Orderly mitosis shapes interphase genome architecture
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eLife Assessment
This valuable study combines microscopy and CRISPR screening in two different cell lines to identify factors involved in global chromatin organization, using centromere clustering as a proxy. Follow-up cell cycle synchronisation studies confirm roles in centromere clustering in mitosis. However, incomplete characterisation of the cell lines used limits the interpretation of the findings. The study will interest researchers studying genome organisation in mitosis.
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Abstract
Abstract
Genomes assume a complex 3D architecture in the interphase cell nucleus. Yet, the molecular mechanisms that determine global genome architecture are only poorly understood. To identify mechanisms of higher order genome organization, we performed high-throughput imaging-based CRISPR knockout screens targeting 1064 genes encoding nuclear proteins in human cell lines. We assessed changes in the distribution of centromeres at single cell resolution as surrogate markers for global genome organization. The screens revealed multiple major regulators of spatial distribution of centromeres including components of the nucleolus, kinetochore, cohesins, condensins, and the nuclear pore complex. Alterations in centromere distribution required progression through the cell cycle and acute depletion of mitotic factors with distinct functions altered centromere distribution in the subsequent interphase. These results identify molecular determinants of spatial centromere organization, and they show that orderly progression through mitosis shapes interphase genome architecture.
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eLife Assessment
This valuable study combines microscopy and CRISPR screening in two different cell lines to identify factors involved in global chromatin organization, using centromere clustering as a proxy. Follow-up cell cycle synchronisation studies confirm roles in centromere clustering in mitosis. However, incomplete characterisation of the cell lines used limits the interpretation of the findings. The study will interest researchers studying genome organisation in mitosis.
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Reviewer #1 (Public review):
Summary:
In this manuscript, Guin and colleagues establish a microscopy-based CRISPR screen to find new factors involved in global chromatin organization. As a proxy of global chromatin organization, they use centromere clustering in two different cell lines. They find 52 genes whose CRISPR depletion leads to centromere clustering defects in both cell lines. Using cell cycle synchronisation, they demonstrate that centromeres-redistribution upon depletion of these hits necessitates cell cycle progression through mitosis.
Strengths:
This manuscript explores the mechanisms of global chromatin organization, which is a scale of chromatin organization that remains poorly understood. The imaging-based CRISPR screen is very elegant, and the use of appropriate positive and negative controls reinforces the solidity of …
Reviewer #1 (Public review):
Summary:
In this manuscript, Guin and colleagues establish a microscopy-based CRISPR screen to find new factors involved in global chromatin organization. As a proxy of global chromatin organization, they use centromere clustering in two different cell lines. They find 52 genes whose CRISPR depletion leads to centromere clustering defects in both cell lines. Using cell cycle synchronisation, they demonstrate that centromeres-redistribution upon depletion of these hits necessitates cell cycle progression through mitosis.
Strengths:
This manuscript explores the mechanisms of global chromatin organization, which is a scale of chromatin organization that remains poorly understood. The imaging-based CRISPR screen is very elegant, and the use of appropriate positive and negative controls reinforces the solidity of the findings.
Weaknesses:
Although the data are generally solid and well interpreted, a control showing that protein depletion works properly in cell-cycle arrested cells is lacking, both when using siRNAs and degron-based depletion.
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Reviewer #2 (Public review):
The authors begin by highlighting the importance of genome organisation in cellular compartmentalisation and identity. They focus their study on centromeres - key chromosomal features required for segregation-and aim to identify proteins responsible for their spatial distribution in interphase nuclei. However, none of the experimental data addresses broader aspects of genome architecture, such as individual chromosome territories or A/B compartments. As such, the title of the article may be misleading and would benefit from being more specific, for example: "Identification of factors influencing centromere positioning in interphase."
Strengths:
One of the strengths of the paper is the comprehensive CRISPR-based screening and the comparative analysis between two distinct cell lines.
Including further …
Reviewer #2 (Public review):
The authors begin by highlighting the importance of genome organisation in cellular compartmentalisation and identity. They focus their study on centromeres - key chromosomal features required for segregation-and aim to identify proteins responsible for their spatial distribution in interphase nuclei. However, none of the experimental data addresses broader aspects of genome architecture, such as individual chromosome territories or A/B compartments. As such, the title of the article may be misleading and would benefit from being more specific, for example: "Identification of factors influencing centromere positioning in interphase."
Strengths:
One of the strengths of the paper is the comprehensive CRISPR-based screening and the comparative analysis between two distinct cell lines.
