ROCK 抑制普遍性基因轉錄之探討

Abstract

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.