Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice

Abstract

The Ten-Eleven-Translocation 2 (TET2) gene encodes a member of TET family enzymes that alters the epigenetic status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies, including myelodysplastic syndromes, myeloproliferative neoplasms, and chronic myelomonocytic leukemia. By analyzing mice with targeted disruption of the Tet2 catalytic domain, we show here that Tet2 is a critical regulator of self-renewal and differentiation of hematopoietic stem cells (HSCs). Tet2 deficiency led to decreased genomic levels of 5hmC and augmented the size of the hematopoietic stem/progenitor cell pool in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multilineage reconstitution and possessed a competitive advantage over wild-type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro, Tet2 deficiency delayed HSC differentiation and skewed development toward the monocyte/macrophage lineage. Our data indicate that Tet2 has a critical role in regulating the expansion and function of HSCs, presumably by controlling 5hmC levels at genes important for the self-renewal, proliferation, and differentiation of HSCs.

Fig. 1.

Tet mRNA expression and 5hmC levels after Tet2 deletion in mice. (A) Expression of Tet1, Tet2, and Tet3 in sorted naïve CD4⁺ (CD4⁺CD8⁻CD44^loCD62L⁺) T cells derived from Tet2^+/+ or Tet2^−/− mice. Cells were isolated from pooled spleen and lymph nodes by flow cytometry, and quantitative RT-PCR analysis was performed. The relative levels of Tet1-3 after normalization to the level of Gapdh mRNA in the same cell population are shown, with the amount in the Tet2^+/+ naïve CD4⁺ population arbitrarily set to 1. Error bars show SD, n = 4. (B) Quantification of 5hmC by anti-CMS dot blot. 5hmC levels in bone marrow, spleen, liver, and kidney in the Tet2^+/+ or Tet2^−/− mice were assessed by dot blot assay with anti-CMS antibody after treatment of genomic DNA with bisulfite. A synthetic oligonucleotide with a known amount of CMS was used as standard. Data are representative of two independent experiments.

Fig. 2.

Tet2 regulates the size of the hematopoietic stem/progenitor pool. (A) Flow cytometric analysis of LSK (Lin⁻c-Kit⁺Sca-1⁺) and LK (Lin⁻c-Kit⁺Sca-1⁻) subsets in the bone marrow of 8- to 12-wk-old Tet2^+/+ or Tet2^−/− mice. The frequency of LSK and LK was assessed by discriminating lineage-negative cells (Lineage: Gr-1, Mac-1, Ter-119, CD3ε, and B220) based on the expression of c-Kit and Sca-1. n = 5–6 per group. P, Student's t test. (B) Absolute number of LSK and LK cells calculated in A. Error bars show SD, n = 5–6 per group. Bone marrow cells were flushed from two femurs and two tibias from each 8- to 12-wk-old mouse. (C) Distribution of myeloid progenitor cell subsets was assessed by the expression of FcγRII/III and CD34 within LK population (n = 3 per group). (D) Absolute number of myeloid progenitor cells calculated in C. CMP, common myeloid progenitor, GMP, granulocyte-monocyte progenitor, MEP, megakaryocyte-erythroid progenitor. Error bars show SD, n = 3 per group. P, Student t test.

Fig. 3.

Cell-intrinsic effect of Tet2 deficiency on the size of the hematopoietic stem/progenitor compartment. (A) Flow cytometric analysis of LSK or LK cells in chimeric mice reconstituted with Tet2^+/+ or Tet2^−/− bone marrow cells. CD45.2⁺ donor-derived (Top), lineage-negative (Middle) cells in the bone marrow of chimeric mice were further subclassified based on expression of c-Kit and Sca-1 (Bottom). A representative result at 12 wk after transplantation is shown (n = 3 per group). (B) Absolute number of cells in CD45.2⁺ donor-derived LSK and LK subsets calculated in Fig. 3A and Fig. S4A. Error bars show SD (for 7–8-wk samples) or range of duplicate (for 12-wk samples). n = 3 for 7–8-wk samples; n = 2 for 12-wk samples. P, Student t test.

Fig. 4.

Augmented hematopoietic repopulation capacity upon Tet2 deficiency. (A) Competitive repopulation assay. CD45.2⁺ bone marrow cells from Tet2^+/+ or Tet2^−/− mice were mixed with CD45.1⁺ competitor cells at different ratios as shown and transplanted into lethally irradiated CD45.1⁺ congenic recipients. At 6 or 12 wk after transplantation, peripheral blood was examined for donor/competitor chimerism. A representative result at 12 wk after transplantation is shown. n = 2–4 per group. (B) Percentage of CD45.2⁺ donor chimerism at 6 or 12 wk after transplantation calculated in Fig. 4A and Fig. S5A. n = 2 for Tet2^+/+ in 5:1 group; n = 4 for the other samples. *P < 0.05, **P < 0.005 (Student's t test.) (C) Percentage of CD45.2⁺ donor chimerism in hematopoietic lineages in peripheral blood at 12 wk after transplantation. The percentage of CD45.2⁺ cells was calculated after gating on myeloid (Mac-1⁺) (Left), T-cell (CD3ε⁺) (Center) and B-cell (CD19⁺) (Right) populations. n = 2 for Tet2^+/+ in 5:1 group; n = 4 for the other samples. *P < 0.05, **P < 0.005 (Student's t test).

Fig. 5.

Tet2-deficient hematopoietic stem cells retain sustained progenitor properties in vitro but preferentially undergo monocyte/macrophage differentiation. (A) Effect of Tet2 deficiency on myeloid differentiation. LSK cells isolated from bone marrow of Tet2^+/+ or Tet2^−/− mice were grown in the presence of cytokine mixtures as shown. After 5 d, flow cytometric analysis was performed for the expression of lineage markers (Upper) and Gr-1 and Mac-1, myeloid markers (Lower). Data are representative of at least two independent experiments (n = 3–5). (B) The cells in A were analyzed for their expression of CD115 (M-CSFR) and F4/80, monocyte/macrophage markers (Upper). The CD115⁺F4/80⁺ cells were further assessed for their expression of myeloid markers (Lower). Data are representative of at least two independent experiments (n = 3–5).