Evaluation of DNA methylation role in regulating cell cycle progression and chromosome segregation

Background: Epigenetic regulation is important to ensure the fitness of the cells. To this regard, DNA methylation has the important role of regulating gene expression and heterochromatinization. DNA methylation pattern changes in cancer and during aging. Specifically, cancer cells are characterised by DNA hypermethylation at promoters of tumour suppressor genes, leading to improved proliferation in different microenvironments. Instead, DNA hypomethylation mostly occurs at repetitive sequences of the genome, which characterise the telomere and the centromere. The centromere sustains chromosome segregation through the recruitment of the kinetochore complex, which allows a strong connection with the microtubules and acts as an anchor to permit the movement of chromosomes. Impairment of chromosome segregation is known to be sensed as a stress by normal cells which undergo cell cycle arrest. Changes at the centromeric DNA methylation correlate with aneuploidy, suggesting the occurrence of alterations in chromosome segregation. However, how DNA methylation regulates the fidelity of chromosome segregation is not yet fully understood. To address this issue, a DNA hypomethylating inducible system was used. Methods: To induce DNA methylation, I used Normal Retinal Pigment Epithelial hTert immortalized (RPE-1) and colon-rectum cancer cell line (DLD-1) engineered to trigger the degradation of the endogenous DNMT1 with an inducible degron. Prolonged DNMT1 absence leads to a global passive DNA methylation loss, which mimics the aging event. The system is not toxic and most importantly, it is reversible. The reversibility permits to discern the effect of the DNA methylation alteration from the combinatory effect achieved when the DNMT1, together with DNA methylation, is lost. To evaluate the effect of prolonged DNMT1 absence and DNA hypomethylation on the fitness of the cells and thus, the cell cycle progression and chromosome segregation, genetics, cell and molecular biology techniques were used. Results: The performed experiments show that DNMT1 absence per se does not induce cell cycle arrest in both normal and cancer cells. Instead, prolonged DNMT1 absence and subsequent DNA hypomethylation are required to trigger p53/p21 cell cycle arrest. In DLD-1 cancer cells, DNMT1 absence also correlates with difficulties in properly congressing the chromosomes, which fail in gaining a bioriented state with subsequent misaligned chromosomes. Moreover, DNMT1 absence or DNA hypomethylation favours the recruitment of CENP-A and CENP-C, which are important for centromere functioning. Ultimately, Aurora B, the kinase required to correct improper kinetochore-microtubule attachments, underwent altered dynamics: 1) increased activity when microtubules are disassembled; 2) reduced recruitment at the metaphase plate, signature of mature end-on attachment; 3) no varied recruitment at misaligned chromosomes. Conclusion: Prolonged DNMT1 absence generates cellular stress, due to the induced DNA hypomethylation. This stress culminates in the cell cycle arrest when the threshold sustainable minimal level of DNA methylation is reached. In cancer cells DNMT1 or DNA methylation regulates the recruitment of CENPs and Aurora B kinase. Interestingly, DNA methylation loss induces enhanced stability of the already in place kinetochore-microtubule attachments, while the misaligned chromosomes seem to be disregarded.