P., Chen L., Lee S., Kawakami K., Guo S. (2007) Development 134, 127–13617164418Berberoglu M. A., Dong Z., Mueller T., Guo S. (2009) Gene Expr. Patterns 9, 411–422PMC274671319524703Rouaux C., Arlotta P. (2010) Nat. Neurosci. 13, 1345–1347PMC420744220953195Hirata T., Suda Y., Nakao K., Narimatsu M., Hirano T., Hibi M. (2004) Dev. Dyn. 230, 546–55615188439Molyneaux B. J., Arlotta P., Hirata T., Hibi M., Macklis J. D. (2005) Neuron 47, 817–83116157277Chen B., Schaevitz L. R., McConnell S. K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 17184–17189PMC128256916284245Shimizu T., Hibi M. (2009) Dev. Growth Differ. 51, 221–23119222525Liu X., Brutlag D. L., Liu J. S. (2001) Pac. Symp. Biocomput. 127–13811262934Roth F. P., Hughes J. D., Estep P. W., Church G. M. (1998) Nat. Biotechnol. 16, 939–9459788350Bailey T. L., Williams N., Misleh C., Li W. W. (2006) Nucleic Acids Res. 34, W369–373PMC153890916845028Chin J., Längst G., Becker P. B., Widom J. (2004) Methods Enzymol. 376, 3–1614975295Anderson B. J., Larkin C., Guja K., Schildbach J. F. (2008) Methods Enzymol. 450, 253–272PMC305135619152864Li Q., Ritter D., Yang N., Dong Z., Li H., Chuang J. H., Guo S. (2010) Dev. Biol. 337, 484–495PMC333082919850031De Val S., Chi N. C., Meadows S. M., Minovitsky S., Anderson J. P., Harris I. S., Ehlers M. L., Agarwal P., Visel A., Xu S. M., Pennacchio L. A., Dubchak I., Krieg P. A., Stainier D. Y., Black B. L. (2008) Cell 135, 1053–1064PMC278266619070576Jeong J. Y., Einhorn Z., Mercurio S., Lee S., Lau B., Mione M., Wilson S. W., Guo S. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 5143–5148PMC145880816549779Baala L., Briault S., Etchevers H. C., Laumonnier F., Natiq A., Amiel J., Boddaert N., Picard C., Sbiti A., Asermouh A., Attié-Bitach T., Encha-Razavi F., Munnich A., Sefiani A., Lyonnet S. (2007) Nat. Genet. 39, 454–45617353897Sessa A., Mao C. A., Hadjantonakis A. K., Klein W. H., Broccoli V. (2008) Neuron 60, 56–69PMC288776218940588Arnold S. J., Huang G. J., Cheung A. F., Era T., Nishikawa S., Bikoff E. K., Molnár Z., Robertson E. J., Groszer M. (2008) Genes Dev. 22, 2479–2484PMC254669718794345Porter F. D., Drago J., Xu Y., Cheema S. S., Wassif C., Huang S. P., Lee E., Grinberg A., Massalas J. S., Bodine D., Alt F., Westphal H. (1997) Development 124, 2935–29449247336Mangale V. S., Hirokawa K. E., Satyaki P. R., Gokulchandran N., Chikbire S., Subramanian L., Shetty A. S., Martynoga B., Paul J., Mai M. V., Li Y., Flanagan L. A., Tole S., Monuki E. S. (2008) Science 319, 304–309PMC249460318202285Seth A., Culverwell J., Walkowicz M., Toro S., Rick J. M., Neuhauss S. C., Varga Z. M., Karlstrom R. O. (2006) Development 133, 725–73516436624Lampe X., Samad O. A., Guiguen A., Matis C., Remacle S., Picard J. J., Rijli F. M., Rezsohazy R. (2008) Nucleic Acids Res. 36, 3214–3225PMC242548918417536Tümpel S., Cambronero F., Sims C., Krumlauf R., Wiedemann L. M. (2008) Proc. Natl Acad. Sci. U.S.A. 105, 20077–20082PMC262934919104046Dong X., Navratilova P., Fredman D., Drivenes Ø., Becker T. S., Lenhard B. (2010) Nucleic Acids Res. 38, 1071–1085PMC283133019969543Shimizu T., Nakazawa M., Kani S., Bae Y. K., Shimizu T., Kageyama R., Hibi M. (2010) Development 137, 1875–188520431123Fordyce P. M., Gerber D., Tran D., Zheng J., Li H., DeRisi J. L., Quake S. R. (2010) Nat. Biotech. 28, 970–975PMC293709520802496Westerfield M. (ed) (1995) The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish, Brachydanio rerio, 3rd Ed., University of Oregon Press, Eugene, ORFujita P. A., Rhead B., Zweig A. S., Hinrichs A. S., Karolchik D., Cline M. S., Goldman M., Barber G. P., Clawson H., Coelho A., Diekhans M., Dreszer T. R., Giardine B. M., Harte R. A., Hillman-Jackson J., Hsu F., Kirkup V., Kuhn R. M., Learned K., Li C. H., Meyer L. R., Pohl A., Raney B. J., Rosenbloom K. R., Smith K. E., Haussler D., Kent W. J. (2011) Nucleic Acids Res. 39, D876–882PMC324272620959295Siepel A., Bejerano G., Pedersen J. S., Hinrichs A. S., Hou M., Rosenbloom K., Clawson H., Spieth J., Hillier L. W., Richards S., Weinstock G. M., Wilson R. K., Gibbs R. A., Kent W. J., Miller W., Haussler D. (2005) Genome Res. 15, 1034–1050PMC118221616024819Krishnan K., Salomonis N., Guo S. (2008) PLoS ONE 3, e3621.PMC257538118978947Chen H., Contreras X., Yamaguchi Y., Handa H., Peterlin B. M., Guo S. (2009) PLoS One 4, e6918.PMC273503319742326Guo S., Brush J., Teraoka H., Goddard A., Wilson S. W., Mullins M. C., Rosenthal A. (1999) Neuron 24, 555–56610595509