Diminished interaction between mutant NOTCH1 and the NuRD corepressor complex upregulates CCL17 in chronic lymphocytic leukemia.
Diminished interaction between mutant NOTCH1 and the NuRD corepressor complex upregulates CCL17 in chronic lymphocytic leukemia.
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突变型 NOTCH1 与 NuRD 辅阻遏物复合物之间的相互作用减弱，可上调慢性淋巴细胞白血病中的 CCL17。
DOI：
10.1038/s41375-019-0526-5
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发表时间：
2019
期刊：
影响因子：
11.4
通讯作者：
Xu Zhenshu
中科院分区：
文献类型：
--
作者：
Wang Shifen;Ge Maolin;Cui Jing;Qiao Zhi;Chen Xiuli;Wu Shunquan;Zhan Rong;Zhou Huarong;Liu Han;Xu Zhenshu
Letter 2951 西方国家成年患者的白血病[1-3]。 NOTCH1 突变与临床侵袭性 CLL 相关，并已成为不良预后的独立预测因素 [3]。 CLL 中约 80% 的 NOTCH1 突变由 2 bp CT 移码缺失 (c. 7541_7542delCT) 组成，该缺失在 C 端 PEST 结构域中产生截短，并延长 NOTCH1 活性形式（即 NOTCH 的胞内结构域 (ICN)）的半衰期[1,2,4–6]。有证据表明，NOTCH1 突变导致截短的蛋白比野生型 (WT) 蛋白更稳定，从而在 CLL 细胞中维持活跃的 NOTCH1 信号传导 [4]。然而，突变体和WT NOTCH1之间是否存在任何功能差异仍然很大程度上未知，并且对于为什么CLL中最常见的NOTCH1突变发生在PEST结构域的了解相对较少。最近的研究表明，CLL 中的 NOTCH1 突变出现在祖细胞或前 B 细胞阶段 [7]，并且可能有助于 CLL 造血祖细胞或前 B 细胞的扩增 [8]。 CLL 造血祖细胞比健康造血祖细胞表现出更高水平的活性 ICN [9, 10]。因此，为了研究突变型和WT NOTCH1在B细胞祖细胞中的功能差异，我们通过在小鼠B细胞祖细胞Baf3细胞系中异位表达WT ICN（ICN）和突变型ICN（ICN-delCT）来构建模拟具有高水平ICN的CLL造血祖细胞的细胞模型[1]，并确定它们是否能够差异调节这些细胞的生物学特性。有趣的是，尽管ICN-delCT的表达水平与ICN相当（补充图1a），但带有ICN-delCT的Baf3细胞增殖速度比带有WT ICN的细胞更快，且呈剂量依赖性（补充图1b），这表明突变型NOTCH1可能比WT NOTCH1导致pro-B细胞更多的扩增。进入细胞核后，ICN 与转录因子 CSL 和类 Mastermind 共激活因子家族形成复合物，激活一系列下游基因（如 HES1 和 DTX1）的转录 [11]。为了确定 WT 和突变 ICN 是否具有不同的转录活性，我们使用 6xCSL-荧光素酶报告基因在这些细胞中进行了荧光素酶报告基因测定。虽然ICN和ICN-delCT均显着激活报告基因，但ICN-delCT显示出比ICN更高的转录活性（图1a）。这些结果表明，增强的pro-B细胞扩增和升高的转录活性不是由NOTCH1活性形式的半衰期延长引起的，而是反映了突变体和WT NOTCH1之间的功能差异。为了确定 pro-B 细胞中突变型和 WT NOTCH1 功能差异的机制，我们试图确定 ICN 和 ICN-delCT 在细胞核中是否具有不同的相关伙伴。使用 NOTCH1 抗体通过免疫沉淀从这些 Baf3 细胞的核提取物中纯化 ICN 及其相关伙伴。质谱分析确定了 ICN 的蛋白质伴侣（图 1b 和补充表 1）。有趣的是，与我们发现 ICN-delCT 显示出更高的转录活性一致，我们发现 MTA2 和 MTA1 蛋白（两者都是核小体重塑和脱乙酰酶 (NuRD) 辅阻遏物复合物的组成部分）与 ICN 共纯化，但不与 ICN-delCT 共纯化（补充表 1）。据报道，参与转录抑制的因素，包括 NuRD 复合物和 PRC1 复合物的成分，与 T 细胞急性淋巴细胞中的 ICN 相关……
Letter 2951 leukemia in adult patients in Western countries [1–3]. NOTCH1 mutations associate with clinically aggressive forms of CLL and have emerged as an independent predictor of adverse prognosis [3]. Approximately 80% of NOTCH1 mutations in CLL consist of a 2-bp CT frameshift deletion (c. 7541_7542delCT) that generates a truncation in the C-terminal PEST domain and prolongs the half-life of the active form of NOTCH1, ie, the intracellular domain of NOTCH (ICN)[1, 2, 4–6]. There is evidence that the NOTCH1 mutation results in a truncated protein more stable than wild-type (WT) protein, thus sustaining active NOTCH1 signaling in CLL cells [4]. However, whether there is any functional difference between mutant and WT NOTCH1 remains largely unknown, and relatively little is known regarding why the most frequent