Dnmt3a is one of the major de novo methylation enzymes required for proper mammalian embryogenesis and brain development (Chen et al. 2003
; Okano et al. 1999
). During neurogenesis, Dnmt3a protein is strongly expressed in neural precursor cells, postmitotic CNS neurons, and oligodendrocytes (Feng et al. 2005
). Previous studies in Dnmt3a−/−
mice brain showed impaired postnatal neurogenesis at two neurogenic zones, including subependymal/subventricular zones (SEZ/SVZ) in the hippocampal dentate gyrus. Further, Dnmt3a mutant mice had fewer Tuj1 positive neurons and more glial cells compared to WT mice (Wu et al. 2010
). These evidences indicate Dnmt3a is an important regulator in neurogenesis and gliogenesis. In this study, we were interested in whether Dnmt3a-deficient neural differentiation can be modeled in vitro
. Our results found that Dnmt3a-deficient embryonic stem cells derived mNSCs showed a substantially greater number of both astrocytes and oligodendrocytes compared to WT cells, suggesting loss of Dnmt3a results in precocious glial cells maturation. Precocious differentiation in Dnmt3a-deficient NSCs appeared to be more robust in the P6 passage, coincident with the onset of gliogenic activity in wild-type NSCs. Thus, Dnmt3a-deficiency in early passage NSCs show more attenuated differentiation and proliferation phenotypes, raising the possibility that other epigenetic events must occur to facilitate more robust precocious differentiation in NSCs in the absence of Dnmt3a. However, neuronal differentiation was not impaired, though it is still unknown whether these Dnmt3a neurons have impaired or altered function. Furthermore, we were able to rescue the Dnmt3a expression in both Dnmt3a−/−
ESC and NSC stages. However, we did notice the effects of our transient rescue in the NSC stage was less pronounced compared to the stable rescue in the ESC stage. One possibility is that Dnmt3a virus is too toxic for non-infected Dnmt3a−/−
mNSCs. Another possibility is that since we performed transient rescue in late passage NSCs, the hypomethylated DNA methylation patterns established in early passage NSCs may already have been permissive for glial differentiation.
Our current study demonstrates DNA methylation is required for proper neural differentiation. Unlike Dnmt1-deficiency, Dnmt3a-deficiency results in mild genome-wide hypomethylation, and can produce more precocious glial cells. This may be due to increased survival of Dnmt3a-deficient NSCs compared Dnmt1-deficient NSCs. Our previous study showed conditional Dnmt1 deletion in NSCs results in precocious astrocyte differentiation (Fan et al. 2005
), though most cells cannot be maintained over continued passage. Loss of Dnmt1 results in severe global hypomethylation, which dramatically reduces cell survival in culture (Fan et al. 2001
). Dnmt1 conditional knockout mouse showed visibly smaller olfactory bulbs (OB) than WT mice (Fan et al. 2001
). Interestingly, Dnmt3a−/−
mice OB size and the number of newborn neurons resembled Dnmt1-KO mice (Wu et al. 2010
). Finally, in the most extreme case, triple-knockout (TKO) mESCs lines lacking all three of DNA methyltransferases cannot be induced toward neural lineage cells and undergo apoptosis upon differentiation (Tsumura et al. 2006
The role of Dnmt3a in cell proliferation can be seen in different human cancers. For example, Dnmt3a mutations in acute myeloid leukemia (AML) has been recently reported by three independent groups (Ley et al. 2010
; Yamashita et al. 2010
; Yan et al. 2011
). These mutations led to select genome hypomethylation (Ley et al. 2010
) and gene de-regulation (Yan et al. 2011
). In addition, other components of the DNA methylation pathway are also found in AML cancers. For example, somatic mutation of TET2, which convert 5-methycytosine to 5-hydroxymethycytosine, was also found in AML individuals (Figueroa et al. 2010
). However, unlike the hematopoietic stem cell system, where DNA methyltransferases were found to be essential for self-renewal but not differentiation, (Tadokoro et al. 2007
) we demonstrated that Dnmt3a regulated both cell self-renewal and differentiation activities in the neural lineage. Interestingly, Dnmt3a deficiency did not impact cell proliferation in embryonic stem cells stage, perhaps due to compensation from the highly expressed Dnmt3b. Together, these data suggests Dnmt3a behave differently and have distinct roles in different cell lineages.
Mutations in Dnmt3a have been identified in other cancer types. Most relevant, studies in glioblastoma cell lines showed an association with decreased Dnmt3a expression and hypomethylation of satellite repeats at pericentromeric regions (Caprodossi et al. 2007). Intriguingly, ectopic expression of Dnmt3a in glioblastoma cell lines can partially rescue repeat hypomethylation. Overall, these results are consistent with the cell proliferation results in our mNSCs differentiation. Our studies implicate a role for the p53 tumor suppressor pathway that contributes to altered cell proliferation. Previous studies have shown that DNA hypomethylation led to chromosomal instability and tumorogenesis (Eden et al. Science. 2003). Thus, p53 pathway may be downregulated as a consequence of hypomethylation in Dnmt3a-deficient cells. Moreover, the effect of cell proliferation might be achieved by cooperation of multiple factors, including abnormal genes mutations (NPM1, FLT3, and UHRF1/2, etc.), epigenetic modifications (Dnmt1/3b, histone methylation/deacetylation and microRNA regulation. etc), and cytokines induction during differentiation. However, how Dnmt3a regulates cell proliferation and apoptosis still need to be addressed in future work.
In many neural trauma and neural degenerative diseases, neural cell transplantation is becoming an increasingly attractive alternative therapy for patients’ treatment. However, one major hurdle to overcome is our inability to control cellular properties of cells once transplanted into human body; these properties include incorporation of neural cells into the neural network and cell proliferation. Dnmt3a may be a critical regulator of cell activities after transplantation in light of the results of this study. DNA methylation is considered to play an important role in graft survival process. For example, recent histone deacetylase inhibitor drug therapy introduced in spinal injury mouse model showed enhanced improvement limb function (Abematsu et al. 2010
). These findings are encouraging and implicate the use of other epigenetic drugs for enhanced transplantation therapy. Our studies will pave the way for clinical application of cell transplantation, like spinal cord injury, stroke, and other CNS trauma.