Direct observation of coordinated assembly of individual native centromeric nucleosomes

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Abstract

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.

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    Reviewer #1 Major comments:

    Figure 2E. This shows the residence time of Cse4 without Ndc10 association. How does this compare to the residence time on mutant CEN3 (Supplemental Figure 1). It looks like Cse4 still binds to CEN3 with some specificity even in the absence of Ndc10. Does this suggest that Cse4 has some intrinsic ability to …

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript "Single molecule visualization of native centromeric nucleosome formation reveals coordinated deposition by kinetochore proteins and centromere DNA sequence" by Popchock and colleagues describes a new high-throughput single-molecule technique that combines both in vitro and in vivo sample sources. Budding yeast centromeres are genetically defined centromeres, which makes them ideal for studying short DNA segments at the single-molecule level. By flowing in whole cell lysates, Cse4 nucleosomes can form under near physiological conditions. Two analytical experiments were performed: endpoint and time-lapse. In the …

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    Referee #2

    Evidence, reproducibility and clarity

    Centromeres drive chromosome inheritance from one cell generation to the next, so understanding their nature is of utmost importance in biology. The assembly of centromeric chromatin is of outstanding interest since defects in this step impair faithful genetic inheritance. Popchock et al investigate the molecular mechanisms of Cse4(CENPA) deposition and stabilization on a native budding yeast centromeric DNA. Using TIRMF (Total Internal Reflection Fluorescent Microscopy) enabled single molecule visualization of de novo Cse4(CENPA) nucleosome formation from a yeast cellular extract. The centromeric DNA used in this study was derived …

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    This work presents a novel experimental setup to explore native centromeric nucleosome formation at a single molecule level with a high degree of temporal resolution. It combines TIRF microscopy with an immobilized fluorescent yeast CEN3 DNA sequence. Binding of centromere proteins was probed by using whole-cell lysates produced from various mutant yeast strains expressing fluorescently tagged centromere proteins. This system was used to investigate the recruitment of Cse4 (yeast CENP-A), the histone H3 variant which defines centromeres, to the native yeast CDE3 sequence. Consistent with previous studies, the authors …