Age-related clonal hematopoiesis (ARCH) is associated with aging and can easily lead to malignant hematological or cardiovascular disease. DNA (cytosine-5)-methyltransferase 3A (DNMT3A) fulfils a variety of different functions during the aging process and is frequently mutated in ARCH and subsequent hematological malignancies. However, the specific molecular mechanism by which DNMT3A influences ARCH is unclear. This project first confirmed that DNMT3A mutations in hematopoietic stem and progenitor cells (HSPCs) were present in clinical samples from a large cohort of healthy volunteers. Secondly, we constructed a Dnmt3a R878H knock-in mouse model and found that Dnmt3a R878H mutation could induce hematopoietic cloning abnormalities in mice over a year later, mainly manifested in the active proliferation of HSPCs, deviations in the functional differentiation trajectory, and micro-environment disorder of immunity. Then, we applied high-throughput chromosomal capture (Hi-C) and single-cell RNA sequencing to explore the effects of aging-associated DNMT3A mutations in HSPCs, together with chromosome spatial structure change and transcriptional regulation and an in-depth investigation of the downstream molecular effects of the DNMT3A mutation on ARCH and the regulation of homeostasis and associated factors in immune cells, providing a new foundation for the early diagnosis and prevention of blood-related diseases induced by clonal hematopoiesis.
Single-cell sequencing has suggested the existence of significant heterogeneity among hematopoietic stem cells, and that Dnmt3a mutations can regulate histone methylation modifications in hematopoietic stem and progenitor cells. Hi-C sequencing has revealed that in hematopoietic stem and progenitor cells, the overall chromatin conformation undergoes relatively subtle changes. However, closer examination of the inter-chromosomal interactions shows that the primary alterations occur between chromosomes 12 and 19. Utilizing advanced bioinformatic techniques to analyze differential Topologically Associating Domains (TADs) and chromatin loops, we identified significant differences in the spatial organization strength located specifically on chromosome 19. This finding suggests that, despite the overall stability of the chromatin landscape, specific regions such as those on chromosome 19 undergo more pronounced structural rearrangements, potentially impacting gene regulation and cellular function in these critical hematopoietic cell populations.
Combining the preliminary Hi-C analysis results from ARCH mice, we identified Apobec3 as one of the significantly dysregulated genes following Dnmt3a mutation. Given the established roles of Apobec3 in DNA repair and RNA editing, our RNA sequencing GSEA analysis in mice suggests that the DNA damage repair pathway is markedly enhanced in Dnmt3a-mutated hematopoietic stem and progenitor cells as well as granulomonocytic cells. This indicates that Dnmt3a mutation may induce increased DNA damage, subsequently activating the DNA repair pathway. Within the DNA repair-related gene clusters, we observed that the expression of Atm gene and protein is significantly lower in mutant mice compared to wild-type mice, suggesting a partial blockade of cellular repair function in mutant mice.
Further validation using patient bone marrow cells and 293 cells overexpressing mutated DNMT3A and APOBEC3F revealed that, among the DNA repair-related gene clusters, all genes except ATM showed increased expression, while ATM expression was notably decreased. Additionally, comet assay studies demonstrated a pronounced tailing phenomenon in cells harboring the mutation. Exome sequencing of mice revealed that with enhanced Apobec gene expression in mutant mice, there was a significant increase in the number of copy number variations (CNVs) and single nucleotide variations (SNVs).
Collectively, these results suggest that Dnmt3a mutation can induce DNA/RNA damage through Apobec3, leading to secondary genetic mutations by interfering with the cellular repair machinery. The compromised repair capacity in turn facilitates the accumulation of additional genetic alterations, underscoring the complex interplay between epigenetic regulators, DNA damage, and repair pathways in the context of hematopoietic malignancies.
No relevant conflicts of interest to declare.
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