NOTCH1 mutations in CLL occur in the PEST domain. Recent studies have demonstrated that NOTCH1 mutations in CLL appear at the progenitor or pro-B cell stages [7], and might contribute to the expansion of CLL hematopoietic progenitors or pro-B cells [8]. CLL hematopoietic progenitors display higher levels of active ICN than their healthy counterparts [9, 10]. Therefore, to investigate the functional difference between mutant and WT NOTCH1 in the B cell progenitors, we generated cell models mimicking CLL hematopoietic progenitors with high levels of ICN by ectopically expressing WT ICN (ICN) and mutant ICN (ICN-delCT) in the murine B cell progenitor Baf3 cell line [1], and determined whether they could differentially regulate the biological characteristics of these cells. Interestingly, even though ICN-delCT was expressed at a level comparable with ICN (Supplementary Fig. 1a), the Baf3 cells with ICN-delCT proliferated faster than those with WT ICN in a dose-dependent manner (Supplementary Fig. 1b), suggesting mutant NOTCH1 may cause more expansion in pro-B cells than WT NOTCH1. Upon entering the nucleus, ICN forms a complex with the transcription factor CSL and the Mastermind-like family of co-activators to activate the transcription of a series of downstream genes such as HES1 and DTX1 [11]. To determine whether WT and mutant ICN possess different transcriptional activities, we performed luciferase reporter assays in these cells using a 6xCSL-luciferase reporter. While both ICN and ICN-delCT significantly activated the reporter, ICN-delCT showed higher transcriptional activity than ICN (Fig. 1a). These results indicate that the enhanced pro-B cell expansion and elevated transcriptional activity are not caused by the prolonged half-life of the active form of NOTCH1, but rather reflect a functional difference between mutant and WT NOTCH1. To identify the mechanisms underlying the functional difference between mutant and WT NOTCH1 in pro-B cells, we sought to determine whether ICN and ICN-delCT had different associated partners in the nucleus. ICN and its associated partners were purified from nuclear extracts derived from these Baf3 cells by immunoprecipitation using NOTCH1 antibody. Mass spectrometry analysis identified protein partners of ICN (Fig. 1b and Supplementary Table 1). Interestingly, consistent with our finding that ICN-delCT showed higher transcriptional activity, we found that MTA2 and MTA1 proteins, both of which are components of the nucleosome remodeling and deacetylase (NuRD) corepressor complex [12], co-purified with ICN but not with ICN-delCT (Supplementary Table 1). Factors involved in transcriptional repression, including components of the NuRD complex and the PRC1 complex, have been reported to be associated with ICN in T-cell acute lymphoblastic …