Abstract
Clonal hematopoiesis (CH), the clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs), is highly prevalent in aging populations and is linked to age-related diseases such as cardiovascular disease and malignancy (Jaiswal et al., N. Engl. J. Med., 2014, 2017). DNA methylation regulators, Ten-Eleven Translocation 2 (TET2) and DNA methyltransferase 3A (DNMT3A), are among the most frequently mutated genes in CH (Genovese et al., N. Engl. J. Med. 2014). Recent studies report the essential role of TET2/DNMT3A in regulating DNA methylation in euchromatin particularly in the regulation of key genes involved in hematopoietic stem cell function and lineage specification. DNA methylation also maintains heterochromatin by silencing transposable elements (TEs). Our recent study reported a distinct role for DNMT3A and TET2 in regulating the spatial relocation of H3K9me3-marked heterochromatin during HSPC aging, which contributes to age-related CH (Hong et al., Nat. Aging, 2023). Our data suggested the upregulation of TEs correlated with the activation of interferon-stimulated genes (ISGs) and contributed to the functional decline of aged HSPCs. This decline was mitigated in TET2-mutant HSPCs, increasing their competitive capacity. However, given HSPC heterogeneity, the cell type specific TEs alterations during aging and in the pathogenesis of CH remain largely unknown.
To further delineate the transcriptional and epigenetic regulation of TEs during aging, we purified Lin- HSPCs from C57BL/6 wild-type (WT) and TET2-knockout (TET2-KO) mice at 7 weeks (7w), 21 months (21m), and 30 months (30m) of age. We then performed single-cell (sc) RNA-seq and scATAC-seq. After preprocessing, cell type labeling, and integration of both scRNA and scATAC datasets, we quantified TE expression and accessibility at both the locus and family levels using MATES, an autoencoder-based methodology (Wang et al., Nat. Commun., 2024). We compared WT and TET2-KO aging by integrative scRNA/scATAC analysis of lineage biases and regulon activities.
Coding-gene and TE expression profiles independently resolved the same HSPC progenitor subpopulations, indicating cell type specific TE expression. This strongly suggested the tissue specific regulation of TEs, consistent with previous publications regarding embryonic development (Miao, et al, Genome Biol 2020). Furthermore, scATAC-seq analysis revealed similar subpopulation specific TE trends, including distinct, smooth trajectories of TE accessibility along myeloid, lymphoid, and megakaryocyte-erythroid lineages. Integrating our 7w and 21m WT scRNA-seq and scATAC-seq, we found that age-dependent gains in TE accessibility and transcription varied across all major progenitor subpopulations. These age-dependent changes in TE accessibility and transcription occurred in all major classes of TEs including LINEs, SINEs and LTRs, but the changes were mitigated in TET2 deficient cells, strongly suggesting distinct regulatory mechanisms in TE during TET2 deficient HSPCs aging. The age-dependent changes in HSCs were strongly exemplified by many TE elements at the subfamily and locus levels in groups such as the MurERV40int and RLTR10 from the ERV class. Furthermore, we revealed a positive correlation between TE expression and inflammatory genes, suggesting the potential involvement of TEs in regulating intracellular innate immune response during aging. In addition to modulating the age-related changes in TEs, loss of TET2 mitigated age-associated chromatin opening and expression of age-related genes such as Stxbp4 and S100a6 (Svendsen et al., Blood, 2021). Additionally, a regulon-based analysis showed age-related decreases in regulons associated with HSC function and maintenance such as Meis1 (Kocabas, et al. Blood, 2012), this age-related decrease was mitigated in TET2-KO. Parallel analysis of aging human HSPC datasets uncovered similar gene and TE activation patterns, suggesting a conserved mechanism (Hua et al., Blood, 2019).
Beyond extrinsic inflammation, age-related TE upregulation may trigger innate immune signaling that impairs HSPC self-renewal and specification. Our multi-omics approach elucidates the epigenetic and transcriptional mechanisms underlying TE dysregulation in aging HSPCs. Our study provides new insights into the molecular underpinnings of age-related hematopoietic dysfunction and identified potential therapeutic targets for mitigating CH and associated diseases.
