HOXB4 Engages in Signaling Pathways Associated with HSC Self Renewal.
Authors: Schiedlmeier Bernhard , Santos Ana , Klump Hannes , Ribeiro Ana , Auer Herbert , Baum Christopher , Mallo Moises 
Published: April 2020

Abstract
Ectopic expression of the transcription factor HOXB4 has been shown to mediate expansion of adult hematopoietic stem cells (HSCs) in vitro and in vivo, and to generate embryonic stem cell (ESC) derived HSCs capable to reconstitute lethally irradiated mice. To identify target genes of HOXB4 in adult HSCs and progenitor cells proliferating in vitro, we transduced murine cells with a retroviral construct that coexpresses EGFP and an inducible form of HOXB4 (HOXB4ER). Upon addition of 4-hydroxytamoxifen (TMX), HOXB4ER translocates from the cytoplasma to the nucleus, thereby being capable to modulate gene expression. Transduced cell populations were expanded for 14 days in the presense of TMX. Gene expression profiles were obtained from FACS-sorted HOXB4ER+ LSK (lineage neagtive, Sca1+, ckit+ ) cells after culture with and without TMX. As a control, profiling was performed with LSK cells expressing unmodified active HOXB4 ± TMX. On the Genechip Mouse Genome 430 2.0 Array (Affymetrix) we observed 110 characterized gene products to be differentially expressed 4 hours after inactivation of HOXB4. Globally, the Gene Ontology categories “signal transduction”, “intracellular signaling cascade”, “Wnt receptor signaling” and “cell differentiation” were significantly over-represented among the group of up-regulated HOXB4 target genes. The list of down-regulated genes revealed a significant overrepresentation of the GO categories “regulation of cell cycle”, “regulation of apoptosis” and “regulation of DNA-dependent transcription”. Importantly, differential expression of genes involved in several signaling pathways known to either stimulate or inhibit HSC self renewal (WNT, Notch, FGF, TGFβ/BMP and TNFα) was observed as well as a high degree of concordance with HSC-specific genes discovered in previous microarray reports. TNF receptor 2 was down-regulated by HOXB4 and an inhibitor of TNF receptor 1, BRE (brain and reproductive organ expressed protein) was up-regulated. This suggested that HOXB4-expressing HSCs may still be capable of undergoing self renewal cell divisions in vitro, even in the presence of TNFα. Thus, LSK cells were transduced with the inducible HOXB4ER vector or with a control vector expressing a truncated form of human CD34 (tCD34), pooled in a 1:1 ratio, and cultivated for 7 days in serum-free medium with and without TNFα ± TMX. In the absence of TNFα and TMX, in vitro cultured HOXB4ER+ as well as tCD34+ cells both maintained high levels of multilineage reconstitution activity in vivo. However, in the presence of TNFα without HOXB4 induction, the ability of reconstitution was almost completely lost. In contrast, in the presence of TNFα, high levels of multilineage reconstitution were achieved with TMX- induced HOXB4ER cells, but not so with tCD34+ control cells. Hence, HOXB4 renders long-term repopulating HSCs insensitive to the negative effects of TNFα. We also analyzed gene expression changes in a murine ES cells containing a tetracycline-inducible HOXB4 gene. Over 700 characterized genes were differentially regulated 48 hours after doxycycline-mediated HOXB4 induction in day 6 embryoid bodies (EB). Remarkably, HOXB4 engaged in the same aforementioned signal transduction pathways during EB differentiation, in part by targeting identical gene products. In summary, our data and functional studies reveal that HOXB4 changes the response of ESC and HSC to extrinsic cues regulating self renewal and differentiation.