NF-Ya Is Essential for Hematopoieic Stem Cell Proliferation.

Abstract 1507

Poster Board I-530

Proliferation, self-renewal and differentiation of hematopoietic stem cells (HSCs) must be tightly regulated in order to sustain hematopoiesis over a lifetime and to prevent uncontrolled expansion of cells. Several genes have been implicated in the regulation of HSC behavior, such as HoxB4, Notch1, Lef-1 and others. Previous studies from our and other laboratories have demonstrated that the heterotrimeric transcription factor NF-Y is a potent inducer of many of these regulatory genes by over-expressing NF-Ya, the regulatory subunit of NF-Y. Furthermore, Bhattarchaya et al. showed that the deletion of NF-Ya in mice leads to lethality before day E8.5, highlighting its importance in mouse development. While there is no doubt that NF-Y plays a role in the progression of the cell cycle in vitro, different groups – targeting different subunits for deletion or silencing – have obtained different results. In order to comprehensively investigate the in vivo function of NF-Y in the hematopoietic system, we utilized a conditional knockout mouse model. We found that the bone marrow (BM) cellularity decreases sharply starting as soon as one day after the ablation of NF-Ya. Our data indicate that the cell loss can be attributed to a combination of cell cycle arrest in G2/M-phase of the cell cycle and apoptosis at 24 hours after the gene deletion.

Since NF-Y has been identified as a master regulator of genes involved in HSCs behavior, we focused on the effects of the NF-Ya deletion within the HSC compartment. We found a down regulation of HoxB4, Notch-1, Lef-1 and Bmi-1 following NF-Ya deletion. However, 24h after induction of NF-Ya deletion the HSC population appeared unaffected and their numbers remained stable, likely due to their predominantly quiescent status.

To investigate the capability of stem cells to progress though the cell cycle, we activated HSCs using the interferon inducer poly IC and observed that, once activated, also HSCs accumulate in the G2/M-phase of the cell cycle. Finally, to test whether the deletion of NF-Ya impairs or absolutely abrogates HSC function, we performed long term experiments including competitive BM transplantation and colony formation assays that demonstrate that NF-Y activity is absolutely essential for HSC function.

Altogether, our data identify NF-Y plays a pivotal role in the survival of hematopoietic cells and the progression of cells though the G2/M-phase of the cell cycle in vivo. Additionally, while we found that NF-Y ablation leads to reduced expression of many genes important for HSC behavior, this had no immediate effects on the maintenance of these cells due to their quiescent nature.