Tadokoro Yuko, Ema Hideo, Okano Masaki, Li En, Nakauchi Hiromitsu
Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
J Exp Med. 2007 Apr 16;204(4):715-22. doi: 10.1084/jem.20060750. Epub 2007 Apr 9.
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.
DNA甲基化是一种对发育至关重要的表观遗传修饰。DNA甲基转移酶Dnmt3a和Dnmt3b在原肠胚形成期胚胎和分化中的生殖系细胞中执行从头DNA甲基化。人们一直认为这些酶通常在调节细胞分化中发挥作用。为了验证这一假设，我们研究了Dnmt3a和Dnmt3b在成体干细胞中的作用。CD34(-/低)、c-Kit(+)、Sca-1(+)、谱系标志物(-)(CD34(-)KSL)细胞，即小鼠骨髓细胞中高度富集造血干细胞(HSCs)的一部分，同时表达Dnmt3a和Dnmt3b。利用逆转录病毒Cre基因转导，我们在从这些基因的功能域两侧带有两个loxP位点的小鼠中纯化得到的CD34(-)KSL细胞中，有条件地破坏Dnmt3a、Dnmt3b或Dnmt3a和Dnmt3b两者(Dnmt3a/Dnmt3b)。我们发现Dnmt3a和Dnmt3b在造血细胞分化过程中作为从头DNA甲基转移酶发挥作用。出乎意料的是，体外集落测定和体内移植测定表明，Dnmt3a、Dnmt3b和Dnmt3a/Dnmt3b缺陷的造血干细胞中髓系和淋巴系分化潜能均得以维持。然而，Dnmt3a/Dnmt3b缺陷的造血干细胞，而非Dnmt3a或Dnmt3b缺陷的造血干细胞，在移植测定中无法进行长期重建。这些发现确立了Dnmt3a和Dnmt3b介导的DNA甲基化在造血干细胞自我更新中的关键作用。
