Ubap2l-Bmi-1-Rnf2 Define a Novel Polycomb Complex Essential For Self-Renewal Of Hematopoietic Stem Cells

The polycomb group protein Bmi-1 is a well known determinant of hematopoietic stem cell function. Bmi-1^-/- mice display severe hematopoietic defects, including progressive loss of hematopoietic cells from the bone marrow. Bmi-1 is dispensable for hematopoietic stem cell specification, but essential for their maintenance, an effect attributable to its ability to promote HSC self-renewal. The mechanism by which Bmi-1 regulates this process is not completely understood. Bmi-1 has been shown to repress the INK4A/ARF locus encoding the cell cycle inhibitors p16^ink4a and p19^arf , to interact with the E4F1 protein and to regulate the DNA damage response pathway, however experimental manipulation of these proteins/pathways only partially rescues the hematopoietic defects of the Bmi-1^-/-mice. It thus appears that the mechanism by which Bmi-1 regulates HSC self-renewal remains to be determined.

Towards this goal, we purified Bmi-1 containing protein complexes from cellular extracts and identified Bmi-1 interaction partners by mass spectrometry. We observed that the protein Ubap2l, which has never been shown to associate with Bmi-1 and for which no link with polycomb group protein function has been described, was consistently found in Bmi-1-containing protein complexes. Immunoprecipitation experiments revealed that Ubap2l indirectly associates with Bmi-1 via an interaction with the polycomb group protein Rnf2. We then evaluated the possibility that Ubap2l might be involved in the regulation of HSC activity. We observed that Ubap2l transcripts are more abundant in primitive HSC populations compared to total BM. CFC assays performed with BM cells infected with Ubap2l shRNAs revealed that Ubap2l knockdown causes a modest and progressive loss of progenitor activity when cells are kept in culture, with multipotent and bipotent progenitors being substantially more affected than unipotent progenitors. We transplanted these cells in mice and observed a gradual decrease in the percentage of donor derived cells expressing Ubap2l shRNAs in the peripheral blood of the recipient mice, with the most striking effect observed 16 weeks post-transplantation in the BM. Bmi-1 has been shown to regulate the proliferative capacity of both progenitor and stem cells, and its deletion in BM cells is known to dramatically reduce the reconstitution activity of these cells at early time points following transplantation. In contrast, Ubap2l appears to preferentially regulate LTR-HSC activity. We tested the effects of Ubap2l silencing on leukemic cells in vivo and observed that a reduction of Ubap2l levels in these cells had an important impact on their ability to reconstitute recipient mice, suggesting that Ubap2l also plays a role in leukemic stem cell activity. We determined if the mechanism by which Ubap2l regulates HSC activity is related to Bmi-1 function by simultaneously introducing Bmi-1 cDNA and Ubap2l shRNAs in BM cells and found that Bmi-1 is able to rescue the long-term reconstitution defect caused by Ubap2l downregulation in these cells. We observed that Ubap2l silencing does not significantly affect the expression of the known Bmi-1 targets p16^ink4a and p19^arf, implying that Ubap2l regulates HSC activity via a Bmi-1-dependent mechanism that does not involve repression of the INK4A/ARF locus. One explanation for the two Bmi-1 dependent mechanisms at play in the regulation of HSC activity could be that Bmi-1 is part of two separate protein complexes, each regulating different aspects of hematopoietic cell function. To test this hypothesis, we fractionated cellular extracts and were indeed able to resolve two distinct Bmi-1 containing protein complexes, distinguishable by the presence of Ubap2l.

Based on the results we obtained, we propose a model in which two different Bmi-1 containing protein complexes regulate hematopoietic stem cell function. An Ubap2l-independent complex, which is most likely involved in the repression of the INK4A/ARF locus, and could be responsible for the effects of Bmi-1 on multipotent progenitors and STR-HSCs, and an Ubap2l-dependent complex, which operates via a yet to be defined mechanism unrelated to p16^Ink4a and p19^Arf, and would account for the effects of Bmi-1 on LTR-HSC activity. These results position Ubap2l as a key regulator of LTR-HSC activity and unveil a novel protein complex mediating the effects of Bmi-1 on LTR-HSCs.