Conditional Inactivation of the Proto-Oncogene Pokemon Results in Block of Differentiation in Multiple Hematopoietic Lineages.

We recently reported a role of the POK transcription factor Pokemon in oncogenesis (Nature. 433, 278–85). To elucidate its function in fetal and adult hematopoiesis, we studied the hematopoiesis in Pokemon knockout mice (Pokemon^−/−) and the conditional knockout mutants (Pokemon^flox/flox) respectively. We generated the Pokemon^{flox/floxMx−1cre+} mice, in which Exon 2 of the Pokemon genomic locus can be excised in vivo upon pIpC (polyinosinic-polycytidylic ribonucleic acid) injections.

<Erythroid> Pokemon^−/− mice died around 16.5 d.p.c due to severe anemia. Flow cytometric analyses (FACS) revealed a significant decrease of mature erythroblasts and profound increase of AnnexinV positive apoptotic TER119⁺CD71⁺ cells. Pokemon is therefore indispensable for definitive erythropoiesis by blocking apoptosis in late differentiation stage. Since the loss of the tumor suppressor Arf reverted the senescence phenotype of Pokemon^−/− MEFs, we next examined whether the loss of Arf rescues the anemia phenotype of Pokemon^−/− mice. Although Arf mRNA level is elevated in Pokemon^−/− TER119⁺CD71⁺ erythroblasts, Pokemon^−/−Arf^−/− embryos were also lethal due to anemia, which indicates that the apoptosis is caused by Arf independent mechanism. Conditional inactivation of Pokemon upon pIpC injections in adult mice resulted in macrocytic anemia. FACS analyses of BM and spleen demonstrated a significant decrease of TER119SP cells. Spleen weights of Pokemon^{flox/floxMx−1cre+} mice were three times more than those of control mice due to the compensatory expansion of TER119⁺CD71⁺ cells.

<Lymphoid> Conditional inactivation of Pokemon in adult mice also resulted in a significant decrease of B220⁺ B lymphocytes in peripheral blood (PB). Furthermore, total B220⁺ splenocytes were severely decreased and immunohistochemical analyses of spleens displayed the lack of mature germinal center formations. The proB cells were barely detectable, while PreProB cells were accumulated in Pokemon^{flox/floxMx−1cre+} mice BM. We therefore concluded that Pokemon plays a key role in early B cell development, especially at the transition stage from PreProB to ProB cells. Regarding T lymphocytes, the numbers of CD4/8 SP cells in Pokemon^{flox/floxMx−1cre+} PB were similar to those of control mice. No major immuno-phenotypical differences were observed in thymocytes.

<Myeloid> Two months after pIpC injections, Pokemon^{flox/floxMx−1cre+} mice displayed a moderate decrease of Gr1⁺Mac1⁺ myeloid lineage cells in PB and BM. However, in vitro colony forming assays, taking advantage of hematopoietic stem cells from Pokemon^{flox/floxMx−1cre+} mice, displayed a clear block of differentiation at the promyelocyte stage in CFU-GM. Furthermore, FACS analyses of progenitor populations showed the decrease of GMPs (granulocyte/macrophage progenitors) in Pokemon^{flox/floxMx−1cre+} mice.

<Conclusion> Our result indicates that the proto-oncogene Pokemon is a master regulator of both fetal and adult hematopoiesis. To further elucidate the mechanism, GeneChip microarray analyses were performed utilizing the RNA prepared from the flow-sorted TER119⁻CD71⁺ 12.5 d.p.c FL erythroblasts. Candidate target genes were found and the results were also confirmed by real-time PCR. The details of possible target genes will be discussed further at the meeting.