近年來，隨著次世代基因定序(Next generation sequence, NGS)技術的發展，細胞內基因轉錄體(transcriptome) 分析變得相對容易且成本降低。所以我們利用次世代定序技術將不同程度抗藥性鼻咽癌細胞株進行RNA定序，並藉由比較鼻咽癌細胞內轉錄體基因表現量的差異，我們從中推論出造成細胞產生抗藥性的可能原因。依據轉錄組分析結果，我們進一步使用PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System進行Statistical overrepresentation test得到基因的Biological Process，推測癌細胞抗藥性的原因可能與染色體的修飾與重組有關。我的論文的第一部分著重在染色體乙醯轉移酶MOF在抗藥性功能的探討。MOF為組蛋白乙醯轉移酶(Histone acetyltransferases, HATs)屬於MYST (Moz-Ybf2 /Sas3 - Sas2 - Tip60)家族，主要參與細胞中的DNA受損反應、DNA 修復作用和DNA複製過程，並在基因特異性轉錄調控扮演重要的角色。在此我們利用具有順鉑(Cisplatin)抗藥性的鼻咽癌細胞株HONE6，將其MOF抑制後，發現細胞對於順鉑藥物的敏感度增加。同時，在DNA修復路徑中的Fanconi anemia (FA)、Post-replication repair (PRR) 和Homologous recombination (HR)相關基因表現量有下降的情形。
我的論文的第二部分是探討在酵母細胞中，MYST家族組蛋白乙醯轉移酶，NuA4參與DNA的雙股斷裂的修復機制。細胞進行代謝活動或外在環境因素都有可能引起細胞損傷，其中對細胞最嚴重且致命的損傷之一，即為DNA的雙股斷裂(DNA double strand breaks, DSBs)。當酵母菌(Saccharomyces cerevisiae)產生雙股斷裂時，組蛋白修飾複合體NuA4會被聚集到雙股斷裂處，並對組蛋白H2A和H4進行乙醯化修飾。當組蛋白被乙醯化修飾後，細胞染色體的結構會變得較為鬆散，使得DNA修復蛋白能順利與DNA結合進行細胞修復。而從先前的實驗我們知道NuA4的次單位蛋白Yng2和Eaf3，分別具有的PHD domain和Chromodomain可以辨認 DSBs 附近的甲基化修飾，幫助NuA4進入DNA雙股斷裂處。然而，目前卻沒有直接證據證明NuA4的確有作用於DSBs位置。所以這邊我們使用染色質免疫沉澱(Chromatin immunoprecipitation, ChIP)實驗，並由結果確定NuA4作用於DNA雙股斷裂處。

In recent years, with the development of next-generation sequencing (NGS) technology, transcriptome analysis has become much easier and cost reduction. Therefore, we used the NGS technology to sequence the cisplatin-resistant nasopharyngeal carcinoma (NPC) cell lines (HONE6 and HONE15), and compared with cisplatin-sensitive nasopharyngeal carcinoma cells (HONE1). Using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System, we classified the biological process (BP) of HONE6 and HONE15 transcriptome. According to the results of transcriptome analysis, we hypothesize that chromatin could be modified and remodeled to confer NPC cells cisplatin-resistant phenotype. MOF belongs to the MYST (Moz-Ybf2/Sas3 -Sas2-Tip60) family of histone acetyl transferases (HATs), mainly involved in the DNA damage response, DNA repair and DNA transcription in cells. Here, we found that the depletion of MOF in HONE6 cells increased the sensitivity to cisplatin and reduced the expression level of several genes in the homologous recombination (HR), Fanconi anemia (FA), and post-replication repair (PRR) pathways.
In addition, we study the function of another MYST protein, NuA4, in yeast Saccharomyces cerevisiae in response to DNA damage. We found that Esa1, a catalytic subunit of NuA4, can be recruited to the site of DNA double strand breaks using chromatin immunoprecipitation (ChIP) experiments. Combined with our previous study, our results suggest that NuA4 can acetylate histone H4 around the DSB sites, presumably to relax the DNA structure for facilitating DSB repair.

Avvakumov, N. and Cote, J., 2007. The MYST family of histone acetyltransferases and their intimate links to cancer. Oncogene 26, 5395-407.
Bao, Y. and Shen, X., 2007. Chromatin remodeling in DNA double-strand break repair. Curr Opin Genet Dev 17, 126-31.
Barbour, L. and Xiao, W., 2003. Regulation of alternative replication bypass pathways at stalled replication forks and its effects on genome stability: a yeast model. Mutat Res 532, 137-55.
Barry, M.A., Behnke, C.A. and Eastman, A., 1990. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol 40, 2353-62.
Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M.B., Bartek, J. and Lukas, J., 2006. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173, 195-206.
