探討MCM複合體與甲基轉移酶G9a之交互作用在癌細胞增生的調控

Abstract

組蛋白離胺酸甲基轉移酶G9a (又稱為Euchromatic Histone Lysine Methyltransferase 2, EHMT2)，廣泛分布於人體的各種組織中，其主要功能是催化組蛋白H3第九個胺基酸離胺酸(H3K9) 的甲基化，使DNA與組蛋白形成較緊密的異染色質進而抑制各種基因表達，並且在細胞增生、分化、老化等機制中有著重要的作用，至今為止在各個文獻中已發現多數癌症組織中的G9a蛋白表現量顯著高於正常組織，並且G9a與細胞增生、腫瘤大小呈現正相關，因此我們以同樣有G9a過表現的乳癌細胞為模型，在初步研究中發現G9a有與微小染色體維持蛋白 (Minichromosome Maintenance protein, MCM protein) 結合的可能性，且從TCGA資料庫分析發現，乳癌中G9a與MCM次單位的mRNA表現量皆呈現正相關；由MCM2-7六個次單位形成的MCM複合體是在DNA複製中最主要的解旋酶，因此本次研究目的是要釐清G9a與MCM複合體之交互作用在癌細胞增生的調控，並探討兩者之間的結合關係。在本次研究中，我們驗證了G9a與MCM和細胞的長、短期增生以及DNA合成皆有正相關性，並且不影響彼此的轉錄表現，G9a雖然不會改變MCM的轉錄表達及次單位間的結合但會增加細胞核中的MCM蛋白含量，而敲低G9a的基因表達或抑制酵素活性都會使MCM與染色質的結合下降，此結果也導致DNA聚合酶的染色質負載也跟著下降；為了深入了解G9a與MCM的結合關係，我們透過多種蛋白質交互作用試驗發現G9a的ANK repeat domain會結合於MCM4的N-C linker，並從已發表文獻及預測甲基化軟體分析結果推測ANK repeat domain可能會藉由辨認N-C linker區域中精氨酸-甲基離胺酸motif結合；綜合以上結果，我們發現G9a可能藉由與MCM4的結合增加MCM複合物在細胞核中的含量及其染色質負載，而加速DNA複製起始的過程並導致細胞增生的現象。

Histone lysine methyltransferase G9a (also known as EHMT2) is widely expressed in various tissue. It catalyzes the mono- and di-methylation of lysine 9 on histone H3 N-tail to form transcriptionally inactive heterochromatin and silence gene expression. The G9a plays an important role in cell proliferation, differentiation, and cellular aging. It is reported that G9a is overexpressed in various cancer types and has a positive correlation with cancer cell proliferation and tumor size. In our initial data, which used a breast cancer cell line also has elevated G9a expression as the cell model, G9a was associated with the component of the Minichromosome Maintenance (MCM) complex. In TCGA database analysis, the G9a has positive correlations with MCMs at the transcription expression in breast cancer. The candidate molecule, MCM complex, is a heterohexamer, which is composed of MCM2-7, and a major helicase in DNA replication which is overexpressed in multiple cancers. The purpose of this study is to clarify the crosstalk between the MCM complex and G9a in the proliferation of the cancer cell and get insight into the binding region among them. First, we verified that G9a, as well as MCM, had a positive correlation with DNA synthesis and short-/long-term cell proliferation. Next, we discovered that G9a increased the nuclear distribution of MCMs but did not affect transcription level and subunits association. In addition, the chromatin loading of MCMs was attenuated after knockdown or inhibition of G9a, and the DNA polymerase epsilon which subsequently binds after the MCM complex was also attenuated. To investigate the interaction between G9a and MCMs, we utilized multiple protein-protein interaction analyses to reveal that the ankyrin (ANK) repeat domain of G9a directly interacted with the N-C linker of MCM4. Based on the literature and the prediction software, we suggested that the arginine-methllysine motif of the MCM4 N-C linker may be recognized by the ANK repeat domain of G9a. In conclusion, we discovered that the G9a increased nuclear distribution and chromatin loading of the MCM complex which results in DNA replication and cell proliferation by interacting with MCM4.