臺灣博碩士論文加值系統

SUMO修飾 (SUMOylation) 是一種可逆的後轉譯修飾，在真核細胞內，這種修飾普遍參與細胞內各種生化反應，包含了調控後基因體 (epigenetics) 的基因轉錄。目前，SUMO修飾在後基因體調控上的細節與影響，尚未完全釐清。
卡波氏肉瘤相關性皰疹病毒 (KSHV) 與其他皰疹病毒一樣，演化出直接或間接方式利用SUMO修飾系統，調控宿主細胞或病毒基因表現，以利病毒本身的複製及生存。特別是KSHV除了間接利用宿主的SUMO修飾系統外，病毒本身基因體就會表現一個SUMO-2/3專一性的SUMO E3連接酶稱為K-bZIP。人類細胞中具有3種不同的SUMO paralogues，分別為SUMO-1，SUMO-2及SUMO-3。為瞭解皰疹病毒活化過程中，不同的SUMO paralogues在基因體上結合的差異，利用能誘發K-Rta產生的BCBL-1細胞株促使病毒活化，使用ChIP-seq進行SUMO-1及SUMO-2/3在基因體上結合位置的分析。發現病毒活化前SUMO-1及SUMO-2/3基因體上之結合呈現相似型態，集中在細胞的轉錄起始點附近，與病毒表現SUMO-2/3專一性SUMO E3連接酶一致，病毒活化後，只有SUMO-2/3在細胞基因體上結合量明顯增加，且主要集中表現量不被改變基因的啟動子 (promoter) 上。KSHV活化前後，啟動子受SUMO修飾的基因，進行基因本體 (Gene Ontology; GO) 分析，發現病毒活化後，SUMO-2/3修飾的基因，主要集中在與免疫與細胞生存相關的基因上，這顯示病毒可能透過增加特定基因啟動子附近調控蛋白或轉譯因子的SUMO-2/3修飾，在病毒活化過中，避免宿主細胞內抗病毒相關基因的活化。暗示著病毒活化的過程中，病毒利用SUMO-2/3修飾基因的啟動子，讓免疫相關的基因表現量不被改變。病毒活化後，病毒基因體上SUMO-2/3修飾也明顯地增加，這顯示可能與病毒本身表現SUMO-2/3專一性的E3連接酶 (K-bZIP) 有關，利用同源重組全病毒基因，產生K-bZIP的L75A (SIM突變) 及ΔK8突變 (缺失突變) 株，發現L75A突變株在產生病毒顆粒的能力變差，且突變株產生的病毒顆粒重新感染細胞的能力也較差。

Small ubiquitin-like modifiers (SUMO) are reversible post-translational modifications involved in virtually all cellular processes in eukaryotes including the epigenetic regulation of transcription. However, the functions of SUMOylation during mammalian epigenetic transcriptional regulation are largely uncharacterized. Herpesviruses, including Kaposi’s sarcoma associated herpesvirus (KSHV) , have evolved a range of mechanisms to directly or indirectly modulate SUMO machinery in order to regulate the expression of cellular and viral genomes, thus advancing their own replication agenda. Interestingly, KSHV encodes a SUMO ligase, K-bZIP, with specificity toward SUMO2/3. To delineate the differential chromatin binding patterns of SUMO paralogues during herpesvirus reactivation, we comprehensively mapped the genomic SUMO-1 and SUMO-2/3 binding sites during K-Rta induced KSHV reactivation of the BCBL-1 cell line using a ChIP-seq assay. In control, the SUMO-1 and SUMO-2/3 peaks have a similar distance distribution across the genome with highly enriched around transcription start sites (TSSs) and near the promoters of highly transcribed genes. Interestingly, we detect that SUMO-2/3, but not SUMO-1, binding was significantly increased in the promoter regions of transcriptionally active genes with unaltered expression during viral reactivation. Gene ontology analysis shows that these SUMO-2/3 targeted genes are primarily involved in cellular immune responses and cell survival. Our results suggest that the virus enhances SUMO-2/3 modification near promoters during reactivation in order to prevent anti-viral related gene activation. During viral reactivation, we also detect a significant increase of SUMO-2/3 binding across viral genome. This data suggest that KSHV SUMO-2/3 specific E3 ligase, K-bZIP, may involve in viral reactivation. To study this, we made recombinant KSHV with SIM domain (L75A) and deletion mutant. The result shows that SUMO E3 ligase activity is important for KSHV reactivation and infection.

目錄
中文摘要 i
英文摘要 ii

壹、緒論 1
一、卡波氏肉瘤相關性皰疹病毒 1
1. 卡波氏肉瘤相關性皰疹病毒 1
2. 卡波氏肉瘤相關性皰疹病毒生活史 2
3. 卡波氏肉瘤相關性皰疹病毒基因 2
二、SUMO (Small Ubiquitin-related Modifier) 4
1. SUMO paralogues 4
2. SUMO修飾的機制 5
3. SUMO非共價鍵結的交互作用 6
三、SUMOylation與病毒 6
四、非編碼型RNA 7


貳、實驗材料與方法 8
一、實驗材料 8
1. 細胞株 8
2. 培養基與培養液 8
3. 質體 9
4. 溶液 10
5. 抗體 11
6. 其他 12
二、實驗方法 12
1. 細胞培養 12
2. 染色質免疫沉澱法及次世代定序 12
3. 即時聚合酶連鎖反應 13
4. 聚合酶連鎖反應 13
5. 反轉錄聚合酶連鎖反應 14
6. 核糖核酸次世代定序 14
7. 轉型作用 14
8. 質體製備 14
9. 抽取病毒DNA 15
10. 細胞轉染作用 15
11. SDS-聚丙烯醯胺凝膠電泳 15
12. 西方墨點法 15
13. 免疫沉澱法 16
14. ChIP-Seq data分析 16
15. RNA-Seq data分析 16

參、實驗結果 17
一、病毒活化過程中基因體上SUMO paralogue修飾的型態與變化 17
1. 病毒活化過程中，全基因體上SUMO的修飾變化 17
2. 病毒活化過程中，SUMO paralogues分佈型態 19
二、探討SUMO-1及SUMO-2/3在轉錄因子結合位置上修飾 19
三、病毒活化過程中，SUMO-2/3潛在轉錄因子 21
四、探討SUMO-2/3在啟動子的修飾與基因表現之關聯性 22
五、探討KSHV中的K-bZIP在病毒活化中扮演的角色 24

肆、討論 26
一、病毒活化過程中基因體上SUMO paralogue結合型態的差異 26
二、病毒活化過程中，SUMO-2/3修飾與基因表現 27
三、病毒活化過程中，SUMO-2/3修飾與轉錄因子 28
四、病毒與SUMO修飾 29

伍、參考文獻 31

圖、表 37

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