De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells - PubMed (original) (raw)

De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells

Yuko Tadokoro et al. J Exp Med. 2007.

Abstract

DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.

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Figures

Figure 1.

Figure 1.

Expression of Dnmt3a and Dnmt3b in hematopoietic cells. (A) Expression of Dnmt1, Dnmt3a, Dnmt3a2, Dnmt3b, Dnmt3L, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was examined by semiquantitative RT-PCR analysis. cDNAs were prepared from CD34− KSL cells, CD34+ KSL cells, lineage marker− cells, Gr-1+ Mac-1+ neutrophils/macrophages, TER119+ erythroblasts, and B220+ B lymphoid cells in the BM; from B220+ B lymphoid cells, Thy-1.2+ T lymphoid cells, and Nk-1.1+ NK/NKT cells in the spleen; and from CD4−CD8−, CD4+CD8+, CD4+CD8−, and CD4−CD8+ T lymphoid cells in the thymus of adult B6 mice. (B) Immunostaining detected Dnmt3a (red) and Dnmt3b (green) in the cytoplasm and nucleus (blue) of CD34− KSL cells. Bars, 4 μm.

Figure 2.

Figure 2.

Status of DNA methylation in hematopoietic cells. (A) Status of DNA methylation in hematopoietic cells was investigated by the detection of 5-methyl-cytosine (5-MeC). DNA methylation level was lower in undifferentiated cells and higher in mature hematopoietic cells, especially B and T lymphoid cells. Bars, 10 μm. (B) Status of DNA methylation in hematopoietic cells was confirmed by PCR using HpaII-digested genomic DNA. When PCR products are detectable, the genomic DNA is methylated low.

Figure 3.

Figure 3.

Deletion of Dnmt3a or Dnmt3b in HSCs. (A) A study design is schematically shown. (B) Methylcellulose colony assay was performed for Dnmt3a2lox/2lox or Dnmt3b2lox/2lox CD34− KSL cells after infection with EGFP or Cre-EGFP retrovirus. 4% of the infected cells were cultured for a total of 14 d. Highly proliferative colony-forming cells, defined as cells capable of forming colonies >1 mm in diameter, were counted. Data are given as mean ± SD (n = 3). (C) EGFP+ colonies were assigned colony types on morphological examination of colony cells. (D) Competitive repopulation was performed using Dnmt3a2lox/2lox or Dnmt3b2lox/2lox CD34− KSL cells after infection with EGFP or Cre-EGFP retrovirus. Transduction efficiencies were estimated by counting EGFP+ and EGFP− colonies in each case in B. Engraftment of Ly5.2+ donor cells was assessed by the ratio of the number of mice engrafted with Ly5.2+ cells to the number of recipient mice, and by the percentage of Ly5.2+ cells in peripheral blood (mean ± SD). Contribution of EGFP+ cells to all test donor-derived cells was assessed by the ratio of the number of mice engrafted with EGFP+ cells to the number of mice engrafted with Ly5.2+ cells, the percentage of EGFP+ cells among Ly5.2+ cells, and the percentage of EGFP+ cells among each lineage marker expressing Ly5.2+ cells (mean ± SD). N.D., not detected. (E) Representative genotyping of BM cells reconstituted with Dnmt3a2lox/2lox or Dnmt3b2lox/2lox cells infected with EGFP or Cre-EGFP retrovirus is shown. EGFP− or EGFP+ cells were isolated by flow cytometry from the BM of recipient mice, and their genomic DNA was examined by PCR. (F) BM cells in reconstituted mice were analyzed by flow cytometry. Only Ly5.2+ EGFP+ cells are displayed to show whether the KSL population (red squares) is derived from transduced cells.

Figure 4.

Figure 4.

Deletion of both Dnmt3a and Dnmt3b in HSCs. (A) A methylcellulose colony assay was performed for Dnmt3a2lox/2lox/Dnmt3b2lox/2lox CD34− KSL cells after infection with EGFP or Cre-EGFP retrovirus. 4% of the infected cells were cultured for a total of 14 d. (B) EGFP+ colonies were assigned colony types. (C) Competitive repopulation was performed using Dnmt3a2lox/2lox/Dnmt3b2lox/2lox CD34− KSL cells after infection with EGFP or Cre-EGFP retrovirus. Transduction efficiencies were estimated by counting EGFP+ and EGFP− colonies in each case in A. N.D., not detected. (D) Genotyping of peripheral blood cells transiently reconstituted with Dnmt3a2lox/2lox/Dnmt3b2lox/2lox cells infected with EGFP or Cre-EGFP retrovirus is shown. EGFP− and EGFP+ cells were isolated by flow cytometry from the peripheral blood of recipient mice, and their genomic DNA was examined by PCR. In the recipient mice transplanted with Cre-EGFP–transduced Dnmt3a2lox/2lox/Dnmt3b2lox/2lox CD34− KSL cells, Ly5.2+/EGFP+ peripheral blood cells were detected in 8 of 10 mice. In Cre-EGFP–transduced Dnmt3a2lox/2lox/Dnmt3b2lox/2lox cells, Dnmt3a1lox/1lox/Dnmt3b1lox/1lox mature cells were detected in five (red, +) of eight recipient mice. (E) BM cells in mice transplanted with transduced cells were analyzed by flow cytometry. Only Ly5.2+ EGFP+ cells are displayed to show whether the KSL population (red squares) is derived from transduced cells.

Figure 5.

Figure 5.

DNA methylation status in Dnmt3a1lox/1lox/Dnmt3b1lox/1lox hematopoietic cells. CD34− KSL cells of each type were cultured for 14 d. Cre-EGFP–transduced Dnmt3a2lox/2lox/Dnmt3b2lox/2lox cells showed a low level of DNA methylation compared with Cre-EGFP–transduced wild-type cells. Bars, 20 μm.

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