探討PHRF1連接甲基化組蛋白以及影響DNA損傷反應中細胞存活能力之機制

Abstract

當細胞受到游離輻射 (ionizing radiation, IR) 或基因毒性藥物處理時會發生DNA雙股斷裂情形，為了維持遺傳物質的正確性，細胞進行DNA損傷反應機制偵測不同的DNA受損型態，整合所有訊息使細胞對於DNA損傷做出反應，包括停止轉錄、調控細胞週期、進行DNA修復和促進細胞凋亡等。在先前的文獻中人類PHD and RING finger domain-containing protein 1 (PHRF1) 蛋白被報導會受到蛋白激酶ataxia telangectasia-mutated (ATM) 或ataxia telangectasia and Rad3-related (ATR) 磷酸化修飾的調控，但PHRF1真正生理功能仍舊未知。為了進一步探討PHRF1蛋白質在DNA損傷反應中所扮演的角色，分別利用紫外線 (ultraviolet light, UV) 及喜樹鹼 (camptothecin, CPT) 處理細胞，發現細胞的存活能力會因為PHRF1蛋白的表現量而有不同結果；缺少PHRF1表現的細胞比正常細胞在UV和CPT處理下引發更嚴重的細胞凋亡；相對的，PHRF1大量表現細胞在UV和CPT處理下較正常細胞的凋亡現象明顯下降，因此得知PHRF1的表現會增加細胞對於DNA傷害的耐受性。 這一個新發現激勵我們深入探討PHRF1是否參與早期DNA損傷反應， 根據目前的研究結果，排除 PHRF1參與在早期的DNA損傷反應和雙股DNA同源性重組修復的可能性，反而是藉由非同源性末端接合 (Non-homologous end joining, NHEJ) 的方式維持基因體的完整性。另外，PHRF1具有特殊可與甲基化組蛋白接合的plant homeodomain (PHD) finger domain，免疫沉澱實驗顯示PHRF1能夠辨認組蛋白H3K36me2、H3K36me3和H4K20me3甲基化修飾，因此我們提出一個假說PHRF1可能透過與組蛋白甲基化的作用去影響DNA雙股斷裂後的非同源性末端接合。

An effective response to damaged or abnormally replicated DNA in the mammalian cell cycle is propagated by the DNA damage checkpoint pathway. The signal transducing kinases, ataxia telangectasia-mutated (ATM), ataxia telangectasia and Rad3-related (ATR), are essential in DNA damage response. Human PHD and RING finger domain-containing protein 1 (PHRF1/KIAA1542) contains a plant homeodomain (PHD) finger, a putative methylated histone binding domain, and is identified as a phosphorylation substrate of ATM/ATR kinase. However, very little is known about its function in DNA damage response. Using different genotoxic treatments, we found that depletion of PHRF1 sensitized cells to apoptosis when cells were exposed to camptothecin (CPT) and UV light. However, PHRF1-overexpressing cells had a higher viability than control cells after genotoxic treatments. These results prompted us to investigate whether PHRF1 is involved in early DNA damage response. However, based on our current studies, PHRF1 does not affect the formation of DNA damage foci consisted of γ-H2AX, RNF8, RNF168, and BRCA1 and the homologous recombination repair. Instead, we found PHRF1 significantly affect the efficiency of non-homologous end-joining. Furthermore, the PHD domain of PHRF1 is responsible for the association of PHRF1 with methylated histones, such as H3K36me2, H3K36me3, and H4K20me3. On the basis of my findings, PHRF1 may link methylated histones to promote non-homologous end-joining through the interaction with H3K36me2, H3K36me3, and H4K20me3, which in turn, may contribute to maintain the genome integrity.