前列腺癌相關之人類基因甲基轉移酶突變體的結構和生化研究

Abstract

哺乳動物的DNA甲基化是一種在調節各種細胞事件中，發揮關鍵作用的表觀遺傳修飾機制。甲基化失調與多種人類疾病，包括癌症有關。在哺乳動物中，DNA甲基化是由三種DNA甲基轉移酶（DNMT1，DNMT3A和DNMT3B）實現的。近年來在轉移性去勢抗性前列腺癌（mCRPC）患者中，識別出兩種DNMT3B突變體（E515D和R545C），這些具有DNMT3B突變體的癌細胞中具有高甲基化的CpG位點。這兩種突變體不位於甲基轉移酶區域（MTase），而是位於ADD區域內。ADD區域可與組蛋白H3的N-端尾部未甲基化的K4（H3K4me0）相互作用。然而，目前尚不清楚這些突變是否會改變DNMT3B的結構與活性，或者它們是否會影響DNMT3B與組蛋白H3之間的相互作用。在本研究中，我們表達並純化了與DNMT3L（胺基酸178-379）結合的野生型和突變型DNMT3B （E515D和R545C）兩種長度的蛋白（胺基酸214-853和412-853），並測量了它們在存在或不存在40個氨基酸組蛋白H3肽的情況下的甲基轉移酶活性。在沒有或有H3肽的情況下，E515D的甲基轉移酶活性與野生型酶類似。然而，R545C突變體的甲基轉移酶活性，在存在H3肽（H3K4me0）的情況下比野生型DNMT3B活性更為增強。小角度X射線散射（SAXS）進一步顯示DNMT3B/3L複合物具有多種構象，包括「自抑制形式」或「活化形式」。相較於野生型，H3K4me0肽能夠將具有R545C突變的複合物更高百分比轉移到活化形式。總結來說，這些發現表明R545C突變增強了DNMT3B/3L複合物的甲基轉移酶活性，使其更容易通過與組蛋白H3尾部的相互作用而採取活性構象。因此，該研究闡明了R545C突變引起的DNMT3B在前列腺癌中異常高甲基化的潛在機制。

Mammalian DNA methylation is an epigenetic modification mechanism that plays key roles in the regulation of various cellular events. Methylation dysregulations are associated with a wide spectrum of human diseases, including cancer. DNA methylation is achieved by three DNA methyltransferases in mammals, DNMT1, DNMT3A and DNMT3B. Two DNMT3B mutants (E515D and R545C) have been identified in the patients of metastatic castration-resistant prostate cancer (mCRPC) with hypermethylated CpG sites. These two mutations are not located in the methyltransferase domain (MTase) but located within the ADD domain, which is involved in the interaction with the histone H3 tail at unmethylated K4 (H3K4me0). However, it remains unknown if these mutations may change the structure and/or activity of DNMT3B, or it may affect the interactions between DNMT3B and histone H3 tail. In this study, we expressed and purified two constructs (residues 412-853 and 214-853) of wild-type and mutated DNMT3B (E515D and R545C) in complex with DNMT3L (residues 178-379) and measured their methyltransferase activities in the presence or absence of a 40-amino acid histone H3 peptide. In the absence or presence of the H3 peptides, the methyltransferase activities of E515D were similar to those of wild-type enzymes. However, the methyltransferase activities of R545C mutants were more strongly stimulated than those of wild-type DNMT3B by the H3 peptides. Small-angle X-ray scattering (SAXS) further shows that DNMT3B/3L complex was flexible with multiple conformations in autoinhibitory or activated forms. The H3 peptides, particularly H3K4me0/K36me3 peptide, can shift higher percentage of the complex with R545C mutation to the active conformation as compared to the wild-type enzyme. Overall, these findings demonstrate that the R545C mutation enhances the methyltransferase activity of DNMT3B/3L complex, making it more susceptible to adopting an active conformation through interactions with the histone H3 tail. This study thus illuminates the underlying mechanism for aberrant hypermethylation caused by the mutation of R545C in DNMT3B in prostate cancer.