Including further investigation into factors that behave differently across these cell lines - particularly in relation to expression levels or the unique "inverted architecture" of RPE cells-would have added valuable depth.
Weaknesses:
The filtering strategy used in the screen imposes significant constraints, as it selects only for non-essential or functionally redundant genes. This is a critical point, as key regulators of chromatin organisation - such as components of the condensin and cohesin complexes-are typically essential for viability. Similarly, known effectors of centromere behaviour (e.g., work by the Fachinetti's lab) often lead to aneuploidy, micronuclei formation, and cell cycle arrest in G1. The implication of this selection criterion should be clearly discussed, as it fundamentally shapes the interpretation of the study's findings.
A major limitation of the study is the lack of connection between centromere clustering and its biological significance. It remains unclear whether this clustering is a meaningful proxy for higher-order genome organisation. Additionally, the study does not explore potential links to cell identity or transcriptional landscapes. Readers may struggle to grasp the broader relevance of the findings: if gene knockouts that alter centromere positioning do not affect cell viability or cell cycle progression, does this imply that centromere clustering - and by extension, interphase genome organisation - is not biologically significant?
Another point requiring clarification is the conclusion that the four identified genes represent independent pathways regulating centromere clustering. In reality, all of these proteins localise to centromeres. For example, SPC24 and NUF2 are components of the NDC80 complex; Ki-67, a chromosome periphery protein, has been mapped to centromeres; and CAP-Hs, a subunit of the condensin II complex that during G1 promotes CENP-A deposition. Given their shared localisation, it would be informative to assess aneuploidy indices following depletion of each factor. Chromosome-specific probes could help determine whether centromere dysfunction leads to general mis-segregation or reflects distinct molecular mechanisms. Additionally, exploring whether Ki-67 mutants that affect its surfactant-like properties influence centromere clustering could provide a more mechanistic insight.
Finally, the additive effects observed in double knockdowns do not necessarily confirm pathway independence. It is possible that mild mis-segregation effects are amplified when two proteins within the same pathway are depleted. This possibility should be considered in the interpretation of the data.
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Reviewer #3 (Public review):
Summary:
In this manuscript, Guin et al. use a CRISPR KO screen of ~1000 candidates in two human cell lines, along with high-throughput image analysis, to demonstrate that orderly progression through mitosis shapes centromere organization. They identify ~50 genes that perturb centromere clustering when depleted in both RPE1 and HCT116 cells and validate many of these hits using RNAi. They then use auxin-mediated acute depletion of four factors (NCAPH2, KI67, SPC24, and NUF2) to demonstrate that their effects on centromere clustering require passage through mitosis. They further suggest that the lack of these factors during mitosis leads to the disorganization of centromeres on the mitotic spindle, and these effects persist in the subsequent interphase. Overall, the manuscript is clear, well-written, the …
Reviewer #3 (Public review):
Summary:
In this manuscript, Guin et al. use a CRISPR KO screen of ~1000 candidates in two human cell lines, along with high-throughput image analysis, to demonstrate that orderly progression through mitosis shapes centromere organization. They identify ~50 genes that perturb centromere clustering when depleted in both RPE1 and HCT116 cells and validate many of these hits using RNAi. They then use auxin-mediated acute depletion of four factors (NCAPH2, KI67, SPC24, and NUF2) to demonstrate that their effects on centromere clustering require passage through mitosis. They further suggest that the lack of these factors during mitosis leads to the disorganization of centromeres on the mitotic spindle, and these effects persist in the subsequent interphase. Overall, the manuscript is clear, well-written, the experiments performed are appropriate, and the data are interpreted accurately. In my opinion, the main strength of this manuscript is the discovery of several hits associated with altered centromere organization. These hits will serve as a solid foundation for future work investigating genome organization in human cells. On the other hand, how the changes in centromere organization relate to other aspects of interphase genome architecture (A/B compartments, chromosome territories, etc) remains unclear and represents the main shortcoming of this manuscript.
Comments:
(1) Given the authors' suggestion that disorderly mitotic progression underlies the changes in centromere clustering in the subsequent interphase, I think it would be beneficial to showcase examples of disorderly mitosis in the AID samples and perhaps even quantify the misalignment on the metaphase plate.
(2) I don't quite agree with the description that centromeres cluster into chromocenters (p4 para 2, p17 para 1, and other instances in the manuscript). To the best of my knowledge, chromocenters primarily consist of clustered pericentromeric heterochromatin, while the centromeres are studded on the chromocenter surface. This has been beautifully demonstrated in mouse cells (Guenatri et al., JCB, 2004), but it is true in other systems like flies and plants as well.
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