Pbx1 Regulates the Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence.
Authors: Ficara Francesca , Murphy Mark J. , Lin Min , Cleary Michael L. 
Published: October 2019

Abstract
Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemia. It codes for a homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. Lack of Pbx1 is associated with multiple patterning malformations, defects in organogenesis, and severe fetal anemia, however embryonic lethality has prevented an assessment of its roles in the adult hematopoietic stem cell (HSC) compartment and in lymphoid differentiation. The objective of this study was to characterize the physiological roles for Pbx1 in the hematopoietic system, specifically in the regulation of cell fate decisions involved in the timing and/or extent of postnatal HSC and progenitor proliferation, self-renewal or differentiation capacity. A genetic approach was employed to conditionally inactivate Pbx1 in the hematopoietic compartment in vivo using Cre recombinase expressed under the control of the Tie2 or Mx1 promoters. A crucial role for Pbx1 in the development of the lympho-hematopoietic system was evidenced by reduced size, cell number, and altered architectures of the thymus and spleen in mutant mice. A marked reduction was observed in the bone marrow (BM) pro- and pre-B cell compartment, as well as a striking reduction (up to 10-fold) in common lymphoid progenitors (CLP), suggesting a role for Pbx1 at a critical stage of lymphoid development where acute leukemia likely originates. Accordingly, abnormal T cell development was observed in the thymus. Common myeloid progenitors (CMP) and Lin-cKit+Sca1+ (LKS, enriched in HSCs) cells were also reduced, as well as long-term stem cells (LT-HSCs, reduced 7-fold on average). Assessment of the proliferation status of LT- and ST (short-term)-HSCs, as well as multi-potent progenitors (MPP), revealed that the reduction of the HSC compartment was associated with a higher number of stem cells exiting the G0 phase, thus losing their quiescent state. Strikingly, Pbx1-deficient BM cells failed to engraft in competitive transplants, but were able to reconstitute congenic recipients in the absence of competition, indicating a profound defect of functional HSCs, which nevertheless retained reconstitution potential. Importantly, Pbx1 deficient HSCs progressively disappeared from primary transplant recipients, and were unable to engraft secondary recipients, demonstrating that Pbx1 is crucial for the maintenance of LT-HSC self-renewal. Microarray studies performed on mutant and wt LT- and ST-HSCs, followed by bioinformatics analysis, showed that in the absence of Pbx1 LT-HSCs are characterized by premature expression of a large subset of ST-HSC genes. The up-regulated differentially expressed transcripts are enriched for cell cycle regulatory genes, consistent with the observed increased cycling activity. Notably, more than 8% of the down-regulated genes are related to the Tgf-beta pathway, which serves a major role in maintaining HSC quiescence. Moreover, B-cell specific genes, which are expressed in the wt LT-HSC compartment, are down-regulated in the absence of Pbx1, suggesting that the observed reduction in CLP and B-cell numbers ultimately arose from a stem cell defect in lymphoid priming. We conclude that Pbx1 is at the apex of a transcriptional cascade that controls LT-HSC quiescence and differentiation, thus allowing the maintenance of their self-renewal potential, crucial for the homeostasis of the lympho-hematopoietic system.