背景:冷凝素,染色体复合体的结构维持家族,已被证明可以调节有丝分裂过程中的染色体压缩和分离。NCAPD3,凝缩蛋白II的热重复亚基,在凝缩蛋白介导的染色体动力学中起主导作用,但在淋巴瘤中仍未被探索。
目的:本研究旨在揭示NCAPD3在弥漫性大B细胞淋巴瘤(DLBCL)中的分子功能和机制。
方法:在公共数据库和临床标本中评估NCAPD3的表达和临床意义。染色体扩散,免疫共沉淀(co-IP),质谱(MS),进行染色质免疫沉淀(ChIP)测定以阐明NCAPD3在DLBCL中的作用和机制。
结果:NCAPD3在DLBCL中高表达,与不良预后相关。NCAPD3缺乏阻碍细胞增殖,诱导细胞凋亡并增加化疗敏感性。相反,NCAPD3过表达促进细胞增殖。体内实验进一步表明靶向NCAPD3抑制肿瘤生长。值得注意的是,NCAPD3缺乏扰乱有丝分裂,触发非整倍体的形成。为了揭示NCAPD3在DLBCL中的功能,进行了染色体传播,表明染色体在NCAPD3过表达后变得紧密,相反,松散,在NCAPD3缺失时扭曲且缺乏轴向刚度。同时,经典的转录激活标记,H3K4三甲基化,在NCAPD3敲除后发现全球上调,提示NCAPD3可能参与染色质重塑和转录调控。MS显示NCAPD3可能与转录因子相互作用,TFIII.进一步的co-IP和ChIP测定证实NCAPD3可以通过TFIII锚定在SIRT1的启动子处,然后通过识别SIRT1启动子上的H3K9单甲基化(H3K9me1)支持SIRT1的转录。功能逆转实验证实NCAPD3在DLBCL中的致癌作用部分由SIRT1介导。
结论:这项研究表明,NCAPD3的失调可以干扰染色体压缩和分离,并以H3K9me1依赖性方式调节SIRT1的转录活性,这为DLBCL的针对性策略提供了新的见解。
BACKGROUND: Condensin, a family of structural maintenance of chromosome complexes, has been shown to regulate chromosome compaction and segregation during mitosis. NCAPD3, a HEAT-repeat subunit of condensin II, plays a dominant role in condensin-mediated chromosome dynamics but remains unexplored in lymphoma.
OBJECTIVE: The study aims to unravel the molecular function and mechanism of NCAPD3 in diffuse large B-cell lymphoma (DLBCL).
METHODS: The expression and clinical significance of NCAPD3 were assessed in public database and clinical specimens. Chromosome spreads, co-immunoprecipitation (co-IP), mass spectrometry (MS), and chromatin immunoprecipitation (ChIP) assays were conducted to untangle the role and mechanism of NCAPD3 in DLBCL.
RESULTS: NCAPD3 was highly expressed in DLBCL, correlated with poor prognosis. NCAPD3 deficiency impeded cell proliferation, induced apoptosis and increased the chemosensitivity. Instead, NCAPD3 overexpression facilitated cell proliferation. In vivo experiments further indicated targeting NCAPD3 suppressed tumor growth. Noteworthily, NCAPD3 deficiency disturbed the mitosis, triggering the formation of aneuploids. To reveal the function of NCAPD3 in DLBCL, chromosome spreads were conducted, presenting that chromosomes became compact upon NCAPD3 overexpression, instead, loose, twisted and lacking axial rigidity upon NCAPD3 absence. Meanwhile, the classical transcription-activated marker, H3K4 trimethylation, was found globally upregulated after NCAPD3 knockout, suggesting that NCAPD3 might participate in chromatin remodeling and transcription regulation. MS revealed NCAPD3 could interact with transcription factor, TFII I. Further co-IP and ChIP assays verified NCAPD3 could be anchored at the promoter of SIRT1 by TFII I and then supported the transcription of SIRT1 via recognizing H3K9 monomethylation (H3K9me1) on SIRT1 promoter. Function reversion assay verified the oncogenic role of NCAPD3 in DLBCL was partially mediated by SIRT1.
CONCLUSIONS: This study demonstrated that dysregulation of NCAPD3 could disturb chromosome compaction and segregation and regulate the transcription activity of SIRT1 in an H3K9me1-dependent manner, which provided novel insights into targeted strategy for DLBCL.