Functional regulation of RNA polymerase I by DNA methyltransferase 3B and cytoskeletal reorganization

• Main Authors: Tse-Hsiang Wu, 吳澤祥
• Other Authors: Zee-Fen Chang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/528nsg

博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 105 === 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.