CTCF is essential for proper mitotic spindle structure and anaphase segregation

Background

Mitosis is the most common type of cell division in higher organisms featuring a distinctive large-scale re-organisation of the overall cellular contents to form two identical daughter cells (1). Failure or errors in this process can lead to genomic instability, subsequently causing aneuploidy, several types of cancers, and chromosomal disorders (2). CCCTC-binding factor, CTCF, is an evolutionarily conserved zinc-finger DNA-binding protein that represses as well as activates genomic transcription (3, 4, 5). The eukaryotic genome is organised into several functional domains that need to be insulated from one another to prevent non-specific interactions and transcriptional dysregulation (6). CTCF is such a “chromatin insulator”; it binds to specific sequences in the human, mouse, and avian MYC promoters and negatively regulates transcription of the MYC protein (7, 8, 9). CTCF also propagates epigenetic markers by forming a complex with the maternal allele of the H19 gene, a parental gene crucial in genomic imprinting (10, 11). In vertebrate mitotic cells, CTCF is primarily localised proximal to the centromeres throughout the cell division cycle (12). Its binding to the centromeric protein, CENP-E, is critical to the latter’s recruitment to the centromere and the timely progression of mitosis. However, there is no evidence so far to show whether CTCF directly impacts mitotic fidelity.

In this preprint, the authors sought to discover the extent to which CTCF impacts the cell division process in higher organisms. They report novel roles of this protein in mitotic fidelity, chromosomal alignment, mitotic progression, and preservation of nuclear integrity during post-mitotic stages.

 

Main findings

1. Intact CTCF plays an indispensable role in correct chromosome segregation and maintenance of mitotic cell integrity.

Complete knockdown of CTCF is lethal for cells, which has consequently prevented investigation of the protein’s role in mitosis. Accordingly, the authors used CRISPR-based CTCF knockdown clones in mouse melanoma cells. Two clones, c13 and c21, expressed CTCF truncations with reduced stability but intact chromatin-binding properties (14), causing an approximately 60-70% decline in overall CTCF levels. Both clones displayed statistically significant increases in mitotic failure events relative to wild type cells. Further classification of these failures into distinct categories based on time of occurrence during the mitotic cycle showed high levels of chromosome misalignment and mis-segregation in cells expressing the CTCF clones. Based on this data, Chiu and colleagues concluded that CTCF is essential for preserving mitotic fidelity, and correct chromosome alignment and segregation.

2. Mitotic rates and the spindle checkpoint signalling remain unaltered in the presence of CTCF truncations.

An active signalling pathway – the spindle assembly checkpoint (SAC) – prevents cell cycle progression until all chromosomes are correctly bound (reviewed in 15). The authors wanted to understand if the SAC fails to correct erroneous attachments in the clones, causing abnormal mitosis. Treatment with Nocodazole (a well-studied microtubule depolymerising agent) led to high rates of mitotic arrest in both CTCF knockdowns, indicating an active spindle checkpoint. Importantly, the rates of cell cycling were comparable between the CTCF clones and parental cells. Therefore, the authors concluded that disruption of SAC or alteration in mitotic rates are not the cause of chromosomal abnormalities in cells expressing the CTCF truncations.

3. CTCF is required for correct DNA positioning and chromosome orientation in mitotic cells.

Live-cell imaging studies revealed that successful cell division cycles in both CTCF knockdowns had a similar duration to wild type cells. However, failed mitoses in the knockdowns stalled at metaphase and the total mitotic duration was significantly prolonged in both the clones. Furthermore, these experiments also showed chromosome segregation errors in cells expressing the CTCF clones, resulting in aneuploidy after cytokinesis. To understand whether prolonged cell division cycles are related to a disrupted mitotic spindle structure, the authors investigated DNA and microtubule positioning in the CTCF knockdowns and wild type cells via immunofluorescence experiments. In contrast to wild type cells with negligible abnormalities in the mitotic spindles, both the CTCF knockdown clones displayed tri-/tetrapolar spindles and/or other abnormal phenotypes in ~20-30% of the total studied mitotic events. Most of these observed abnormalities were associated with the misplacement of chromosomal DNA; around 13-20 % of spindles in the CTCF knockdowns showed DNA positioning behind the spindle poles, whereas no such observation was reported for wild type cells. Taken together, the authors inferred that intact CTCF is critical for correct chromosomal orientation and spindle organisation during mitosis.

4. Absence of full-length endogenous CTCF can lead to abnormalities in nuclear sizes post mitosis.

Finally, the authors reported that nuclear size in CTCF knockdowns shows an approximately two-fold increase in comparison to wild type nuclei. The enhanced nuclear size is, however, not associated with overall nuclear morphology (circularity or nuclear blebbing) in interphase or mitotic cells. Surprisingly, both CTCF clones displayed reduced nuclear circularity roughly 30 minutes post mitosis. Based on this evidence, the authors deduced that although nuclear phenotypes related to loss of CTCF can be masked during mitosis, the subtleties in overall nuclear morphology and subsequent cell fate possibly become evident only during post-mitotic stages.

