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bioRxiv · 10.1101/2023.05.10.539634

Defining a core configuration for human centromeres during mitosis

Gupta, A. S.·Seidel, C.·Tsuchiya, D.·McKinney, S. A.·Yu, Z.·Smith, S. E.·Unruh, J.·Gerton, J.
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Abstract

The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including cohesin, the centromere-specific H3 variant CENP-A, and centromeric DNA. Centromeric DNA is highly variable between chromosomes yet must accomplish a similar function. Moreover, how the 50 nm cohesin ring, proposed to encircle sister chromatids, accommodates inter-sister centromeric distances of hundreds of nanometers on the metaphase spindle is a conundrum. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We used ChIP-seq and super-resolution microscopy to examine the geometry of essential centromeric components on human chromosomes. ChIP-seq demonstrates that cohesin subunits are depleted in -satellite arrays where CENP-A nucleosomes and kinetochores assemble. Cohesin is instead enriched at pericentromeric DNA. Structured illumination microscopy of sister centromeres is consistent, revealing a non-overlapping pattern of CENP-A and cohesin. We used single particle averaging of hundreds of mitotic sister chromatids to develop an average centromere model. CENP-A clusters on sister chromatids, connected by -satellite, are separated by [~]562 nm with a perpendicular intervening [~]190 nM wide axis of cohesin. Two differently sized -satellite arrays on chromosome 7 display similar inter-sister CENP-A cluster distance, demonstrating different sized arrays can achieve a common spacing. Our data suggest a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes at the outer edge of extensible -satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation for future studies of additional components required for centromere function.

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Gupta, A. S., Seidel, C., Tsuchiya, D., McKinney, S. A., Yu, Z., Smith, S. E., Unruh, J., Gerton, J.. 2023-05-12. Defining a core configuration for human centromeres during mitosis. https://doi.org/10.1101/2023.05.10.539634

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