A new paradigm for centromere biology: Evolution and mechanism of CenH3-independent chromosome segregation in holocentric insects

Projektinformationen

CENEVO

ID Finanzhilfevereinbarung: 758757

DOI

10.3030/758757

Projekt abgeschlossen

EK-Unterschriftsdatum 27 Oktober 2017

Startdatum 1 April 2018

Enddatum 30 November 2024

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 1 497 500,00

EU-Beitrag

€ 1 497 500,00

Koordiniert durch

INSTITUT CURIE
France

Periodic Reporting for period 4 - CENEVO (A new paradigm for centromere biology:Evolution and mechanism of CenH3-independent chromosome segregation in holocentric insects)

Berichtszeitraum: 2022-10-01 bis 2024-03-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Chromosome segregation is essential to all eukaryotic life, yet, many components involved in this process are highly divergent across organisms. In the CENEVO proposal, we aim to characterize a novel pathway for kinetochore and centromere formation in insects that occurs chromosome-wide (also termed “holocentric” in contrast to the “monocentric” organization where centromeric activity is restricted in one chromosomal region) and independent of CenH3/CENP-A, a component previously thought to be the cornerstone for chromosome segregation. Our project will provide insights into the evolutionary plasticity of kinetochore assembly and centromere identity. Given that kinetochore and centromere alterations are frequently associated with cancer development, studying these naturally occurring variations can also provide insights into such malignant states.

In the first part, we are applying proteomic and genomic analyses in lepidopteran cell lines as a new experimental model system to study how the function of CenH3/CENP-A in kinetochore assembly and centromere identity became redundant in these organisms.

In the second part, we are taking a comparative genomic approach across all insects to analyze the interplay between centromeric alterations and the chromosome segregation machinery.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

During the first 30 months we made seminal progress on both aims of our proposal.

For Aim 1 about the characterization of CenH3/CENP-A-independent kinetochore and centromere formation in Lepidoptera we have established tools and protocols for an unconventional model system, cells derived from the silk moth Bombyx mori, to study its kinetochore composition and centromere regulation.

Using proteomic analyses combined with remote homology predictions, we have identified previously unknown kinetochore components and studied their contribution to mitotic progression and kinetochore assembly. In this context, we have identified a divergent homolog of CENP-T, a DNA binding component found in vertebrates and fungi, which we are currently testing to what extend it contributes to connect the CenH3/CENP-A-deficient kinetochore to chromatin. This work has been published in Current Biology earlier this year. Using the tools that we have established we are following up on additional kinetochore components to complement these first insights into the composition and regulation of CenH3/CENP-A-deficient kinetochore assembly in B. mori.

The identification of CENP-T and other kinetochore components also allowed us to apply genomic approaches to map centromere sites along B. mori chromosomes and understand the regulation of its CenH3/CENP-A-deficient holocentromere. This revealed a new mode of centromere specification that appears to be dependent on the chromosome-wide chromatin landscape rather than the presence of CenH3/CENP-A. The paper was under revision at the end of the reporting period and has recently been published by Current Biology.

For Aim 2 about the evolution of CenH3/CENP-A-deficient holocentromeres across insects, we are mapping kinetochore components across mono- and holocentric insect species to test for a recurrent evolution to similar CenH3/CENP-A-independent kinetochore assembly pathways. Part of these results are included in our recent publication.
We have also identified homologs of various spindle and checkpoint associated proteins in collaboration with Dr. Phong Tran at Institut Curie. We will follow-up on their functionality in B. mori cell lines and profile their conservation across insects to test for any correlation with the type of centromere.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

We believe that we made progress beyond the state of the art at several points of our research, in particular in understanding how chromosome segregation can function independently of CenH3/CENP-A:

CenH3/CENP-A was thought to act as the cornerstone for kinetochore assembly in most eukaryotes. We have provided the first insights into a metazoan kinetochore assembly pathway that occurs in the absence of CenH3/CENP-A. More specifically, we have identified homologs of kinetochore components conserved in other eukaryotes including humans. We have characterized one specific component, CENP-T that might contribute to compensate for the role of CenH3/CENP-A in connecting the kinetochore to chromatin. Using the tools that we have established in our lepidopteran cell line system in addition to ongoing biochemical analyses, we expect to complete our understanding on CenH3/CENP-A-independent kinetochore assembly until the end of the project. These analyses will determine how the otherwise conserved kinetochore complex got rewired to allow the loss of CenH3/CENP-A.

CenH3/CENP-A was thought to epigenetically mark centromeres in most eukaryotes. We identified a new mode centromere regulation that is independent of CenH3/CENP-A. The identified dependency on the chromosome-wide chromatin landscape for kinetochore formation provided insights into the plasticity of centromere specification.

Finally, we profiled kinetochore components across mono- and holocentric insects providing insights into kinetochore conservation beyond its model organisms Drosophila melanogaster. We have also identified components involved in spindle formation and checkpoint signaling in B. mori. We will follow those up in functional assays and map their conservation across of mono- and holocentric insect organisms. These studies will reveal insights into the interplay between centromeric architecture and the chromosome segregation machinery.

Aim1

Aim2