臺灣博碩士論文加值系統

甲基轉化酶SMYD3高度表現在許多種類的癌症中，SMYD3藉由甲基化組蛋白或是非組蛋白的受質來調控包括染色質重組、訊息傳導和控制細胞週期等動作。由於在癌症細胞中SMYD3有不正常的高度表現，且研究已指出SMYD3過表現會促進癌症細胞增生且連結到癌症預後不良，因此在許多種類的癌症SMYD3中被認為是一個預後指標。為了研究SMYD3的新功能，我利用SMYD3進行ChIP-seq實驗，並將其結果和先前發表的微陣列晶片資料庫做比對，實驗結果顯示一群和DNA損壞反應相關的基因會受到調控。進一步研究指出SMYD3會調控另一個導致癌症的路徑，DNA修復。此調控是藉由促進許多參與在同源重組的基因之表現。缺乏SMYD3的細胞表現出對於DNA損壞壓力的高度敏感，DNA斷裂及染色體重組的程度增加、修復蛋白聚集的情形降低，這些都導致同源重組功能的損害。若是在缺乏SMYD3的細胞中外源性的表現SMYD3則可以彌補這種缺陷。此外，這個對同源重組相關基因的調控是藉由在其啟動子甲基化H3K4。這些實驗結果顯示，SMYD3除了經由已知的機制促進癌症生成，也透過調控同源重組蛋白質貢獻於維持基因體的完整性。

SET and MYND domain containing-3 (SMYD3) is a methyltransferase highly expressed in many types of cancer. SMYD3 methylates various histone and non-histone targets to regulate distinct roles in chromatin remodeling, signal transduction and cell cycle control. SMYD3 is linked to increased cell proliferation and poor prognosis in human cancers. Due to its abnormal expression in tumors, SMYD3 is considered as prognostic markers in various cancers. To explore novel functions of SMYD3, we performed ChIP-seq experiments and compared the results with previously published microarray data. A group of DNA damage response genes were called. Further study showed that SMYD3 modulates another hallmark of cancer, DNA repair, by stimulating transcription of genes involved in multiple steps of homologous recombination (HR). Deficiency of SMYD3 induces DNA-damage hypersensitivity, increases levels of DNA breaks and chromosomal rearrangement, decreases levels of repair foci, and leads to impairment of homologous recombination. The complementation of exogenous SMYD3 is able to restore the defects caused by SMYD3 depletion. Moreover, the regulation of homologous recombination-related genes is via the methylation of H3K4 at the target gene promoters. These data imply that, besides its reported oncogenic abilities, SMYD3 may maintain genome integrity by ensuring expression levels of HR proteins to cope with the high demand of restart of stalled replication forks in cancers.

口試委員審定書 i
誌謝 ii
中文摘要 iii
Abstract. iv
Table of Contents. v
Chapter 1. Introduction. 1
SMYD3 and its biological functions. 1
DNA double-strand breaks repair pathways. 2
SMYD3 is involved in homologous recombination. 4
Chapter 2. Results. 5
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) analysis reveals a role of SMYD3 in DNA repair. 5
Microarray data analysis identifies SMYD3-regulated expression of DNA repair machinery. 6
SMYD3 mediates the HR pathway. 8
SMYD3 knockdown downregulates HR gene expressions. 9
SMYD3 controls the expression of HR genes through methylating histone H3K4. 11
Chapter 3. Discussion. 12
Chapter 4. Materials and methods. 15
Cell lines, plasmid construction, and gene knockdown 15
ChIP assay. 15
ChIP-seq assay and data analysis. 16
Data analysis of gene expression microarray. 17
Cell fixation and immunofluorescence assays. 17
Colony formation assay. 18
Nuclear/cytosol fractionation. 18
Western blot analysis. 19
Comet assay. 19
Micronuclei counts. 20
RNA analysis and quantitative real-time polymerase chain reaction (qRT-PCR). 20
HR assay. 21
Plasmid based end-joining assay. 21
Plasmid based MMEJ assay. 22
Statistical analysis. 22
Chapter 5. Figures and Figure Legends. 23
Figure 1. ChIP analysis. 23
Figure 2. SMYD3 may be involved in DNA repair. 25
Figure 3. SMYD3 is required for DNA repair machinery. 28
Figure 4. SMYD3-depleted cells are hypersensitive to IR stress. 29
Figure 5. SMYD3 deficiency increases the ratio of DNA damage after IR treatment. 30
Figure 6. SMYD3 mediates the HR pathway. 33
Figure 7. The knockdown efficiency of each knockdown clones used. 35
Figure 8. Complementation of SMYD3 in shSMYD3 cells is able to recover its HR deficiency. 36
Figure 9. SMYD3 knockdown downregulates HR gene expressions. 38
Figure 10. SMYD3 knockdown downregulates the expression of DNA repair foci. 39
Figure 11. SMYD3 regulates the expression of MDC1 through methylating histone H3K4. 40
Figure 12. SMYD3 activates the expression of EXO1 and RAD54B through methylating histone H3K4. 41
Figure 13. SMYD3 methylates histone and non-histone substrates to regulate different pathways that are important for hallmarks of cancer. 43
Chapter 6. Table. 44
Table 1. Down-regulated genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences) cross-referenced with SMYD3 ChIP-seq data. 44
Table 2. Up-regulated genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences) cross-referenced with SMYD3 ChIP-seq data. 46
Table 3. Down-regulated genes in H2A.Z.1WT/H2A.Z.1K101Q array data (genes with < 0.5 fold differences) cross-referenced with H2A.Z.1K101me2 ChIP-seq data. 48
Table 4. Down-regulated genes in H2A.Z.1WT/H2A.Z.1K101Q array data (genes with < 0.5 fold differences) cross-referenced with H2A.Z.1K101me2 ChIP-seq data. 49
Table 5. GO analysis of down-regulated genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences) cross-referenced with SMYD3 ChIP-seq data. 50
Table 6. Down-regulated DNA damage stimulus response genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences). 51
Table 7. Down-regulated HR genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences). 53
Table 8. Oligo sequences for shRNA-mediated gene knockdown 54
Table 9. Primers used in this study 55
Chapter 7. References. 56
Chapter 8. Appendix. 62
Figures Contributed by Other Authors. 62
Appendix Figure 1. Function of nuclear SMYD3. 63
Appendix Figure 2. SMYD3 location and expression are not modulated by DNA damage insults. 64
Appendix Figure 3. SMYD3 deficiency increases the ratio of micronuclei after IR treatment. 66
Appendix Figure 4. SMYD3 knockdown downregulates HR gene expressions. 67
Appendix Figure 5. SMYD3 deficiency does not affect the formation of 53BP1 foci after IR treatment. 68
Appendix Figure 6. BRCA1 is reduced in SMYD3-depleted cells. 69

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