Direct observation of coordinated assembly of individual native centromeric nucleosomes

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.

Automated summary

Eukaryotic chromosome segregation requires a large machine called the kinetochore, which contains CENP-A and is responsible for nucleosome formation. This study investigates the functions of the chaperone protein HJURP in CENP-A deposition and the conservation of high AT DNA content at centromeres. Through a microscopy assay, it is found that CENP-A can arrive at centromeres without HJURP but stable incorporation requires HJURP and other DNA-binding proteins. Homopolymer AT runs in yeast centromeres are also essential for efficient CENP-A deposition. These findings provide insights into nucleosome formation and lay the foundation for future studies on kinetochore complexes.