Functional analysis of the Human Pbx Interacting Protein (HPIP) in Human Hematopoiesis

The recognition of novel proteins that regulate human hematopoietic stem cell and early progenitor cell fate is a prime objective in experimental and clinical hematology. Human hematopoietic PBX interacting protein (HPIP), with no significant homology to known proteins, is a 731 amino acid protein, discovered as a novel interacting partner of the PBX homeodomain protein. HPIP has been implicated as a nuclear-cytoplasmic shuttle molecule and shown to have the capacity to bind to the cytoskeleton. It also inhibits the ability of PBX-HOX heterodimers to bind to target sequences and strongly inhibits the transactivation activity of E2A-PBX1 [t(1;19) translocation, which occurs in 25% of pediatric pre-B cell acute lymphocytic leukaemia] (Abramovich C. et al JBC, 2000; Oncogene, 2002). It is highly expressed in human CD34⁺ progenitor cells, but is silenced in differentiated cells. To gain further insights into the possible functional role of HPIP and its domains and its possible role in a common pathway with HOX transcription co-factor PBX1, HPIP cDNA was cloned in pMSCV-IRES-YFP cassette. Umbilical cord blood enriched with CD34⁺ population of stem cells was obtained to perform in vitro and in vivo experiments. Mutants, with deletions of the microtubule binding region (ΔMBR-HPIP), and nuclear receptor and PBX1 interacting motif (ΔNRPID-HPIP) were generated and tested in vitro and in vivo. The constitutive expression of HPIP wt and ΔMBR-HPIP in human cord blood cells (CD34⁺) enhanced erythroid colony formation in CFC assay (p=0.008, n=6) while the ΔNRPID-HPIP mutant nullified the effect. Both mutants of HPIP augmented significantly, the formation of primitive colonies (GEMM and GM) in methylcellulose assay (p≤0.01, n=6) as compared to YFP control and HPIP wt. In replating CFC assays ΔNRPID-HPIP showed an increased number of myeloid colonies (p≤0.01, n=6) and GM (p=ns) colonies but a decrease in granulocytic colonies (p≤0.05, n=6) compared to YFP control and HPIP wt