PRMT1和其相關的組蛋白修飾在DNA損傷後對p21基因表現的轉錄調控

真核生物的基因要表現，首先要做的就是把核小體(nucleosome)的結構鬆開，這樣轉錄因子才能結合至裸露的DNA上，開啟基因表現。在細胞中，這項工作的調控主要可以透過兩種機制來達成：一種是透過一些蛋白複合體水解ATP所獲的能量來改變組蛋白與DNA的纏繞狀態；另一種則是透過組蛋白的修飾酵素來改變組蛋白的化學性質，進而影響它與DNA的結合力。其中負責後項機制機制的酵素主要有五大類：組蛋白－乙醯轉移酶(HAT)，去乙醯基酶(HDAC)、激酶(HK)、甲基轉移酶(HMT)和泛素轉移酶(ubiquitinylase)。目前已經知道這些酵素所負責的組蛋白修飾彼此之間會相互影響，而且有先後次序，同時會召集其它不同的蛋白質到基因的啟動子，進而活化或是抑制基因的表現。這種組蛋白修飾與基因調控的複雜關係稱為組蛋白密碼(Histone Codes)。 Protein arginine methyltransferase 1 (PRMT1) 是HMT裡的一員，它可以對組蛋白H4上的第三個精胺酸(arginine)進行甲基化。已經有研究指出：PRMT1、p300和CARM1這三個組蛋白修飾酵素在p53所調控的基因活化過程中，是非常重要的，而且這三者是有一定的作用次序，其中PRMT1是扮演一個起始者的角色。所以我的研究主題可分為兩個：一個是研究PRMT1在DNA受到損傷後的表現變化。另一個則是觀察當DNA受到損傷時，在p21基因的啟動子上，PRMT1所負責的組蛋白H4R3甲基化與其它修飾之間的先後次序，以及對於p21基因活化的影響。從實驗結果中得知，當MCF7細胞受到藥物處理所造成的DNA損傷時，PRMT1的mRNA表現量並沒有太大的改變，但蛋白質卻有逐漸累積的情形。另外，利用不同時間點的染色質免疫沉澱方法(Time Course Chromatin immunoprecipitation)，我發現到在p21基因的啟動子上，PRMT1所負責的H4R3甲基化是早於H4的乙醯基化，令人意外的是，它也早於p53結合到p21啟動子的時間。這代表PRMT1所造成的H4R3甲基化可能並不完全需要p53的帶領。在最後的實驗中，我成功地利用siRNA的方式，抑制細胞內PRMT1的表現，這對於未來研究PRMT1在p21基因活化上所扮演的角色，將有所幫助。

關鍵字

組蛋白； DNA損傷；轉錄調控

並列摘要

In eukaryotes, it is important to modify the nucleosome structure to expose DNA for the binding of various transcriptional regulatory factors during transcription. Currently, there are two distinct mechanisms are uncovered in eukaryotic cells. One mechanism involves ATP-dependent remodeling complexes that change the physical interaction between the histones and DNA. The other is mediated by histone modifying enzymes that change the chemical properties of histones by covalent modifications. The latter mechanism mainly comprises five kinds of enzymes: histone acetyltransferase (HAT), histone deacetylase (HDAC), histone kinase (HK), histone methyltransferase (HMT) and ubiquitinylase. It has been demonstrated that histone modifications catalyzed by these enzymes can generate signature patterns to recruit different proteins to the promoters that result in gene activation or repression. This complex relationship between histone modifications and gene regulation is called “Histone Codes”. Protein arginine methyltransferase 1 (PRMT1) is a member of HMT family and it can methylate histone H4 arginine 3 (H4R3). It has been shown that the ordered cooperative functions of PRMT1, p300 and CARM1 are important to transcriptional activation by p53. Thus, my studies have been focused on two aspects: one is to examine the expression level of PRMT1 after DNA damage. The other is to investigate the ordered transcriptional effects of PRMT1-mediated histone H4R3 methylation and the other histone modifications at p21 promoter after DNA damage. Based on my observations, I have found that PRMT1 protein accumulated after DNA damaged by drug treatment in MCF7 cells, however, the expression level of PRMT1 mRNA remained unchanged by semi-quantitative RT-PCR analysis. In addition, by using the time course chromatin immunoprecipitation (ChIP) assay, I found PRMT1-mediated histone H4R3 methylation occurred in the early phase of DNA damage and p300-mediated histone H4 acetylation increased in the late phase of DNA damage. Unexpectedly, the histone H4R3 methylation occurred even earlier than the recruitment of p53, suggesting PRMT1-mediated histone H4R3 methylation may not totally depend on p53 recruitment. Furthermore, I have successfully knocked down the endogenous PRMT1 by siRNA. The future application of the silenced PRMT1 expression will aid in the experiments to clarify the effects of PRMT1 on p21 transcriptional activation.

並列關鍵字

PRMT1 ； DNA damage

參考文獻

An, W., Kim, J., and Roeder, R. G. (2004). Ordered cooperative functions of PRMT1, p300, and CARM1 in transcriptional activation by p53. Cell 117, 735-748.

Boisvert, F. M., Dery, U., Masson, J. Y., and Richard, S. (2005). Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control. Genes Dev 19, 671-676.

Brugarolas, J., Moberg, K., Boyd, S. D., Taya, Y., Jacks, T., and Lees, J. A. (1999). Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation. Proc Natl Acad Sci U S A 96, 1002-1007.

Carey, M. (2005). Chromatin marks and machines, the missing nucleosome is a theme: gene regulation up and downstream. Mol Cell 17, 323-330.

Chang, B. D., Swift, M. E., Shen, M., Fang, J., Broude, E. V., and Roninson, I. B. (2002). Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Proc Natl Acad Sci U S A 99, 389-394.

國際替代計量

PRMT1和其相關的組蛋白修飾在DNA損傷後對p21基因表現的轉錄調控

全文下載

主題瀏覽