臺灣博碩士論文加值系統

Rho-associated kinase (ROCK)調控的細胞膜皺縮在PMA誘發D2細胞凋亡過程中扮演了決定性的角色，該群細胞有細胞核結構改變、核染色質(chromatin)濃縮和RNA合成減少的現象。藉由冷光酵素活性系統(luciferase reporter system)分析第一、二和三型聚合脢所從屬的基因表現，我發現第一、二和三型聚合脢所從屬的基因表現皆會被ROCK(CAT)抑制，說明ROCK的過度活化足以抑制普遍性的基因轉錄。由於ROCK造成的細胞皺縮不會使核染色質過度包裹到無法被微球菌核酸脢(micrococcal nuclease)切割的狀態，顯示ROCK所造成轉錄的抑制不是導因於核染色質更高度秩序(higher-order)的濃縮。藉由分析組蛋白的修飾得知在HEK 293T細胞中ROCK(CAT)的表現會造成組蛋白H3的K9三甲基化增加及S10磷酸化減少，而不影響組蛋白H2B的S14磷酸化。我同時利用核染色質免疫沈澱法(ChIP)證實在表現ROCK(CAT)的HEK 293T以及處理PMA的D2細胞中，位於rRNA啟動子區域的組蛋白H3有K9三甲基化增加的現象。已知在核染色質重組所造成的轉錄抑制機轉中，組蛋白H3的K9甲基化扮演一關鍵性的角色，因此我認為透過活化ROCK所造成的細胞皺縮可能傳遞一個促進組蛋白H3 K9甲基化的訊息。有趣的是，利用siRNA降低組蛋白甲基轉移脢G9a的表現可以減緩ROCK(CAT)所造成的轉錄抑制以及組蛋白H3 K9三甲基化增加。因此我推論細胞質中ROCK所引發的皺縮訊息可傳遞到細胞核，透過G9a的作用促進核染色質重組導致基因轉錄的抑制。

In phorbol ester-induced pro-apoptotic D2 cells, the Rho-associated kinase (ROCK) plays a determining role in membrane contraction, which concomitantly results in nuclear shape change, chromatin condensation and transcription repression. Using various luciferase reporter systems, I found that pol I, II and III-dependent transcriptions are all suppressed by ectopic expression of dominant active form of ROCK(CAT), indicating that too much ROCK activation is sufficient to shut-off general transcription. Here, I showed that ROCK-mediated contraction does not make chromatin too packed to be accessed by micrococcal nuclease digestion, indicating that ROCK-mediated transcription repression is not a result of a higher-order chromatin condensation. By analyzing histone modifications, I found that expression of ROCK(CAT) in HEK 293T cells increased K9 tri-methylation of histone H3 with decreased extent of S10 phosphorylation of histone H3, while S14 phosphorylation of histone H2B remained unaffected. Consistently, results from ChIP assays showed that K9-methylation of H3 in the rRNA promoter region was increased in both HEK 293T expressing ROCK(CAT) and PMA-induced pro-apoptotic D2 cells. Since it is well known that K9 methylation of H3 plays a crucial role in chromatin remodeling for transcriptional repression, it is very likely that ROCK-mediated contraction is capable of conferring signals for K9 methylation of H3 in nuclei. Of interest, depleted expression of G9a, a histone methyltransferase, restored transcription capacity in cells expressing ROCK(CAT) with a concurrent reduction in extent of K9 methylation of histone H3. Accordingly, I hypothesized that ROCK-mediated contraction in cytoplasm transmits signal to nucleus, where function of G9a is upregulated to remodel chromatin for transcriptional repression.

中文摘要………………………………………………………2
Abstract………………………………………………………...3
Introduction…………………………………………………….4
Materials and Methods………………………………………..14
Results…………………………………………………………25
Discussion……………………………………………………..33
Figures and Legends…………………………………………..37
References……………………………………………………..55

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