PHRF1參與DNA損害反應

Abstract

當細胞受到外在環境的刺激進而影響到基因體的完整性時，細胞內會有許多的反應，例如細胞週期的調控，轉錄作用的調控，DNA修補作用，或是細胞凋亡，其目的是避免基因體的完整性遭到破壞，稱之為DNA損害反應。DNA 損害反應中，ATM和ATR扮演重要的角色，Matsuoka等人利用可辨識ATM和ATR受質的抗體進行免疫沉澱後將所獲得的樣品進行質譜儀分析，發現有七百多種蛋白質會被ATM和ATR磷酸化，PHRF1被發現是ATM和ATR磷酸化的受質，但功能未知。 我們的研究以PHRF1為核心，根據胺基酸分析， PHRF1上有許多重要的蛋白質功能區域，包含PHD domain、Ring finger、SQ/TQ cluster domain。以這些資訊為出發點，我們想了解PHRF1在DNA損害反應中所扮演的角色。免疫螢光染色結果顯示PHRF1是一個核蛋白；當細胞遭受基因毒性壓力時， PHRF1會從細胞核質移動到染色質，而將內生性缺乏ATM表現的細胞株進行細胞分層萃取，發現PHRF1受到基因毒性壓力後由核質到染色質的移動能力消失，因此 ATM對PHRF1磷酸化會調控PHRF1移動到染色質的能力。 在2011年，Fnu等人發現H3K36me2會促進非同源末端黏合（non-homologous end-joining，NHEJ）。藉由免疫沉澱， PHRF1分別可以跟H3K36me2、Nbs1及Ku70交互作用，而末端黏合實驗更進一步證實PHRF1可以影響NHEJ，因此我提出假說認為PHRF1是連接H3K36me2和Nbs1及Ku70調控細胞在DNA斷裂時負責非同源末端黏合修補。

The role of histone methylation in double-strand break repair by non-homologous end-joining (NHEJ) is not well defined. Previous studies indicate that H3K36me2 links Nbs1 and Ku70 to promote NHEJ, but which protein is responsible for this connection is unknown. Human PHRF1/KIAA1542 contains a plant homeodomain (PHD), 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. Immunofluorescence and subcellular fractionation results revealed that PHRF1 mainly localized in the nucleus prior to genotoxic stress, but PHRF1 increased onto chromatin upon DNA damage in an ATM-dependent manner. Immunoprecipitation suggested that PHRF1, Nbs1, Ku70, and dimethylated H3K36 (H3K36me2) were in the same immunocomplex. In vivo end-joining assay using a linearized luciferase reporter suggested that end-joining efficiency was decreased in PHRF1 knockdown HEK293 cells but significantly increased in PHRF1 overexpressing U2OS cells, suggesting that the presence of PHRF1 may link H3K36 methylation to non-homologous end joining by interaction with Nbs1 and Ku70.