 

What I liked about this work

This work provides the first piece of evidence for the mitosis-specific role of CTCF. I particularly enjoyed the use of the two CRISPR-knockdown clones, c13 and c21, throughout the study. These systematically proved that intact CTCF is indispensable for proper chromosome segregation and mitotic progression. Additionally, the authors have categorised mitotic failures in the knockdown cells into four primary phenotypes, instead of generalising all abnormal mitotic events under the same label. In my opinion, this helps readers to really grasp the extent of the damage caused by the loss of this protein and its direct correlation with several known/studied disorders. Lastly, nuclear deformations have been shown to be the cause of several human pathologies, including neurodevelopmental disorders, muscular dystrophy, and cancers (reviewed in 15). Therefore, in-depth analysis of nuclear morphologies carried out in this work could contribute towards the possible application of CTCF as a therapeutic target in biomedical and clinical research.

 

Questions to the authors

1. You mentioned that the B16 mouse melanoma cell line shows naturally decreased nuclear circularity, making it difficult to analyse differences between the clones and wild type cells during mitosis. I was wondering if you tried other mammalian cell lines to check whether the observations pertaining to nuclear phenotypes can be reproduced?
2. Quantitatively, the c13 clone caused metaphase failure in ~40% of the studied cells, whereas c21 displayed such errors in only ~10% of the cell population. Do you think the more severe phenotype seen in c13 might help narrow down to a specific amino acid stretch of CTCF that is more crucial for its functionality?

 

References

1. Yanagida M. (2014). The role of model organisms in the history of mitosis research. Cold Spring Harbor perspectives in biology, 6(9), a015768. https://doi.org/10.1101/cshperspect.a015768
2. Song, X., Conti, D., Shrestha, R. L., Braun, D., & Draviam, V. M. (2021). Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation. Nature communications, 12(1), 7010. https://doi.org/10.1038/s41467-021-27131-9
3. Filippova G. N. (2008). Genetics and epigenetics of the multifunctional protein CTCF. Current topics in developmental biology, 80, 337–360. https://doi.org/10.1016/S0070-2153(07)80009-3
4. Wan, L. B., Pan, H., Hannenhalli, S., Cheng, Y., Ma, J., Fedoriw, A., Lobanenkov, V., Latham, K. E., Schultz, R. M., & Bartolomei, M. S. (2008). Maternal depletion of CTCF reveals multiple functions during oocyte and preimplantation embryo development. Development (Cambridge, England), 135(16), 2729–2738. https://doi.org/10.1242/dev.024539
5. Bell, A. C., West, A. G., & Felsenfeld, G. (1999). The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell, 98(3), 387–396. https://doi.org/10.1016/s0092-8674(00)81967-4
6. Bell, A. C., & Felsenfeld, G. (2000). Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature, 405(6785), 482–485. https://doi.org/10.1038/35013100
7. Lobanenkov, V. V., Nicolas, R. H., Adler, V. V., Paterson, H., Klenova, E. M., Polotskaja, A. V., & Goodwin, G. H. (1990). A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5′-flanking sequence of the chicken c-myc gene. Oncogene, 5(12), 1743–1753.
8. Klenova, E. M., Nicolas, R. H., Paterson, H. F., Carne, A. F., Heath, C. M., Goodwin, G. H., Neiman, P. E., & Lobanenkov, V. V. (1993). CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms. Molecular and cellular biology, 13(12), 7612–7624. https://doi.org/10.1128/mcb.13.12.7612-7624.1993
9. Ohlsson, R., Renkawitz, R., & Lobanenkov, V. (2001). CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends in genetics : TIG, 17(9), 520–527. https://doi.org/10.1016/s0168-9525(01)02366-6
10. Pant, V., Mariano, P., Kanduri, C., Mattsson, A., Lobanenkov, V., Heuchel, R., & Ohlsson, R. (2003). The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. Genes & development, 17(5), 586–590. https://doi.org/10.1101/gad.254903
11. Schoenherr, C. J., Levorse, J. M., & Tilghman, S. M. (2003). CTCF maintains differential methylation at the Igf2/H19 locus. Nature genetics, 33(1), 66–69. https://doi.org/10.1038/ng1057
12. Rubio, E. D., Reiss, D. J., Welcsh, P. L., Disteche, C. M., Filippova, G. N., Baliga, N. S., Aebersold, R., Ranish, J. A., & Krumm, A. (2008). CTCF physically links cohesin to chromatin. Proceedings of the National Academy of Sciences of the United States of America, 105(24), 8309–8314. https://doi.org/10.1073/pnas.0801273105
13. Kaczmarczyk, L. S., Levi, N., Segal, T., Salmon-Divon, M., & Gerlitz, G. (2022). CTCF supports preferentially short lamina-associated domains. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 30(1), 123–136. https://doi.org/10.1007/s10577-022-09686-5
14. Cheeseman, I. M., & Desai, A. (2008). Molecular architecture of the kinetochore-microtubule interface. Nature reviews. Molecular cell biology, 9(1), 33–46. https://doi.org/10.1038/nrm2310
15. Kalukula, Y., Stephens, A. D., Lammerding, J., & Gabriele, S. (2022). Mechanics and functional consequences of nuclear deformations. Nature reviews. Molecular cell biology, 23(9), 583–602. https://doi.org/10.1038/s41580-022-00480-z

 

 

Posted on: 23 March 2023

doi: https://doi.org/10.1242/prelights.34176

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