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

ABSTRACT: Dnmt3a is the most recurrently mutated gene in clonal hematopoiesis (CH) and it is a critical regulator of hematopoietic stem cells (HSCs). Conditional deletion of Dnmt3a in mouse HSC results in enhanced self-renewal but impaired differnetiation. Dnmt3a encodes for a de novo DNA methyltransferase enzyme but both mouse and human cells with loss of Dnmt3a show minimal change in DNA methylation levels which do not correlate with gene expression differences. To understand if these changes are due to non-canonical function of Dnmt3a, we generated varying levels of DNA methyaltion-impared Dnmt3a mouse models. Our data depicts that DNA methyaltion-impaired Dnmt3a phenocopy wild-type in serial transplant. And Dnmt3a methyltransferase-deficient HSCs showed differntially methylated regions (DMRs) and gene expression pattern that overlapped with Dnmt3a-null HScs. Suggesting that Dnmt3a has important non-canonical function that partially regulates HSC fate. Dnmt3a-null HSC increased HSC self-renwal and can be transplanted indefinitely and increase longevity with no erosion of telomere length. Dnmt3a-null HSCs showed increased telomere at baseline and maintained overa serial transplant and increased telomerase activity. The role of Dnmt3a in telomere maintenance was not strictly linked to elongated telomere length but in regulation of DNA damage response that occurs at stressed telomeres. These data show a unidentified role of Dnmt3a in HSC telomere maintenance that is note related to DNA methylation function.

ORGANISM(S): Mus musculus

PROVIDER: GSE272913 | GEO | 2024/09/10

REPOSITORIES: GEO

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Project description:Hematopoietic stem cells (HSCs) with certain somatic mutations, most commonly in the DNA methyltransferase DNMT3A, gain a clonal growth advantage leading to the development of clonal hematopoiesis (CH). The distinct functional differences that allow DNMT3A-mutant HSCs to gain a fitness advantage and outcompete wild-type HSC in the context of aging are not fully elucidated. We recently discovered that HSC aging is initiated by decline in local production of insulin-like growth factor 1 (IGF1). Here, we used a mouse model of DNMT3A-mutant CH (Dnmt3aR878H/+) to investigate the extent to which decline in IGF1 alters the selective advantage of Dnmt3aR878H/+ HSCs. Upon transplant into IGF1-deficient recipient mice, Dnmt3aR878H/+ HSCs gained enhanced selective advantage over wild-type HSCs and maintained lineage balanced blood production. As IGF1/mTOR signaling is well understood to regulate energy metabolism, we investigated underlying metabolic differences between Dnmt3aR878H/+ and wild-type HSCs. Dnmt3aR878H/+ HSPCs had similar glycolytic capacity as wild-type HSCs but enhanced mitochondrial reserve capacity and mitochondrial activation potential. To evaluate whether mitochondrial function is a targetable dependency of Dnmt3aR878H/+ HSCs, we administered the mitochondrial-targeted molecule MitoQ resulting in the depletion of their mitochondrial reserve capacity. We find that MitoQ reduces the competitive advantage of Dnmt3aR878H/+ hematopoiesis. To identify the mechanism(s) by which MitoQ alters Dnmt3aR878H/+ phenotypic expansion we evaluated transcriptional changes after MitoQ treatment and find altered response to Igf1/mTOR signaling compared to wild-type HSC. The altered response to Igf1/mTOR signaling in part mediates the Dnmt3aR878H/+ hematopoietic selective advantage. Taken together, our work supports that mitochondrial metabolic regulation is a key mechanism by which DNMT3A-mutant HSCs gain a selective advantage. Targeting this mechanism may maintain polyclonal hematopoiesis during aging and reduce the risk of CH-associated disease.

Project description:Since DNMT3A is a de novo DNA methyltransferase, transcriptional differences in WT versus Dnmt3a-/- HSCs upon infection suggest a possible role for epigenetic regulation in the HSC transcriptional response to inflammation. Therefore, we wanted to investigate if differences in stress and presence of Dnmt3a affect the methylome overall, and specific IFNg responses genes. 100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/ Dnmt3-/-_x0001_ mice (n = 7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after ‘‘methylated’’ adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylationlightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/ sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads). Global methylation of Dnmt3a-/- cells was more divergent upon infection compared to WT. There were vastly more hyper- and hypomethylated regions in infected versus naive Dnmt3a-/- HSCs compared to WT. Among the altered regions, both WT and Dnmt3a-/- HSCs showed slightly more hypomethylated than hypermethylated regions, although changes in both directions were present. In the WT background, regions of hyper- and hypomethylation were mostly restricted to CpG islands. By contrast, highly significant differences in hypomethylation were found in the Dnmt3a-/- HSCs in CpG islands, shores, enhancers, and promoters DNMT3A is highly active in HSCs during infection and that the loss of DNMT3A function results in significant global changes in methylation, including both hyper- and hypomethylation, in genome regions that likely affect gene transcription. we examined the methylation of Batf2, Jun, and Fos. All 3 of these prodifferentiation genes showed increased methylation in the promoter region in the Dnmt3a-/- background as compared to the WT background. Hypermethylation of these promoter regions is consistent with their transcriptional repression.

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

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