HOXB4's road map to stem cell expansion

Department of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2007; 104(43):16952-7. DOI: 10.1073/pnas.0703082104
Source: PubMed


Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

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    • "Among these was the Wilms tumor 1 gene (WT1; P < 10−123); NOTCH1 (p < 10−25), a known modulator of lineage-specific events in hematopoiesis16; and various genes of the homeobox gene cluster A (particularly HOXA5: p < 10−35; Fig. 2b, Supplementary Fig. S4a). Hypermethylation was also observed in the HOXB cluster (particularly HOXB3; p < 10−17), but not in HOXB4 which has previously been implicated in in vitro expansion of HPCs (Supplementary Fig. S4b)1718. Other relevant genes with hypermethylated CpG sites include the myeloid translocation gene 16 (MTG16; also known as CBFA2T3; p < 10−150) that has been implicated in the maintenance of stem cell quiescence; the retinoid X receptor alpha (RXRA; p < 10−91), whose down-regulation is essential for neutrophil development and the adenosine a2a receptor (ADORA2A; p < 10−109), which inhibits neutrophil degranulation; and the epigenetic regulators histone deacetylase 9 (HDAC9; p < 10−4; Supplementary Fig. S4c) and DNMT3A (p < 10−46). "
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    ABSTRACT: Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses stemness during culture. In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs. Culture conditions of CD34+ cells - either with or without mesenchymal stromal cells (MSCs) - had relatively little impact on DNAm, although proliferation is greatly increased by stromal support. However, all cultured HPCs - even those which remained CD34+ - acquired significant DNA-hypermethylation. DNA-hypermethylation occurred particularly in up-stream promoter regions, shore-regions of CpG islands, binding sites for PU.1, HOXA5 and RUNX1, and it was reflected in differential gene expression and variant transcripts of DNMT3A. Low concentrations of DNAm inhibitors slightly increased the frequency of colony-forming unit initiating cells. Our results demonstrate that HPCs acquire DNA-hypermethylation at specific sites in the genome which is relevant for the rapid loss of stemness during in vitro manipulation.
    Scientific Reports 11/2013; 3:3372. DOI:10.1038/srep03372 · 5.58 Impact Factor
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    • "Overexpression studies have reported that HOXB4 could enhance mHSCs expansion and mESCs differentiation [68], [69]. Furthermore, overexpression of HOXB4 up-regulates the transcriptional expression of BRE in mHSCs [68]. In our microarray study, however, we did not observe a significant change of HOXB4 expression in response to BRE-silencing. "
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    ABSTRACT: BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-β/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.
    PLoS ONE 07/2013; 8(7):e67896. DOI:10.1371/journal.pone.0067896 · 3.23 Impact Factor
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    • "Similar leukemic transformation was observed in transgenic mice carrying Nup98-Hoxd13 [14] and Nup98-Hoxa9 [15]. Although many attempts have been made to identify the down-stream target genes for Hox gene derivatives [16],[17],[18],[19], the molecular role of these derivatives in hematopoiesis and leukemogenesis remains largely unknown. "
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    ABSTRACT: Hoxb4, a 3'-located Hox gene, enhances hematopoietic stem cell (HSC) activity, while a subset of 5'-located Hox genes is involved in hematopoiesis and leukemogenesis, and some of them are common translocation partners for Nucleoporin 98 (Nup98) in patients with leukemia. Although these Hox gene derivatives are believed to act as transcription regulators, the molecular involvement of the Hox gene derivatives in hematopoiesis and leukemogenesis remains largely elusive. Since we previously showed that Hoxb4 forms a complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component and functions as an E3 ubiquitin ligase activator for Geminin, we here examined the E3 ubiquitin ligase activities of the 5'-located Hox genes, Hoxa9 and Hoxc13, and Nup98-Hoxa9. Hoxa9 formed a similar complex with the Roc1-Ddb1-Cul4a component to induce ubiquitination of Geminin, but the others did not. Retroviral transduction-mediated overexpression or siRNA-mediated knock-down of Hoxa9 respectively down-regulated or up-regulated Geminin in hematopoietic cells. And Hoxa9 transduction-induced repopulating and clonogenic activities were suppressed by Geminin supertransduction. These findings suggest that Hoxa9 and Hoxb4 differ from Hoxc13 and Nup98-Hoxa9 in their molecular role in hematopoiesis, and that Hoxa9 induces the activity of HSCs and hematopoietic progenitors at least in part through direct down-regulation of Geminin.
    PLoS ONE 01/2013; 8(1):e53161. DOI:10.1371/journal.pone.0053161 · 3.23 Impact Factor
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