探討PML及PML-RARα的小類泛素修飾對Daxx結合之專一性

Abstract

骨髓性淋巴癌(PML)和類色素酸接受體α(RARα)的融合造成了急性前骨髓性 白血病（acute promyelocytic leukemia, APL）。類色素酸活性調控下游基因表現影響 了血球的發育，在APL 細胞中，許多corepressor，像NcoR, SMRT, HDACs 與PML、 RARα相互作用而更加抑制了RARα下游基因。此外，最近的研究報導Daxx 與 PML 作用會促使更多的co-repressor 結合到PML-RARα上，此作用是藉著PML 上 的Lys160 site 的小類泛素修飾蛋白（small ubiquitin-like modifier, SUMO）與Daxx 上的特定胺基酸的SUMO interacting motif (SIM)結合，但Daxx 如何和sumoylated 的PML 專一性結合的機制尚未清楚。 在這篇論文中， 我們藉由不同的方法， 得知PML/PML-RARα 上三個 SUMO-acceptor Lys（Lys65, Lys160, Lys490）都會與Daxx 相互作用。並探討了PML 及PML-RARα上三個Lys residue 受SUMO 修飾的不同，訂出了每一個Lys 所代表 的修飾位置，並發現這些Lys 受SUMO 修飾是相互影響的，將Lys 突變成Arg， 會影響其他site 的SUMO 修飾，顯示了PML 上每個Lys 的sumoylation 不是獨立 的。 我們還發現了外加與Daxx SUMO interacting motif (SIM)序列一樣的peptide， 可以有效的回復Daxx 所造成的轉錄抑制，促使RAR 下游基因的表現。將此peptide 加至來自APL 的細胞：NB4 中，可以有效的降低NB4 不正常的生長速度。綜合言 之，本篇論文詳加瞭解了PML 及PML-RARα上的sumoylation 和Daxx 的專一性 結合，更證明了Daxx-SIM 可以當作一個有效抑制APL transformarion 的治療方法。

PML (promyelocytic leukemia gene) fused to retinoic acid receptor α (RARα), derived from the t(15;17) translocation, causes acute promyelocytic leukemia (APL). Several transcriptional corepressors such as NcoR, SMRT and HDACs were shown to associate with PML-RARα, leading to transcriptional repression. Besides, recent reports indicated that Daxx could contribute to transcriptional repression of PML/RARα-targeted genes via interacting with SUMO modification at Lys160 of PML portion. The specificity of how Daxx binds to sumoylated PML has not been well characterized. In this study, we used different approaches to show that all three SUMO-acceptor Lys sites of PML and PML-RARα are important for Daxx interaction. In addition, we found that sumoylation level of PML at each Lys acceptor site is affected by each other when individual sumoylation site was mutated, indicating that PML sumoylation at each Lys acceptor is not independent. In addition, we found exogenous Daxx SUMO-interacting motif (SIM) peptide could reverse Daxx-elicited repression on PML-RARα-mediated reporter activity and could affect APL cell line-NB4 cells proliferation rate. Together, our studies elucidate the sumoylation-mediated interaction between PML or PML-RARα and Daxx and provide the evidence that Daxx-SIM peptide may be considered a potential agent for blocking APL-mediated transformation phenotype.