# Mechanisms of Kinetochore Assembly

> **NIH NIH R01** · STANFORD UNIVERSITY · 2024 · $334,123

## Abstract

Project Summary
The process of cell division requires that each cell receive an identical complement of the genome. Errors in
chromosome segregation give rise to chromosomal aneuploidies that are causative for human genetic disease
such as Down syndrome and promote the progression of most human cancers. The work in this proposal is
focused on understanding the functions of the centromere and kinetochore, the primary site on the
chromosome that attaches each chromosome to the mitotic spindle for proper segregation during division. The
centromere and kinetochore serve as the primary control center for chromosome segregation through their
roles in microtubule attachment, force coupling for chromosome movement, and error correction when
chromosome attachment or alignment on microtubules is perturbed. Underlying the centromere is a specialized
chromatin domain that is delineated by replacement of histone H3 in nucleosomes with a centromere specific
histone variant termed CENP-A. The position of CENP-A defines where the centromere and kinetochore will
form and defects in CENP-A assembly or maintenance cause centromere and kinetochore loss which result in
chromosome missegregation. In this proposal we study three different aspects of CENP-A chromatin. In our
first Aim we work to understand how the assembly of CENP-A chromatin is controlled so that the centromere is
replenished during each cell cycle and maintained through replication and cell division. We focus on three
important factors for CENP-A assembly, CENP-C, HJURP and the Mis18 complex that are essential for
building new CENP-A nucleosomes at centromeres. Our second aim studies the problem of how the presence
of CENP-A in chromatin dictates the sites of new CENP-A assembly. We apply a new method we have
developed called DiMeLo-seq that overcomes existing limitations in studying the complex repetitive
centromeres of vertebrates. This approach uses directed methylation to mark the sites where proteins are
bound to chromatin and then uses long read sequencing to map the binding sites through the repetitive
centromere sequences. We use this approach to study the density, distribution, and sites of regeneration of
CENP-A through the cell cycle. In our third aim we study how centromeres are condensed and organized by
the activities of CENP-A nucleosome binding proteins. We discovered that two centromere proteins (CENP-C
and CENP-N) can bridge CENP-A nucleosomes and condense centromeric chromatin. We use human cells to
test the role of this condensation activity in centromere condensation, resistance to force, and chromosome
segregation. Overall, our proposal will elucidate the regulatory mechanisms and structural organization of
vertebrate centromeres and how those properties ensure accurate chromosome segregation.

## Key facts

- **NIH application ID:** 10825404
- **Project number:** 2R01GM074728-18
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Aaron F Straight
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $334,123
- **Award type:** 2
- **Project period:** 2005-09-01 → 2027-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10825404

## Citation

> US National Institutes of Health, RePORTER application 10825404, Mechanisms of Kinetochore Assembly (2R01GM074728-18). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10825404. Licensed CC0.

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