Topoisomerase II (cid:2) Negatively Modulates Retinoic Acid Receptor (cid:3) Function: a Novel Mechanism of Retinoic Acid Resistance (cid:1)
Topoisomerase II (cid:2) Negatively Modulates Retinoic Acid Receptor (cid:3) Function: a Novel Mechanism of Retinoic Acid Resistance (cid:1)
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拓扑异构酶 II (cid:2) 负向调节视黄酸受体 (cid:3) 功能：视黄酸抗性的新机制 (cid:1)
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通讯作者：
J. L. Goodman
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作者：
M. Klein;S. F. Hayes;J. L. Goodman
视黄酸 (RA) 受体 (cid:1) (RAR (cid:1) ) 和共调节因子之间的相互作用在协调基因转录和骨髓分化中发挥着关键作用。在急性早幼粒细胞白血病 (APL) 患者中，RAR (cid:1) 基因通过 t(15;17) 易位与早幼粒细胞白血病 (PML) 基因融合，导致 PML/RAR (cid:1) 融合蛋白的表达。在此，我们报告拓扑异构酶 II beta (TopoII (cid:2) ) 与 RAR (cid:1) 转录活性相关并负向调节，并且 TopoII (cid:2) 水平的增加及其相关性会导致 APL 细胞系对 RA 产生耐药性。 TopoII (cid:2) 的敲低能够通过允许 RA 诱导的分化和增加 RA 基因表达来克服耐药性。 RA 敏感细胞系克隆中 TopoII (cid:2) 的过度表达通过减少 RA 诱导的靶基因表达和分化而产生抗性。染色质免疫沉淀分析表明，TopoII (cid:2) 与 RA 反应元件结合，并且抑制 TopoII (cid:2) 会导致组蛋白 3 在赖氨酸 9 处过度乙酰化并激活转录。我们的结果确定了转录功能。我们的研究表明，TopoII (cid:2) 与 RAR (cid:3) 相互作用，并负向调节 RAR (cid:3) 活性，并且 TopoII (cid:2) 水平的增加会导致 RA 诱导的粒细胞分化受阻。支持该模型的证据包括以下观察结果。 (i) TopoII (cid:2) 在体外和体内与 RAR (cid:3) 和 PML/RAR (cid:3) 的 RAR (cid:3) 部分相关。 (ii) 我们观察到 RA 抗性细胞系 NB4-MR2 中 TopoII (cid:2) 水平增加且相互作用。 (iii) TopoII (cid:2) 的表达引起了几种细胞系中 (cid:2) RARE-tk-CAT 报告基因的抑制。 (iv) shRNA或抑制剂ICRF-193下调TopoII (cid:2)，导致RA诱导的基因表达和粒细胞分化增加，并恢复NB4-MR2细胞中的RA敏感性。 (v) 在 RA 敏感细胞系 NB4 的克隆中过表达 TopoII (cid:2) 可赋予 RA 抗性，并显着减少 RA 诱导的分化和 RA 靶基因 RAR (cid:2) 的诱导。 (vi) ChIP实验表明TopoII (cid:2)与RAR (cid:2)的启动子相关，并且抑制TopoII (cid:2)导致该启动子区域的组蛋白3赖氨酸9乙酰化增加。
Interactions between retinoic acid (RA) receptor (cid:1) (RAR (cid:1) ) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RAR (cid:1) gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the expression of a PML/RAR (cid:1) fusion protein. Here, we report that topoisomerase II beta (TopoII (cid:2) ) associates with and negatively modulates RAR (cid:1) transcriptional activity and that increased levels of and association with TopoII (cid:2) cause resistance to RA in APL cell lines. Knockdown of TopoII (cid:2) was able to overcome resistance by permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoII (cid:2) in clones from an RA-sensitive cell line conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicated that TopoII (cid:2) is bound to an RA response element and that inhibition of TopoII (cid:2) causes hyperacetylation of histone 3 at lysine 9 and activation of transcription. Our results identify a transcriptional function. Our studies show that TopoII (cid:2) interacts with RAR (cid:3) and negatively modulates RAR (cid:3) activity and that increased levels of TopoII (cid:2) lead to a block in RA-induced granulocytic differentiation. Evidence supporting this model consists of the following observations. (i) TopoII (cid:2) associates with RAR (cid:3) and the RAR (cid:3) portion of PML/RAR (cid:3) in vitro and in vivo. (ii) We observed increased levels of TopoII (cid:2) and interaction in the RA-resistant cell line NB4-MR2. (iii) Expression of TopoII (cid:2) caused inhibition of a (cid:2) RARE-tk-CAT reporter gene in several cell lines. (iv) Downregulation of TopoII (cid:2) , by shRNA or the inhibitor ICRF-193, caused an increase in RA-induced gene expression and granulocytic differentiation and restored RA sensitivity in NB4-MR2 cells. (v) Overexpression of TopoII (cid:2) in clones of the RA-sensitive cell line NB4 conferred RA resistance with a significant reduction in RA-induced differentiation and induction of the RA target gene, RAR (cid:2) . (vi) ChIP experiments indicated that TopoII (cid:2) is associated with the promoter of RAR (cid:2) , and inhibition of TopoII (cid:2) leads to increased histone 3 lysine 9 acetylation at this promoter region.