Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice

Centromere protein A (Cenpa for mouse, CENP-A for other species) is a histone H3-like protein that is thought to be involved in the nucleosomal packaging of centromeric DNA. Using gene targeting, we have disrupted the mouse Cenpa gene and demonstrated that the gene is essential. Heterozygous mice are healthy and fertile whereas null mutants fail to survive beyond 6.5 days postconception. Affected embryos show severe mitotic problems, including micronuclei and macronuclei formation, nuclear bridging and blebbing, and chromatin fragmentation and hypercondensation. Immunofluorescence analysis of interphase cells at day 5.5 reveals complete Cenpa depletion, diffuse Cenpb foci, absence of discrete Cenpc signal on centromeres, and dispersion of Cenpb and Cenpc throughout the nucleus. These results suggest that Cenpa is essential for kinetochore targeting of Cenpc and plays an early role in organizing centromeric chromatin at interphase. The evidence is consistent with the proposal of a critical epigenetic function for CENP-A in marking a chromosomal region for centromere formation.

着丝粒蛋白A（Centromere protein A，小鼠中简称Cenpa，其余物种简称CENP-A）是一种类组蛋白H3样（histone H3-like）蛋白，被认为参与着丝粒DNA的核小体包装过程。本研究采用基因靶向技术敲除小鼠的Cenpa基因，证实该基因为必需基因。杂合子小鼠表型健康且可育，而纯合缺失突变体无法在受孕后6.5天存活。受影响的胚胎出现严重的有丝分裂异常，包括微核与巨核形成、核桥与核起泡、染色质碎片化及过度浓缩。对受孕后5.5天的间期细胞进行免疫荧光分析，结果显示Cenpa完全缺失，着丝粒蛋白B（Cenpb）呈弥散性灶点分布，着丝粒上无明确的着丝粒蛋白C（Cenpc）信号，且Cenpb与Cenpc在整个细胞核内弥散分布。上述结果表明，Cenpa对于着丝粒蛋白C（Cenpc）的动粒（kinetochore）靶向定位是必需的，并在间期着丝粒染色质组装过程中发挥早期作用。该研究结果与CENP-A通过关键表观遗传（epigenetic）功能标记着丝粒形成的染色体区域这一假说一致。