Kocsisova Zuzana, Kornfeld Kerry, Schedl Tim
Department of Developmental Biology, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO, 63108, USA.
Department of Genetics, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO, 63108, USA.
BMC Dev Biol. 2018 May 30;18(1):12. doi: 10.1186/s12861-018-0171-7.
The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E.
To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development.
In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.
增殖细胞核抗原（在秀丽隐杆线虫中为PCNA或PCN-1）是DNA聚合酶δ的一种必需的持续合成因子，已被广泛用作S期的标志物。在秀丽隐杆线虫早期胚胎中，PCN-1的积累是周期性的，在S期定位于细胞核，在细胞周期的其余阶段定位于细胞质。秀丽隐杆线虫的幼虫和成虫生殖系是研究细胞周期调控的重要模型系统，据观察，细胞周期调节因子细胞周期蛋白E（在秀丽隐杆线虫中为CYE-1）在该组织中表现出非周期性的连续积累模式。PCN-1在幼虫和成虫生殖系中的积累模式尚未得到很好的界定，本研究的目的是确定其积累模式是像在其他细胞和生物体中那样呈周期性，还是像细胞周期蛋白E那样呈连续模式。
为了研究从其天然位点表达的PCN-1在幼虫和成虫生殖系中的积累情况，我们使用CRISPR/Cas9技术构建了一个新的pcn-1等位基因，该等位基因编码一种带有表位标签的蛋白质。使用EdU核苷酸掺入法标记S期细胞核，并通过抗体染色检测FLAG::PCN-1。所有祖细胞区细胞核，包括那些不在S期的细胞核（因为它们对EdU染色呈阴性）都显示出PCN-1的积累，这表明PCN-1在生殖系祖细胞区的所有细胞周期阶段都有积累。从转基因表达的GFP::PCN-1融合蛋白也观察到了相同的结果。分析了pcn-1功能丧失突变，发现pcn-1对强大的生育力和胚胎发育是必需的。
在秀丽隐杆线虫早期胚胎以及其他生物体中，PCN-1仅在S期在细胞核中积累。相比之下，在秀丽隐杆线虫生殖系的祖细胞区，PCN-1在所有细胞周期阶段都在细胞核中积累。这种模式类似于细胞周期蛋白E的积累模式。这些观察结果支持了这样一种模型，即生殖系干细胞和祖细胞中的有丝分裂细胞周期调控与体细胞不同，因为它不太依赖于经典细胞周期蛋白的周期性积累。
