RNA 聚合酶I 藉由DNA 甲基轉移酶3B 以及細胞骨架重組所進行的功能性調節

Abstract

Chapter I DNA methyltransferase 3b (DNMT3b)已知在表徵遺傳學上的修飾作用扮演重要的調節角色。它主要的功能在於產生de novo 的DNA 甲基化作用(methylation)，這個功能對於細胞生長(cell growth)以及維持基因組的穩定(genome stability)是必須的。利用HCT116 細胞，我們發現DNMT3b 剔除(knockout)的細胞表現高量的DNA 損傷訊號 (DNA damage signal)，以H2AX foci 形式表現，經由抑制RNA polymerase I(Pol I)的轉錄作用，可以明顯降低這些DNA 損傷訊號。雖然Pol I 主要的功能是核醣體RNA(ribosomal RNA)轉錄作用，但這些DNA 損傷訊號的位置並不在rRNA 基因上，除非細胞週期同步於有絲分裂期(mitotic progression)。我們進一步觀察到Pol I 的抑制作用可以減少在BKO 細胞中的基因組不穩定性(genome instability)。將原生型(wild-type)以及失去酵素活性(catalytic-dead)的DNMT3b 表現於BKO 細胞中，可以降低BKO 細胞中的DNA 損傷訊號以及基因組不穩定性，表示DNMT3b 在預防Pol I 所引導的DNA 損傷訊號所扮演的角色，不需要DNA 甲基化作用(DNA methylation)的參與。研究結果也顯示，PolI 可以和BLM 結合，並且防止轉錄作用所引導的R loop 形成。此外，我們利用ChIP-re-ChIP 的實驗來證明DNMT3b 缺乏會導致BLM 結合到 Pol I 調節的rDNA 基因上的數量減少。大量表現RNaseH1 在BKO 細胞中，可移除RNA/DNA hybrid 以及減弱DNA 損傷訊號。根據這些發現，我們推測在HCT116 細胞中的DNMT3b 主要的功能角色是防止Pol I 轉錄作用所導致的R-loop 形成，進而維持基因組的穩定性。 Chapter II 目前已知ribosomal RNA(rRNA) 的合成作用是受到細胞能量(cellular energy)以及細胞增生狀態(proliferation status)來調節。在本篇研究中，我們發現到rRNA 基因轉錄作用會受到細胞骨架壓力(cytoskeletal stress)的影響。我們的結果顯示HeLa細胞外型被等向性(isotropic)的micropattern所限制住的時候，會導致rRNA轉錄作用顯著的減少，而這個機制是依賴ROCK才能完成；此一現象在長條形(elongated)的micropattern則不會發生。在細胞中表現一個活化型的ROCK也可以導致rRNA轉錄作用被抑制。等向性的限制與ROCK過度活化所形成的異常F-actin結構有很大的不同，但它們在rRNA轉錄作用的抑制作用卻是極為相似的，並且都可以藉由histone deacetylase (HDAC)的抑制作用或者過度表現Nesprin來回復rRNA轉錄作用。Nesprin是藉由類似護盾的機制將從actin filament傳遞到細胞核之間的作用力阻斷。我們進一步顯示在ROCK過度表現的情況下，HDAC1結合到rDNA基因的程度會增加，進而減少H3K9/14 乙醯化作用(acetylation)以及抑制轉錄作用。我們的結果證實一個表徵遺傳學控制活化rDNA基因的機制，是藉由接收到細胞骨架壓力進而抑制rRNA轉錄作用。

Chapter I DNA methyltransferase 3b (DNMT3b) is an important regulator in epigenetic modification by de novo DNA methylation that is essential for cell growth and genome stability. Using HCT-116 cells, we found that DNMT3b knockout increases DNA damage signal indicating by H2AX foci, which are markedly reduced by inhibition of RNA polymerase I (Pol I) transcription repression. Although the major function of Pol I is ribosomal RNA transcription, H2AX was not associated with rRNA genes, unless cells were synchronized for mitotic progression. We further observed that Pol I inhibition was able to decrease genome instability in these BKO cells. Expression of wild-type and catalytic-dead DNMT3b in BKO cells abolished DNA damage signal and genome instability, suggesting the role of DNMT3b in preventing Pol I dependent DNA damage is independent of its DNA methylation function. It has been shown that Pol I is associated with BLM to prevent transcription-mediated R loop formation. The ChIP-re-ChIP analysis demonstrated that DNMT3b deficiency decreased the amount of BLM associated with Pol I-bound rDNA genes. Overexpression of RNase H1 that removes RNA/DNA hybrid diminished DNA damage signal in BKO cells. According to these findings, we proposed that DNMT3b in HCT116 might has a functional role in preventing polI transcription-mediated R-loop formation to maintain genome stability. Chapter II It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of dominant active ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress.