Researchers publishing in Cell Stem Cell have investigated the function of the gene DNMT3A and found that it has wide-ranging effects beyond methylation.
More effects than expected
Clonal hematopoiesis (CH), which occurs when stem cells create large numbers of cells with the same mutation, is linked to blood cancers [1]. DNMT3A is the most commonly mutated gene in CH [2], it has been directly linked to cancer itself [3], and we have previously reported on DNMT3A mutants being able to outcompete unmutated cells in the body.
These researchers note that as DNMT3A has been described as a methyltransferase enzyme due to its function in embryonic stem cells [4], most work has been done on its relationship to methylation. However, other evidence suggests that this function may not be relevant in the context of CH, cancer, and other age-related diseases; the methylation directly affected by DNMT3A may have little to do with the actual downstream consequences of its mutations [5]. The researchers of this study, therefore, wanted to know what this gene does, and does not, actually do in adult organisms.
Methylation-deficient variants still have strong effects
The mouse gene Dnmt3L works with Dnmt3a to methylate DNA. Therefore, in their first experiment, the researchers used lentiviral transduction to overexpress Dnmt3L in blood stem cells (HSPCs) derived from mice along with another group that overexpresses Dnmt3a as well as a control group. As expected, the Dnmt3L group had very different methylation from either group, and overexpressing Dnmt3L in Dnmt3a-deficient cells yielded no statistically significant difference, showing that its effects are dependent on Dnmt3a.
Changing the expression of Dnmt3a, on the other hand, yielded completely different results. Death by apoptosis significantly increased in cells that overexpressed this gene. Cells that did not express Dnmt3a reproduced out of control, creating far more colony-forming units. By creating Dnmt3a variants that had impaired methylation activity, the researchers ascertained that its restraining effects are not due to methylation. Cells that had one of these variants had far less methylation than cells with normal Dnmt3a, yet their reproduction was equally controlled.
Mouse experiments found that, while some level of DNA methylation is required for normal embryonic development, the low-methylation variants did not have significant amounts of CH compared to wild-type cells when given as transplants to irradiated mice. Further experimentation involving transplants found that cells that don’t express Dnmt3a at all were significantly tilted towards self-renewal, while the methylation-affected variants were more restrained.
Concordant with their other experiments, the researchers analyzed the effects of these variants and found that function and methylation had no significant correlations. This even extended to gene expression, which would theoretically be directly related; instead, gene expression and methylation had no significant correlation in this experiment.
Effects on telomeres and reproduction
Previous work has also found that Dnmt3a loss is linked to longer telomeres in mice [7] and that DNMT3A mutations are linked to lengthened telomeres in cancers in people [8]. Therefore, the researchers examined the telomeres of their transplanted cells and found that this is indeed the case; both telomere length and the telomere-lengthening gene Tert were increased in cells that do not express Dnmt3a. This length was also increased in the short term in the methylation-impaired variants but was reduced over time, suggesting both methylation-related and unrelated mechanisms.
A lack of Dnmt3a was also found to keep cells proliferating when telomere-related mechanisms suggested that they should not. The DNA damage response is activated when telomeres become too short, causing senescence; however, a lack of Dnmt3a was found to cause these cells to continue to proliferate, and this had nothing to do with methylation. Furthermore, both telomerase and alternative mechanisms of lengthing telomeres were caused by a lack of Dnmt3a.
This study illuminates key facts about cellular senescence and proliferation. Uncontrolled growth is perhaps even more immediately dangerous in the context of aging than unwelcome senescence. The proliferation of unhealthy cells is a driving force behind cancer and other age-related diseases, and this study may be followed up by future work that seeks to restore DNMT3A in order to stop runaway clonal expansion of mutated cells.
Literature
[1] Jaiswal, S., Fontanillas, P., Flannick, J., Manning, A., Grauman, P. V., Mar, B. G., … & Ebert, B. L. (2014). Age-related clonal hematopoiesis associated with adverse outcomes. New England Journal of Medicine, 371(26), 2488-2498.
[2] Challen, G. A., & Goodell, M. A. (2020). Clonal hematopoiesis: mechanisms driving dominance of stem cell clones. Blood, The Journal of the American Society of Hematology, 136(14), 1590-1598.
[3] Ley, T. J., Ding, L., Walter, M. J., McLellan, M. D., Lamprecht, T., Larson, D. E., … & Wilson, R. K. (2010). DNMT3A mutations in acute myeloid leukemia. New England Journal of Medicine, 363(25), 2424-2433.
[4] Okano, M., Xie, S., & Li, E. (1998). Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nature genetics, 19(3), 219-220.
[5] Spencer, D. H., Russler-Germain, D. A., Ketkar, S., Helton, N. M., Lamprecht, T. L., Fulton, R. S., … & Ley, T. J. (2017). CpG island hypermethylation mediated by DNMT3A is a consequence of AML progression. Cell, 168(5), 801-816.
[6] Jia, D., Jurkowska, R. Z., Zhang, X., Jeltsch, A., & Cheng, X. (2007). Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature, 449(7159), 248-251.
[7] Gonzalo, S., Jaco, I., Fraga, M. F., Chen, T., Li, E., Esteller, M., & Blasco, M. A. (2006). DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nature cell biology, 8(4), 416-424.
[8] Myllymäki, M., Redd, R., Reilly, C. R., Saber, W., Spellman, S. R., Gibson, C. J., … & Lindsley, R. C. (2020). Short telomere length predicts nonrelapse mortality after stem cell transplantation for myelodysplastic syndrome. Blood, The Journal of the American Society of Hematology, 136(26), 3070-3081.
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