Here, we have examined effects of conditional deletion of Dnmt1 on the hematopoietic system. Mature hematopoietic lineages were unaffected by loss of Dnmt1, however, this enzyme was required for HSC self-renewal, niche retention and differentiation. In addition, Dnmt1 deletion resulted in increased cycling and differentiation of the myeloid restricted progenitor pool. DNA methylation and global gene expression analyses revealed that Dnmt1 regulates distinct patterns of methylation and gene expression in HSCs, multipotent progenitors and lineage-restricted progenitors.
Mature cells of the hematopoietic system were found to be stable following deletion of Dnmt1, however, we observed impaired hematopoiesis when Dnmt1 was deleted following transplantation and when WT cells were transplanted into Dnmt1 deficient recipients. There are at least two explanations that may account for this; (1) The timepoint at which we assessed steady state effects (12 weeks post pIpC) may not be long enough to observe modest self-renewal and differentiation defects of HSCs and progenitor cells in the absence of competing WT cells, and (2) the DNA methyltransferases Dnmt3a and 3b may be able to partially compensate for loss of Dnmt1 certain contexts. This is consistent with our observation of increased methylation of the proximal promoter of Car1 in Dnmt1Δ/Δ myeloid restricted progenitors.
The hematopoietic phenotype observed following Dnmt1 deletion is complex and appears to involve most of the primitive hematopoietic compartment including HSCs, multipotent and myeloid lineage restricted progenitor cells. Defects in HSC engraftment are accounted for by decreased niche retention, decreased self-renewal capacity and differentiation defects. The enhanced proliferative activity of Dnmt1 deficient myeloid progenitors coupled with differentiation indicates stress on the system to produce more mature myeloid cells. While all of these functional consequences of Dnmt1 deletion will certainly compound and contribute to one another in an in vivo setting, it was interesting to see the wide variety of gene expression changes and different methylation patterns following acute loss of Dnmt1 in LT-HSCs, ST-HSC/MPPs and myeloid progenitors. Within the ST-HSC/MPP population, in the case of both IAP and Car1, loss of methylation was associated with activation of gene expression. It is possible then that the ST-HSC/MPP population is most directly regulated by Dnmt1 or by loss of methylation maintained by Dnmt1, as opposed to the LT-HSC or myeloid progenitor populations. Comprehensive methylation profiling in the presence and absence of Dnmt1, Dnmt3a and Dnmt3b, coupled with global gene expression analysis, will provide a means to systematically classify gene loci that are regulated by the various Dnmts in hematopoietic stem and progenitor populations.
Certainly, our evidence suggests that epigenetic regulation, at least with respect to DNA methylation, of adult stem cells is distinct from embryonic stem cells and other somatic cell types. For example, there are clear distinctions between functional roles of de novo
methylation and maintenance methylation. De novo
methylation induced by Dnmt3a and Dnmt3b is necessary for LT-HSC self-renewal but not differentiation (Tadokoro et al., 2007
). In contrast, we have observed that maintenance methylation induced by Dnmt1 is especially important for HSC and progenitor cell state transitions, such as the stepwise differentiation of HSCs to ST-HSC/MPPs, ST-HSC/MPPs to myeloid progenitors, mobilization, and regulating cell cycle entry. We suggest that these unique roles of Dnmt1 may be a common mechanism by which maintenance methylation regulates cell state transitions of adult somatic stem cells.