Camahort Raymond, Shivaraju Manjunatha, Mattingly Mark, Li Bing, Nakanishi Shima, Zhu Dongxiao, Shilatifard Ali, Workman Jerry L, Gerton Jennifer L
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Mol Cell. 2009 Sep 24;35(6):794-805. doi: 10.1016/j.molcel.2009.07.022.
The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4-containing nucleosomes is a subject of debate. Using unbiased biochemical, cell-biological, and genetic approaches, we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the nonhistone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating that Scm3 is not essential for the formation of functional centromeric chromatin. We also find that octameric Cse4 nucleosomes can be reconstituted in vitro. Furthermore, Cse4-Cse4 dimerization occurs in vivo at the centromeric nucleosome, and this requires the predicted Cse4-Cse4 dimerization interface. Taken together, our experimental evidence supports the model that the Cse4 nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.
出芽酵母着丝粒组蛋白变体Cse4定位于着丝粒核小体，是动粒组装和染色体分离所必需的。含Cse4的着丝粒核小体的确切组成一直存在争议。我们使用无偏差的生化、细胞生物学和遗传学方法，对含Cse4的核小体的组成进行了测试。通过微球菌核酸酶处理的染色质，我们发现Cse4与组蛋白H2A、H2B和H4相关，但与H3或非组蛋白Scm3无关。Cse4的过表达挽救了scm3缺失的致死性，表明Scm3对于功能性着丝粒染色质的形成并非必不可少。我们还发现八聚体Cse4核小体可以在体外重建。此外，Cse4-Cse4二聚化在体内着丝粒核小体处发生，这需要预测的Cse4-Cse4二聚化界面。综上所述，我们的实验证据支持Cse4核小体是一种八聚体的模型，其包含Cse4、H2A、H2B和H4各两个拷贝。
