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. 2020 Apr 14;14(4):551-560.
doi: 10.1016/j.stemcr.2020.02.011. Epub 2020 Mar 26.

Divergent Effects of Dnmt3a and Tet2 Mutations on Hematopoietic Progenitor Cell Fitness

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Free PMC article

Divergent Effects of Dnmt3a and Tet2 Mutations on Hematopoietic Progenitor Cell Fitness

Elizabeth L Ostrander et al. Stem Cell Reports. .
Free PMC article

Abstract

The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt3ITD) than loss of Dnmt3a, which is associated with a more rapid expansion of committed progenitor cells. The effect of Tet2 mutation manifests more profound myeloid lineage skewing in committed hematopoietic progenitor cells rather than long-term HSCs. Molecular characterization revealed divergent transcriptomes and chromatin accessibility underlying these functional differences.

Keywords: DNMT3A; TET2; clonal hematopoiesis; hematopoietic stem cell.

Figures

Figure 1
Figure 1
Loss of Dnmt3a and Tet2 Enhance Self-Renewal in HSCs to Different Degrees (A) HSC serial transplantation schematic. In descending column order—contribution of 200 ControlMx1, Dnmt3a-KOMx1 (3aKO), and Tet2-KOMx1 (T2KO) HSCs to peripheral blood, lineage chimerism, HSC frequency, and HSC number in (B) primary (CNT n = 28; 3aKO n = 24; T2KO n = 22), (C) secondary (CNT n = 27; 3aKO n = 19; T2KO n = 21), and (D) tertiary (CNT n = 33; 3aKO n = 23; T2KO n = 19) transplants. (E) Self-renewal and (F) differentiation quotients of indicated HSC genotypes after each transplant. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001. Mean ± SEM is shown.
Figure 2
Figure 2
Tet2 and Dnmt3a Loss of Function Divergently Influence Rate of Transformation from Same Co-operating Mutation (A) Kaplan-Meier plot comparing time to morbidity between ControlVav (n = 30), Flt3ITD (n = 20), Dnmt3a-KOVav (n = 15), Tet2-KOVav (n = 11), Dnmt3a-KOVavFlt3ITD (n = 16), and Tet2-KOVavFlt3ITD (n = 12) mice. (B) White blood cell count of day 600 ControlVav and moribund mice of indicated genotypes. (C) Pathological diagnosis of moribund mice. (D) Representative flow cytometry plots of moribund mice demonstrating expansion of MPP3 (red box) and depletion of HSCs (purple box) in Flt3ITD genotypes. (E) Frequency and number of HSCs and MPP3 in moribund mice. (F–J) (F) Frequency and number of HSCs and MPP3 in 8-week-old ControlVav (n = 18), Flt3ITD (n = 14), Dnmt3a-KOVav (n = 18), Dnmt3a-KOVavFlt3ITD (n = 10), Tet2-KOVav (n = 15), and Tet2-KOVavFlt3ITD (n = 9) mice. Pathological analysis of young adult mice showing (G) WBC counts, (H) peripheral blood myeloid cells, (I) spleen weights, and (J) spleen myeloid cells. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001. Mean ± SEM is shown.
Figure 3
Figure 3
Tet2 Mutation Does Not Impart Ectopic Self-Renewal to Hematopoietic Progenitors, but Skews Myeloid Differentiation of Committed Progenitor Cells (A) Donor-derived peripheral blood cells and 16-week lineage chimerism in recipients of 200 MPP1 from ControlVav (n = 8), Dnmt3a-KOVav (n = 7), and Tet2-KOVav (n = 5) mice. (B) Frequency of MPP1-transplanted mice with >1% donor-derived engraftment in myeloid, B cell, and T cell lineages. (C) Representative plots showing donor-derived MPP1 and HSCs in recipients of Dnmt3a-KOVav MPP1. (D) Frequency and chimerism of donor-derived HSCs and MPP1 in recipients of 200 MPP1. (E) Representative immunophenotyping of in vitro differentiated progenitor cells. (F) Immunophenotypic populations produced via in vitro differentiation of progenitor cells from ControlVav and Tet2-KOVav mice (n = 4 per population of each genotype). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001. Mean ± SEM is shown.
Figure 4
Figure 4
Dnmt3a and Tet2 Loss of Function Alter Hematopoietic Progenitor Function through Distinct Molecular Mechanisms (A) Principal-component analysis plot of gene expression in ControlVav HSCs (n = 3) and MPP3 (n = 4), Dnmt3a-KOVav HSCs (n = 3), and Tet2-KOVav HSCs (n = 4) and MPP3 (n = 4). (B) Venn diagrams displaying DEG overlap in Dnmt3a-KOVav and Tet2-KOVav HSCs compared with ControlVav HSCs. (C) Heatmaps displaying DEGs (p < 0.05, fold-change >1 or <−1) between ControlVav and Tet2-KOVav HSCs and MPP3. (D) Gene set enrichment analysis showing differentially regulated pathways between ControlVav and Tet2-KOVav MPP3. (E) Gene score enrichment plot of “Hallmark TNFα Signaling via NFkB” gene set in Tet2-KOVav MPP3. (F) ATAC-seq heatmaps from ControlVav, Dnmt3a-KOVav, and Tet2-KOVav mice. Signals displayed are peaks 1 kb up- and downstream of transcription start sites of protein coding genes. (G) Multi-dimensional scaling plot with distances approximating the largest log2 fold-changes in the top 500 peaks between ATAC-seq samples.

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