Human disease including cancer arises from disfunction of essential processes within a cell. One essential cellular process is the copying of the genomic DNA—the road map of cells—followed by separation of this newly made DNA into two cells, each encased by a membrane barrier. This process of DNA duplication and separation into two daughter cells is referred to as cell division. While the detailed process of cell division was initially described in 1882, our understanding of “how” this occurs remains incomplete. Dr. Sue Biggins, the Director of Basic Sciences Division at Fred Hutchinson Cancer Center has devoted her lab’s work to understanding this intricate process that is an essential part of life. Their most recent work investigated the granular details of how proteins stabilize key structures on chromatids—condensed or compact DNA—to enable the separation of the duplicated DNA structure into a new cell in budding yeast, a model system for studying cell division. They discovered that despite relatively weak binding by an essential protein—histone variant CENP-A/Cse4—to DNA, other proteins are recruited to one specific site in the center of the chromosome (picture the spring location on a clothespin) and stabilize the weak binding between CENP-A/Cse4 and the chromosomal DNA. These findings published in the EMBO Journal bring into focus the complex regulation of protein:DNA affinity that is critical for cells to divide. Furthermore, these results can shed light on how this process is dysregulated in diseases including cancer by inhibiting cell growth or by driving uncontrolled growth.
“This manuscript took advantage of a recently developed high-throughput single molecule imaging analysis to identify previously unknown mechanisms that ensure centromeric nucleosomes are stably assembled,” shared Dr. Andrew Popchock, a former research fellow from the Biggins lab. Other approaches require creating stabilizing mutations for these inherently weak but stable protein:DNA interactions to form, but the approach developed in the Biggins’ lab was able to assemble these complexes de novo without additional stabilizing techniques. The researchers applied this method to yeast cells to study cell division since this system retains, with high conservation, the mechanisms at play in dividing human cells. When yeast chromosomes are ready to separate and form new cells—referred to as budding—regulated processes are needed to separate the chromosomes from their tethered core or centromeric junction. “Counterintuitively, centromeric DNA, which must efficiently recruit specialized nucleosomes [complexes of DNA wrapped around histone proteins] in cells to ensure chromosome segregation, is a poor template for nucleosome assembly in vitro due to its lack of bendability and its high AT content,” explained Dr. Popchock. For this reason, “we set out to identify the factors that ensure it assembles in vivo and identified previously unknown regulatory mechanisms that stabilize the nucleosome.”
The new capability of stably assembling centromeric nucleosomes—DNA wrapped around proteins at the ‘spring site’ of the chromosome ‘clothespin’—provided the ideal system to ask the following question: Can previously identified proteins—Okp1/Ame1 and chaperone Scm3—stabilize the binding between the CENP-A/Cse4 protein and centromeric DNA? The researchers genetically altered CENP-A/Cse4 and the centromeric DNA sequence along with other proteins in this complex and discovered that the other proteins indeed stabilized the binding of CENP-A/Cse4 to the centromeric DNA by separately interacting with the CENP-A/Cse4 essential N-terminal domain. Phosphorylation of this domain by Ipl1/Aurora B also aided the stability of assembled chaperone Scm3, CENP-A/Cse4 and centromeric DNA. These findings emphasize the importance of the N-terminal domain of CENP-A/Cse4 and interactions with a chaperone protein and another kinetochore complex plus a kinase to stabilize assembly of centromeric nucleosomes prior to chromosome separation in dividing cells.
“While this represents a significant step forward in the resolution that we can monitor this process in real time, it still remains unclear how this process occurs so rapidly and with such high fidelity in cells,” shared Dr. Biggins. “We will continue to use our single molecule imaging technique to identify additional factors that stabilize the centromeric nucleosome in vivo. In addition, we are using the assay to expand our work to identifying additional mechanisms that ensure kinetochore assembly.” Kinetochore assembly is another process during cell division that contributes to the high-fidelity nature of cell division. “We will temporally map the assembly of all kinetochore components for the first time as well as identify previously unknown key regulatory events in the assembly process,” explained Dr. Biggins. The Fred Hutchinson Cancer Center Cellular Imaging Core facility resources were essential for the single molecule imagine technique experiments as was computational support from Image Analysis Staff Scientist Dr. Julien Dubrulle.
The spotlighted research was funded by the National Institutes of Health, the Burroughs Wellcome Fund, the Pew Charitable Trusts, and Howard Hughes Medical Institute.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Charles Asbury and Sue Biggins contributed to this work.
Popchock AR, Hedouin S, Mao Y, Asbury CL, Stergachis AB, Biggins S. 2025. Stable centromere association of the yeast histone variant Cse4 requires its essential N-terminal domain. EMBO J. 44(5):1488-1511.
Science spotlight writer Annabel Olson is a postdoctoral research fellow in the Nabet lab at Fred Hutchinson Cancer Center. Her research focuses on studying the mechanisms that drive cancer development for both genetic and virus-associated cancers. A key tool in her research is the use of targeted protein degradation to dissect dysregulated signaling pathways in cancer and to double as a relevant pre-clinical therapeutic platform.
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