Bertone, P., Stolc, V., Royce, T.E., Rozowsky, J.S., Urban, A.E., Zhu, X., Rinn, J.L., Tongprasit, W., Samanta, M., Weissman, S., Gerstein, M. and Snyder, M., 2004. Global identification of human transcribed sequences with genome tiling arrays. Science 306, 2242-6.
Bird, A.W., Yu, D.Y., Pray-Grant, M.G., Qiu, Q., Harmon, K.E., Megee, P.C., Grant, P.A., Smith, M.M. and Christman, M.F., 2002. Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair. Nature 419, 411-5.
Burden, D.A. and Osheroff, N., 1998. Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim Biophys Acta 1400, 139-54.
Burgess, D.J., 2012. Genomic instability: close-up on cancer copy number alterations. Nat Rev Genet 13, 5.
Campos, E.I. and Reinberg, D., 2009. Histones: annotating chromatin. Annu Rev Genet 43, 559-99.
Cheng, J., Kapranov, P., Drenkow, J., Dike, S., Brubaker, S., Patel, S., Long, J., Stern, D., Tammana, H., Helt, G., Sementchenko, V., Piccolboni, A., Bekiranov, S., Bailey, D.K., Ganesh, M., Ghosh, S., Bell, I., Gerhard, D.S. and Gingeras, T.R., 2005. Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308, 1149-54.
Ciccia, A., McDonald, N. and West, S.C., 2008. Structural and functional relationships of the XPF/MUS81 family of proteins. Annu Rev Biochem 77, 259-87.
Doyon, Y. and Cote, J., 2004. The highly conserved and multifunctional NuA4 HAT complex. Curr Opin Genet Dev 14, 147-54.
Eastman, A., 1987. Cross-linking of glutathione to DNA by cancer chemotherapeutic platinum coordination complexes. Chem Biol Interact 61, 241-8.
Faucher, D. and Wellinger, R.J., 2010. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway. PLoS Genet 6.
Galanski, M., 2006. Recent developments in the field of anticancer platinum complexes. Recent Pat Anticancer Drug Discov 1, 285-95.
Gottesman, M.M., 2002. Mechanisms of cancer drug resistance. Annu Rev Med 53, 615-27.
Gupta, A., G.G. Sharma, C.S. Young, M. Agarwal, E.R. Smith, T.T. Paull, J.C. Lucchesi, K.K. Khanna, T. Ludwig, and T.K. Pandita. 2005. Involvement of human MOF in ATM function. Molecular and cellular biology. 25:5292-5305.
Hampton, G.M. and Frierson, H.F., 2003. Classifying human cancer by analysis of gene expression. Trends Mol Med 9, 5-10.
Holt, R.A. and Jones, S.J., 2008. The new paradigm of flow cell sequencing. Genome Res 18, 839-46.
Howlett, N.G., Taniguchi, T., Olson, S., Cox, B., Waisfisz, Q., De Die-Smulders, C., Persky, N., Grompe, M., Joenje, H., Pals, G., Ikeda, H., Fox, E.A. and D'Andrea, A.D., 2002. Biallelic inactivation of BRCA2 in Fanconi anemia. Science 297, 606-9.
Klarer, A.C. and McGregor, W., 2011. Replication of damaged genomes. Crit Rev Eukaryot Gene Expr 21, 323-36.
Kouzarides, T., 2007. Chromatin modifications and their function. Cell 128, 693-705.
Lee, K.Y. and Myung, K., 2008. PCNA modifications for regulation of post-replication repair pathways. Mol Cells 26, 5-11.
Lennon, G.G. and Lehrach, H., 1991. Hybridization analyses of arrayed cDNA libraries. Trends Genet 7, 314-7.
Liu, Y. and West, S.C., 2004. Happy Hollidays: 40th anniversary of the Holliday junction. Nat Rev Mol Cell Biol 5, 937-44.
Mardis, E.R., 2008. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9, 387-402.
Mitchell, L., Lambert, J.P., Gerdes, M., Al-Madhoun, A.S., Skerjanc, I.S., Figeys, D. and Baetz, K., 2008. Functional dissection of the NuA4 histone acetyltransferase reveals its role as a genetic hub and that Eaf1 is essential for complex integrity. Mol Cell Biol 28, 2244-56.
Morozova, O. and Marra, M.A., 2008. Applications of next-generation sequencing technologies in functional genomics. Genomics 92, 255-64.
Mutz, K.O., Heilkenbrinker, A., Lonne, M., Walter, J.G. and Stahl, F., 2013. Transcriptome analysis using next-generation sequencing. Curr Opin Biotechnol 24, 22-30.
