Differential gene expression between human Cord blood transduced with HPIP-wt, mutant NRPID and control YFP

Background Homeobox gene associated regulatory networks are among the key determinants of early hematopoietic development. Previously, the hematopoietic PBX interacting protein (HPIP) has been identified as a novel interacting partner of the TALE homeodomain protein PBX1, forming a microtubule signalling complex. Expression of HPIP has been associated with increased tumorigenicity of the MCF7 breast cancer cell line. We now demonstrate that HPIP is a novel regulatory protein in human hematopoiesis: constitutive expression of HPIP in human umbilical cord blood derived CD34+ cells increased the absolute number of clonogenic progenitors in liquid expansion culture as well as in methylcellulose assays with a significantly enhanced formation of erythroid colonies compared to the control (p0.01, n=6). Limiting dilution LTC-IC assays confirmed the hematopoietic activity of the protein on primitive human progenitor cells with an over 5fold increase in the absolute number of LTC-ICs compared to non-transduced cells (n=8; p<0.05). In vivo HPIP expression induced a significant shift towards myeloid engraftment (n=8;p<0.05) and doubled the proportion of hCD34+CD38+ human cells in transplanted mice (p0.05, n=8). Structure function analyses identified the C - terminal nuclear receptor/PBX interacting domain (NRPID; LXXLL domain) as a critical domain for the hematopoietic activity of HPIP. Gene expression data by microarray and Q-RT-PCR analysis demonstrated that HPIP induced particularly differential expression of genes involved in the MAPK pathway and cytokine-cytokine interaction. Taken together, these data demonstrate that proteins involved in the organization of microtubular signalling complexes such as HPIP can act as regulators of early human hematopoiesis.

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