Centromeres are essential for faithful chromosome segregation, serving as sites for the recruitment of kinetochore proteins that mediate attachment to spindle microtubules during mitosis and meiosis1. In addition to being the basis for kinetochore assembly, centromeres have more recently been shown to have a fundamental role in 3D chromosome organization, both in interphase and mitosis2,3. To better understand this function, we harness the power of biological diversity and explore the genome architecture of organisms exhibiting a drastic evolution in centromere layout: species in the Lepidoptera order. These species are known to be holocentric, i.e., having centromeres distributed over the entire length of chromosomes instead as restricted to a single region. Among the many questions raised by this puzzling linear chromosome organization, our initial aim was to understand whether and how the spatial genome organization has changed to accommodate the chromosome-wide distribution of centromeric sites in holocentric organisms.

We initiated our study on the silkworm, Bombyx mori, and described its unconventional 3D genome organization in interphase4. At the genome-wide scale, the research reveals highly distinct chromosome territories, with very few interchromosomal contacts. The absence of a detectable specific signal in trans is a strong signature of the absence of regional monocentromere. At the chromosomal scale, chromosomes do not only segregate between two compartments (A: active, B: inactive) as in other eukaryotes. Instead, we identified a third type of compartment, encompassing more than 15% of the genome and segregating away from both A and B. Indeed, this compartment makes very frequent short-range self-contacts and rarely engages in long-range contacts, even with other domains of this same compartment. As this was unprecedented in the literature, and to reflect and describe its observed behavior, we named this newly identified compartment S, for “secluded”.

To follow up on our initial description, now we would like to understand i) how this peculiar spatial genome organization is established ii) the interplay between centromere organization and 3D chromosomes architecture. The silkworm was the model of choice when we initiated our analysis, however, we now feel that it has too many limitations to perform the further characterization envisioned. To leverage the difficulties, we are moving towards a new lepidopteran model which is the pantry moths, Plodia interpunctella (hereafter Plodia). This model meets all criteria to circumvent difficulties encountered with B. mori. It has a short life cycle (25 days at 28°C), an ease of culture on a low-cost artificial diet and a mass synchronized egg-laying ability. Additionally, most useful genetic manipulations, like genomic insertion of transgenes and CRISPR KOs have already been proven by our collaborator Arnaud Martin (The George Washington University) 5,6.

Biophysical modeling and preliminary characterization of S using our Hi-C data suggest a high degree of compaction resulting from elevated loop extrusion activities. We thus propose to investigate the role of architectural proteins, and in particular SMCs (Structural Maintenance of Chromatin) complexes, in organizing the genome's spatial architecture in Lepidoptera, using Plodia as our animal model. We will systematically investigate the roles of cohesin, condensin, and insulators through Hi-C and ChIP-seq experiments in both normal and perturbed conditions.

To understand the interplay between centromere organization and genome conformation, we need to characterize functional centromeric regions during mitosis. For this, we will perform chromatin immunoprecipitation on metaphase chromosomes using a kinetochore protein as a bait. This information will then be put in light of the 3D architecture of chromosomes as explored by Hi-C analysis, both in interphase and mitosis.
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Centromeres are essential for faithful chromosome segregation, serving as sites for the recruitment of kinetochore proteins that mediate attachment to spindle microtubules during mitosis and meiosis1. In addition to being the basis for kinetochore assembly, centromeres have more recently been shown to have a fundamental role in 3D chromosome organization, both in interphase and mitosis2,3. To better understand this function, we harness the power of biological diversity and explore the genome architecture of organisms exhibiting a drastic evolution in centromere layout: species in the Lepidoptera order. These species are known to be holocentric, i.e., having centromeres distributed over the entire length of chromosomes instead as restricted to a single region. Among the many questions raised by this puzzling linear chromosome organization, our initial aim was to understand whether and how the spatial genome organization has changed to accommodate the chromosome-wide distribution of centromeric sites in holocentric organisms.

We initiated our study on the silkworm, Bombyx mori, and described its unconventional 3D genome organization in interphase4. At the genome-wide scale, the research reveals highly distinct chromosome territories, with very few interchromosomal contacts. The absence of a detectable specific signal in trans is a strong signature of the absence of regional monocentromere. At the chromosomal scale, chromosomes do not only segregate between two compartments (A: active, B: inactive) as in other eukaryotes. Instead, we identified a third type of compartment, encompassing more than 15% of the genome and segregating away from both A and B. Indeed, this compartment makes very frequent short-range self-contacts and rarely engages in long-range contacts, even with other domains of this same compartment. As this was unprecedented in the literature, and to reflect and describe its observed behavior, we named this newly identified compartment S, for “secluded”.

To follow up on our initial description, now we would like to understand i) how this peculiar spatial genome organization is established ii) the interplay between centromere organization and 3D chromosomes architecture. The silkworm was the model of choice when we initiated our analysis, however, we now feel that it has too many limitations to perform the further characterization envisioned. To leverage the difficulties, we are moving towards a new lepidopteran model which is the pantry moths, Plodia interpunctella (hereafter Plodia). This model meets all criteria to circumvent difficulties encountered with B. mori. It has a short life cycle (25 days at 28°C), an ease of culture on a low-cost artificial diet and a mass synchronized egg-laying ability. Additionally, most useful genetic manipulations, like genomic insertion of transgenes and CRISPR KOs have already been proven by our collaborator Arnaud Martin (The George Washington University) 5,6.

Biophysical modeling and preliminary characterization of S using our Hi-C data suggest a high degree of compaction resulting from elevated loop extrusion activities. We thus propose to investigate the role of architectural proteins, and in particular SMCs (Structural Maintenance of Chromatin) complexes, in organizing the genome's spatial architecture in Lepidoptera, using Plodia as our animal model. We will systematically investigate the roles of cohesin, condensin, and insulators through Hi-C and ChIP-seq experiments in both normal and perturbed conditions.

To understand the interplay between centromere organization and genome conformation, we need to characterize functional centromeric regions during mitosis. For this, we will perform chromatin immunoprecipitation on metaphase chromosomes using a kinetochore protein as a bait. This information will then be put in light of the 3D architecture of chromosomes as explored by Hi-C analysis, both in interphase and mitosis.
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Début de la thèse : 01/10/2025

Funding category: Contrat doctoral

Concours pour un contrat doctoral