Dnmt3a Controls Hematopoietic Stem Cells Via DNA Methylation-Independent Regulation of Telomeres

Mutations in DNMT3A are the most common variants in age-related clonal hematopoiesis and recurrent initiating events in a range of human blood disorders. This DNA methyltransferase enzyme is a crucial epigenetic regulator that maintains the balance between hematopoietic stem cell (HSC) self-renewal and differentiation. Competitive transplantation of Dnmt3a-null HSCs results in accumulation of HSCs in the bone marrow but impaired differentiation. Despite the dramatic phenotype of HSCs deficient for Dnmt3a, there are minimal overall changes in total DNA methylation levels, questioning the role of Dnmt3a-mediated DNA methylation in regulating HSC fate decisions. Here, we explore the possibility that Dnmt3a regulates HSCs through DNA methylation-independent functions.

We engineered a series of mice with variants in the endogenous locus of Dnmt3a, altering its methyltransferase activity to different degrees: Dnmt3a^E752A (0% DNA methyltransferase capacity) and Dnmt3a^R832A (˜20% DNA methyltransferase capacity). Crossing these mice with Dnmt3a^fl/fl ensured that the mutant protein was the only expressed Dnmt3a copy after Cre-recombination. We hypothesized that if Dnmt3a's DNA methylation function is crucial for HSC regulation, Dnmt3a variant HSCs would resemble Dnmt3a^fl/fl. Conversely, if Dnmt3a has non-canonical functions, Dnmt3a variant HSCs may phenocopy Dnmt3a^+/+ or Dnmt3a^fl/+ HSCs. In competitive transplantation, Dnmt3a variant HSCs showed similar impairment of peripheral blood chimerism as Dnmt3a^fl/fl HSCs, suggesting Dnmt3a-mediated DNA methylation is important for HSC differentiation. However, clonal expansion of Dnmt3a^fl/fl HSCs (fl/fl: 32330±4758, E752A: 16984±2616) was rescued by Dnmt3a protein deficient for DNA methyltransferase activity. Despite the dramatically different phenotypes, the DNA methylation profiles of Dnmt3a^E752A and Dnmt3a^fl/fl HSCs were essentially overlapping, indicating DNA methylation profile is not predictive of HSC functional potential. The enhanced self-renewal of HSCs following loss of Dnmt3a appears independent of DNA methyltransferase function, implying this protein has other important regulatory roles dictating HSC fate.

Considering possible non-canonical functions of Dnmt3a, we revisited our finding that Dnmt3a-null HSCs can propagate in vivo indefinitely. This observation suggests that Dnmt3a loss circumvents inherent mechanisms that normally constrain longevity of HSCs, such as telomere erosion. We hypothesized that Dnmt3a might serve as a previously unrecognized regulator of telomeres as a non-canonical function that regulates HSC function. Dnmt3a-null BM showed longer telomere length over serial transplantation and increased telomerase activity compared to control cells. Human embryonic stem (ES) cells engineered for DNMT3A loss-of-function also showed increased telomerase expression and activity when subject to hematopoietic differentiation. To examine the effects of Dnmt3a loss-of-function on telomeres in a model that facilitates telomere shortening, we crossed Vav-Cre:Dnmt3a^fl/fl mice to Terc knockout mice to generate Vav-Cre:Dnmt3a^fl/fl;Terc^-/- (=DT^KO). Deleting Dnmt3a in a Terc-null background mitigated some of the self-renewal defects of telomerase-deficient HSCs, though they were still reduced compared to Dnmt3a^KO HSCs. Thus, while loss of Dnmt3a could partially rescue the function of Terc-deficient HSCs, telomerase activity is required to sustain the clonal expansion of Dnmt3a^KO HSCs. However, DT^KO BM cells showed retention of telomere length in quaternary transplants, suggesting that in the absence of Dnmt3a, a telomerase-independent mechanism of telomere maintenance is activated. Dysfunctional telomeres are identified as DNA double-strand breaks and are targeted by the DNA damage response (DDR). We observed significant levels of gH2AX in late passage transplant Terc^KO BM, which was rescued by loss of Dnmt3a. There was a substantial increase in telomere dysfunction-induced foci (TIF) in Terc^KO HSCs, which was rescued by the loss of Dnmt3a. Our findings suggest that in Dnmt3a deficient BM cells, the induction of DDR upon telomere shortening is suppressed, directly influencing repopulation capacity. Overall, our study provides the first evidence of DNA methylation-independent function of Dnmt3a in HSC and its role in regulating DDR at sites of dysfunctional telomeres.

Parsons:Silence Therapeutics: Consultancy. Young:Pairidex Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder. Challen:Pairidex Inc.: Current equity holder in private company, Other: Scientific Advisory Board; Incyte: Consultancy, Research Funding; Ajax Therapeutics: Consultancy, Research Funding; ReNAgade Therapeutics Management: Consultancy, Research Funding.