DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells

Abstract

DNA methylation is essential for development and in diverse biological processes. The DNA methyltransferase Dnmt1 maintains parental cell methylation patterns on daughter DNA strands in mitotic cells; however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. Defects were observed in Dnmt1-deficient HSC self-renewal, niche retention, and in the ability of Dnmt1-deficient HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had specific impact on myeloid progenitor cells, causing enhanced cell cycling and inappropriate expression of mature lineage genes. Dnmt1 regulates distinct patterns of methylation and expression of discrete gene families in long-term HSCs and multipotent and lineage-restricted progenitors, suggesting that Dnmt1 differentially controls these populations. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment.

Figure 1. Conditional deletion of Dnmt1 results in primitive hematopoietic cell defects

(A) Experimental scheme. (B) Genomic PCR from control (Dnmtfl/fl Cre⁻) or Dnmt1^Δ/Δ (Dnmtfl/fl Cre⁺) BM for Dnmt1 and (β-actin. (C) Western blot analysis of Dnmt1 in control and Dnmt1^Δ/Δ BM before and 6 weeks after pIpC. (D) Differential PB counts in control and Dnmt1^Δ/Δ mice before and after pIpC. Data expressed as mean±SD; n≥3 mice per genotype. (E) Average PB chimerism of control or Dnmt1^Δ/Δ BM cells after transplant into lethally irradiated recipients. Data expressed as mean±SD; n=4 recipients per group; *p<0.05, **p<0.01. (F) Average BM chimerism of control or Dnmt1^Δ/Δ cells at 20 weeks post-transplant. Data expressed as mean+SD; n=4 recipients per group; *p<0.05, **p<0.01. (g) Average BM chimerism within secondary transplant recipients at 16 weeks post-transplant. Data expressed as mean+SD; n=3 recipients per group; *p<0.05, **p<0.01.

Figure 2. Dnmt1 is necessary for HSC self-renewal

(A) Experimental scheme. (B) Average PB chimerism of control or Dnmt1^Δ/Δ BM cells following competitive transplantation into lethally irradiated recipients. Data expressed as mean±SD; n=3 recipients per group; **p<0.01, ***p<0.001. (C) Average BM chimerism of control or Dnmt1^Δ/Δ cells at 20 weeks post-transplant. Data expressed as mean+SD; n=3 recipients per genotype; **p<0.01. (D) PB chimerism of control or Dnmt1^Δ/Δ BM cells in individual, mature hematopoietic lineages in primary transplant recipients. Data points represent individual mice; *p<0.05, **p<0.01. (E) PB chimerism following secondary competitive transplantation. Data points represent individual mice.

Figure 3. Dnmt1 deletion results in BM niche retention defects

(A) Representative FACS plots of recipient BM at 16 hours post-transplant of CFSE-labeled control or Dnmt1^Δ/Δ Lin⁻ Sca-1⁺ BM cells. Data expressed as mean±SEM; n=3 recipient mice per genotype. (B) Experimental scheme. (C) Representative FACS plots of BM from control or Dnmt1^Δ/Δ mice (CD45.2⁺) at 12 weeks post non-ablative transplant of WT BM or LT-HSCs (CD45.1⁺CD45.2⁺). A mock-injected recipient is shown as a control for background staining. (D) Average number of LT-HSCs and ST-HSC/MPPs in control or Dnmt1^Δ/Δ BM (femurs+tibiae). Data expressed as mean+SD; n=3 per genotype. (E) Heat map of HSC self-renewal and mobilization-related genes demonstrating fold expression changes (log₂) in Dnmt1^Δ/Δ LT-HSCs, ST-HSC/MPPs and myeloid progenitors compared to control.

Figure 4. Dnmt1 deletion impacts cycling and transcription of myeloid progenitors

(A) Total number of colony-forming units (CFU) generated from 5000 control or Dnmt1^Δ/Δ LT-HSCs or ST-HSC/MPPs; n=3 mice per genotype; ***p<0.0001. (B) Frequency of CMPs, GMPs, and MEPs in lineage-depleted control or Dnmt1^Δ/Δ BM; n=3 per genotype; *p<0.05, **p<0.01. (C) Representative FACS plot of BM cells in G0/G1, S or G2/M cell cycle phases based on incorporation of BrdU and 7AAD. (D) Average frequency of control or Dnmt1^Δ/Δ CMPs, GMPs and MEPs in S phase. Data expressed as mean+SD; n=3 mice per genotype; *p<0.05, **p<0.01. (E) Real-time PCR analysis of CyclinD1 in control or Dnmt1^Δ/Δ myeloid progenitors. Data expressed as mean+SD; n=3 mice per genotype. (F) GO analysis of immunoglobulin-related genes upregulated in Dnmt1^Δ/Δ LT-HSCs, ST-HSC/MPPs and myeloid progenitors. GO terms with a p value less than 1e⁻⁴ are considered significantly enriched and indicated by asterisks. (G) Real-time PCR analysis of H2-D1, H2-T22 and CD81 transcripts in control or Dnmt1^Δ/Δ myeloid progenitors. Data expressed as mean+SD; n=3 mice per genotype.