The Hematopoietic Activity of the Homeobox Gene HOXB4 Depends on a N-Terminal Proline-Rich Region In Vitro and In Vivo.

HOXB4 belongs to the family of homeobox transcription factors, which play a key role in hematopoietic development. The expression of HOXB4 induces a significant increase of long-term repopulating stem cells (SC) in human and mouse models, without perturbing hematopoietic differentiation. So far the underlying mechanisms of the SC amplificatory impact of HOXB4 are poorly understood. In an attempt to understand the unique characteristics of HOXB4, we performed a mutational study by deleting its proline - rich N-terminus, which has been described to act as a transcriptional activation domain in many other proteins, like non-homeobox genes (e.g. p53, AP2) and other homeobox genes (e.g. HOXD4 and HOXA13). We performed in vitro and in vivo experiments transducing murine 5-FU enriched HSCs with the pMSCV-IRES-GFP based retroviral vector harbouring the HOXB4 wild-type (wt) and several mutants, including a Δproline HOXB4 mutant (ΔP), where the proline-rich sequence between the aminoacidic positions 71–123 is deleted.

In vitro the ΔP-HOXB4 mutant led to a 3.2fold decrease in the number of hematopoietic cells harvested after 1 week of liquid expansion compared to the HOXB4 wt (n=3; p<0.05). Furthermore, deletion of the proline - rich region resulted in a 7.5fold decrease in the total number of CFCs per 500 cells plated (n=3; p<0.03), compared to the HOXB4 wt. The ΔP-HOXB4 mutant also generated less secondary colonies (18.5 versus 103 per 500 cells initially plated; n=2; p<0.0001) compared to the HOXB4-wt. Of note and in contrast to the ΔP-HOXB4 mutant, the deletion of the PBX interacting domain (Pbx-HOXB4, aa W→A) or deletion of the C-terminal stretch (aa 221 −251; cDel-HOXB4) did not reduce the formation of primary colonies compared to HOXB4 wt (n=3; 178 and 124 versus 226 colonies per 500 cells initially plated, respectively). On the level of the short-term repopulating stem cells constitutive expression of HOXB4 wt (n=3) induced on average a 1,565fold increase in the frequency of ΔCFU-S in comparison to the GFP control (n=11) (10,645 versus 6.8 colonies/45,000 input cells; p<0.0000001). The deletion on the N-terminal proline-rich region of HOXB4 (n=14) led to a significant decrease in the Δ-CFU-S frequency in comparison to the HOXB4-wt (mean 42fold; p<0.000001), while it still generated significantly more Δ-CFU-S compared to the GFP control (252 versus 6.8/45,000 input cells; p<0.001).

Taken together, these results characterize the N-terminal proline - rich stretch between aminoacidic positions 71–123 as a domain, which is highly relevant for the hematopoietic activity of HOXB4.