Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with the Centromere through CENP-B and Regulates Chromosome Segregation
Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with the Centromere through CENP-B and Regulates Chromosome Segregation
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DOI：
10.1074/jbc.m115.685511
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发表时间：
2015-11-20
影响因子：
4.8
通讯作者：
Band, Vimla
中科院分区：
文献类型：
--
作者：
Mohibi, Shakur;Srivastava, Shashank;Band, Vimla
ADA 3（alternation/deficiency in activation 3）是转录辅激活因子和组蛋白乙酰转移酶（FIAT）复合物的保守组分。最近，我们产生了Ada 3基因敲除小鼠，并证明了Ada 3的缺失导致早期胚胎死亡。使用Ada 3（FL/FL）小鼠胚胎成纤维细胞，使用腺病毒Cre缺失Ada 3，显示了ADA 3在通过有丝分裂的细胞周期进展中的关键作用。在这里，我们证明了ADA 3与人类X染色体着丝粒上a-卫星区域的高阶重复区域的关联，这与其在有丝分裂中的作用一致。鉴于着丝粒蛋白（CENP）在有丝分裂中的作用，我们接下来分析了ADA 3是否通过CENP与着丝粒关联。体内邻近连接试验和免疫荧光研究都证实了ADA 3与CENP-B蛋白的关联，CENP-B蛋白是一种高度保守的着丝粒蛋白，可与α-卫星DNA上的17-bp DNA序列结合。缺失分析表明，ADA 3通过其N端直接与CENP-B结合，并且ADA 3的CENP-B结合缺陷突变体不能拯救细胞增殖。值得注意的是，敲低ADA 3降低了CENP-B与着丝粒的结合，表明ADA 3是CENP-B加载到着丝粒上所必需的。最后，我们表明，Ada 3（FL/FL）小鼠胚胎成纤维细胞Ada 3的缺失表现出各种染色体分离缺陷。总之，我们证明了一种新的ADA 3与CENP-B-着丝粒的相互作用，这可能是其先前已知的有丝分裂功能的原因。这项研究，连同其在维持基因组稳定性方面的已知功能及其在癌症中的错误定位，表明ADA 3在有丝分裂中的重要作用。
ADA3 (alteration/deficiency in activation 3) is a conserved component of several transcriptional co-activator and histone acetyltransferase (FIAT) complexes. Recently, we generated Ada3 knock-out mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. The use of Ada3(FL/FL) mouse embryonic fibroblasts with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with the higher order repeat region of the a-satellite region on human X chromosome centromeres that is consistent with its role in mitosis, Given the role of centromere proteins (CENPs) in mitosis, we next analyzed whether ADA3 associates with the centromere through CENPs. Both an in vivo proximity ligation assay and immunolluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein that binds to the 17-bp DNA sequences on a-satellite DNA. Deletional analysis showed that ADA3 directly associates with CENP-B through its N terminus, and a CENP-B binding-deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting that ADA3 is required for the loading of CENP-B onto the centromeres. Finally, we show that deletion of Ada3 from Ada3(FL/FL) mouse embryonic fibroblasts exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study, together with its known function in maintaining genomic stability and its mis-localization in cancers, suggests an important role of ADA3 in mitosis.