Oliver, T.G., Mercer, K.L., Sayles, L.C., Burke, J.R., Mendus, D., Lovejoy, K.S., Cheng, M.H., Subramanian, A., Mu, D., Powers, S., Crowley, D., Bronson, R.T., Whittaker, C.A., Bhutkar, A., Lippard, S.J., Golub, T., Thomale, J., Jacks, T. and Sweet-Cordero, E.A., 2010. Chronic cisplatin treatment promotes enhanced damage repair and tumor progression in a mouse model of lung cancer. Genes Dev 24, 837-52.
Pfeiffer, P., Goedecke, W. and Obe, G., 2000. Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis 15, 289-302.
Pfister, S., Rea, S., Taipale, M., Mendrzyk, F., Straub, B., Ittrich, C., Thuerigen, O., Sinn, H.P., Akhtar, A. and Lichter, P., 2008. The histone acetyltransferase hMOF is frequently downregulated in primary breast carcinoma and medulloblastoma and constitutes a biomarker for clinical outcome in medulloblastoma. Int J Cancer 122, 1207-13.
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. and Bonner, W.M., 1998. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273, 5858-68.
Sale, J.E., 2013. Translesion DNA synthesis and mutagenesis in eukaryotes. Cold Spring Harb Perspect Biol 5, a012708.
San Filippo, J., Sung, P. and Klein, H., 2008. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77, 229-57.
Sanger, F., Nicklen, S. and Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463-7.
Sartori, A.A., Lukas, C., Coates, J., Mistrik, M., Fu, S., Bartek, J., Baer, R., Lukas, J. and Jackson, S.P., 2007. Human CtIP promotes DNA end resection. Nature 450, 509-514.
Sharma, S. and Raghavan, S.C., 2010. Nonhomologous DNA end joining in cell-free extracts. J Nucleic Acids 2010.
Shendure, J. and Ji, H., 2008. Next-generation DNA sequencing. Nat Biotechnol 26, 1135-45.
Su, W.P., S.H. Hsu, L.C. Chia, J.Y. Lin, S.B. Chang, Z.D. Jiang, Y.J. Lin, M.Y. Shih, Y.C. Chen, M.S. Chang, W.B. Yang, J.J. Hung, P.C. Hung, W.S. Wu, K. Myung, and H. Liaw. 2016. Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks. 202:77-92.
Su, W.P., Hsu, S.H., Wu, C.K., Chang, S.B., Lin, Y.J., Yang, W.B., Hung, J.J., Chiu, W.T., Tzeng, S.F., Tseng, Y.L., Chang, J.Y., Su, W.C. and Liaw, H., 2014. Chronic treatment with cisplatin induces replication-dependent sister chromatid recombination to confer cisplatin-resistant phenotype in nasopharyngeal carcinoma. Oncotarget 5, 6323-37.
Sultan, M., Schulz, M.H., Richard, H., Magen, A., Klingenhoff, A., Scherf, M., Seifert, M., Borodina, T., Soldatov, A., Parkhomchuk, D., Schmidt, D., O'Keeffe, S., Haas, S., Vingron, M., Lehrach, H. and Yaspo, M.L., 2008. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 321, 956-60.
Sung, P., Krejci, L., Van Komen, S. and Sehorn, M.G., 2003. Rad51 recombinase and recombination mediators. J Biol Chem 278, 42729-32.
Sykes, S.M., Mellert, H.S., Holbert, M.A., Li, K., Marmorstein, R., Lane, W.S. and McMahon, S.B., 2006. Acetylation of the p53 DNA-binding domain regulates apoptosis induction. Mol Cell 24, 841-51.
Taipale, M., Rea, S., Richter, K., Vilar, A., Lichter, P., Imhof, A. and Akhtar, A., 2005. hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells. Mol Cell Biol 25, 6798-810.
Thorslund, T. and West, S.C., 2007. BRCA2: a universal recombinase regulator. Oncogene 26, 7720-30.
Ulrich, H.D., 2005. The RAD6 pathway: control of DNA damage bypass and mutagenesis by ubiquitin and SUMO. Chembiochem 6, 1735-43.
Ulrich, H.D., 2009. Regulating post-translational modifications of the eukaryotic replication clamp PCNA. DNA Repair (Amst) 8, 461-9.
Wang, Z., Gerstein, M. and Snyder, M., 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10, 57-63.
Wu, L. and Hickson, I.D., 2006. DNA helicases required for homologous recombination and repair of damaged replication forks. Annu Rev Genet 40, 279-306.
Xia, B., Sheng, Q., Nakanishi, K., Ohashi, A., Wu, J., Christ, N., Liu, X., Jasin, M., Couch, F.J. and Livingston, D.M., 2006. Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2. Mol Cell 22, 719-29.
Zhao, S., Fung-Leung, W.P., Bittner, A., Ngo, K. and Liu, X., 2014. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS One 9, e78644.