2014-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/701553摘要：PHRF1具有多個蛋白質作用區域，包含可辨認甲基化組蛋白的plant homeodomain (PHD)、可對受質進行泛素化的RING domain和7個可被ATM和ATR磷酸化的SQ/TQ motif，先前研究發現PHRF1是ATM和ATR磷酸化的受質，藉由對TGIF泛素化的作用影響cPML進入細胞質進而調控TGF-b訊息傳遞。我們投稿中的研究成果顯示PHRF1主要分布在細胞核，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；利用紫外線及喜樹鹼 (camptothecin, CPT) 處理細胞時，缺少PHRF1表現的細胞在UV和CPT處理下會引發細胞凋亡，PHRF1大量表現的細胞在處理UV和CPT下的凋亡現象較正常細胞明顯下降；進一步實驗發現PHRF1並不會影響早期DNA損傷反應和同源性重組修復(Homologous recombination, HR)，反而藉由影響非同源性末端接合 (Non-homologous end joining, NHEJ) 的方式維持基因體的完整性。 由於PHRF1具有可與甲基化組蛋白作用的PHD domain，免疫沉澱實驗顯示PHRF1能夠辨認組蛋白H3K36me2和H3K36me3甲基化修飾，同時PHRF1利用被磷酸化的pSDpTE motif和NBS1作用，將內生性缺乏NBS1表現的細胞株進行細胞分層萃取時發現PHRF1受到基因毒性壓力後由核質到染色質的現象會消失，另外我們也證實PHRF1可對PARP1泛素化進而降解PARP1，綜合以上結果，我們推測PHRF1連接H3K36甲基化和Nbs1並對PARP1泛素化使得PHRF1在DNA雙股斷裂時調控細胞的非同源末端黏合修補。 <br> Abstract: Methylated histone readers are critical for chromatin dynamics, transcription, DNA recombination and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain which ubiquitinates substrates. A recent study shows that PHRF1 is a tumor suppressor that promotes the TGF-b cytostatic signaling through TGIF ubiquitination. Also, PHRF1 has been identified as a phosphorylation substrate of ATM/ATR kinase. Here we provide evidence for a novel functional role of PHRF1 in non-homologous end-joining (NHEJ). Responses to genotoxic insults in PHRF1-depleted and -overexpressing cells imply an indispensable role of PHRF1 in cellular viability. Selective ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, while PHRF1 overexpression leads to elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verified that PHRF1 is constitutively bound to di- and tri-methylated H3K36 (H3K36me2 and H3K36me3) via its PHD domain. Substitution of SDTE to ADAE in PHRF1 decreases its affinity for NBS1 and reduces the NHEJ efficiency. PHRF1 is mainly localized in the nucleus before genotoxic stress, but undergoes chromatin translocation upon DNA damage in an NBS1-dependent manner. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. Taken together, we provide evidence for a novel integrative role of PHRF1 in linking H3K36 methylation, NBS1, and PARP1 to NHEJ.PHRF1H3K36me3NBS1PARP1NHEJPHRF1H3K36me3NBS1PARP1NHEJ