The Histone Demethylase Fbxl10/Jhdm1b/Kdm2b Maintains Self-Renewing Hematopoietic Stem Cells.

Abstract 1608

Post-translational modification of histones, including methylation, acetylation, phosphorylation, and ubiquitination, are central to the epigenetic regulation of transcription, replication and repair. Among these histone modifications, histone methylation marks play a major role in the regulation of development, differentiation, and cell fate decision. Histone methylation marks are reversible and dynamically regulated by two antagonizing groups of enzymes that add and remove methyl groups to histone proteins. Recent identification of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. However, their roles in hematopoietic stem cells (HSCs) remain poorly understood. Gene-expression analysis of 26 histone demethylases in various hematopoietic populations from mice demonstrated that Fbxl10 (F-box and leucine-rich repeat protein 10)/Jhdm1b/Kdm2b/Ndy1, which is a histone H3 lysine 4 and lysine 36 demethylase, shows the highest levels of expression in CD34^-c-Kit⁺Sca-1⁺Lineage^- (CD34^-KSL) HSCs and CD34⁺KSL multipotent progenitors, but is markedly down-regulated during differentiation in bone marrow, particularly in TER119⁺ erythroblasts and Gr-1⁺ granulocytes. Based on these data, we hypothesized that Fbxl10 plays a role in the maintenance of self-renewal capacity or multipotency of HSCs. To address this question, we examined the effects of forced expression of Fbxl10 in CD34^-KSL HSCs. CD34^-KSL HSCs were transduced with a GFP control or an Fbxl10 retrovirus and then further incubated in the presence of SCF and TPO. At day 14 of culture, although the cumulative cell numbers of the two groups were comparable, the percentage of KSL cells was higher in the Fbxl10 culture than in the GFP control culture. At day 10 of culture, the Fbxl10 culture contained approximately 2-fold more colony-forming cells (CFCs) than the GFP control culture. In particular, the number of high proliferative potential-CFCs (HPP-CFCs), which are defined as colonies with diameter greater than 1mm, was markedly increased in the Fbxl10 culture. Morphological evaluation of the HPP colonies revealed that the Fbxl10 culture contained approximately 20-fold more colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (nmEM) compared with the GFP control, indicating that forced expression of Fbxl10 in CD34^-KSL HSCs expands colony-forming cells with multi-lineage differentiation potential during ex vivo culture. Next, we performed competitive repopulation assays using 10-day ex vivo cultured cells corresponding to 30 initial CD34^-KSL HSCs. Unexpectedly, the chimerism of donor-derived cells overexpressing Fbxl10 in peripheral blood was similar to that of the GFP controls in both primary and secondary transplantation. However, Fbxl10-expressing cells retained significantly higher repopulating capacity in the tertiary transplantation than the GFP control cells, indicating that forced expression of Fbxl10 in CD34^-KSL HSCs prevents exhaustion of the long-term repopulating potential of HSCs following serial transplantation. Target genes of Fbxl10 in HSCs have not been documented. The Ink4a-Arf-Ink4b locus is one of the major targets of Fbxl10 reported in mouse embryonic fibroblasts and this locus was also tightly repressed in KSL cells expressing Fbxl10. Chromatin immunoprecipitation (ChIP) assays confirmed the direct binding of flag-tagged Fbxl10 to the Ink4a-Arf-Ink4b locus in transduced cells. Of interest, Fbxl10 still retained positive effects on HSCs even in an Ink4a-Arf-deficient background, suggestive of additional targets of Fbxl10. Taken together with the evidence that the Fbxl10 complex includes the polycomb-group proteins, Ring1b and Bmi1, our findings indicate that Fbxl10 is a novel epigenetic regulator of HSCs which potentially collaborates with the polycomb-group proteins.