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Abstract

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited

Runx1 binds DNA in cooperation with CBFβ to activate or repress transcription, dependent upon cellular context and interaction with a variety of co-activators and co-repressors. Runx1 is required for emergence of adult hematopoietic stem cells (HSC) during embryonic development and for lymphoid, myeloid, and megakaryocyte lineage maturation from HSC in adult marrow. Runx1 levels vary during the cell cycle, and Runx1 regulates G1 to S cell cycle progression. Both Cdk and ERK phosphorylate Runx1 to influence its interaction with co-repressors, and the Wnt effector LEF-1/TCF also modulates Runx1 activities. These links likely allow cytokines and signals from adjacent cells to influence HSC proliferation versus quiescence and the rate of progenitor expansion, in response to developmental or environmental demands. J. Cell. Physiol. 219: 520–524, 2009. © 2009 Wiley-Liss, Inc.

Runx1, and the related Runx2 or Runx3, bind DNA via their Runt DNA-binding domains (Ogawa et al., 1993; Wang et al., 1993). Interaction of the Runt domain with CBFβ strengthens DNA affinity (Ogawa et al., 1993; Wang et al., 1993). The consensus Runx1 binding site is 5′-PuACCPuCA-3′ (Kamachi et al., 1990; Meyers et al., 1993). Runx1 expression predominates in hematopoietic cells, with the exception of the erythroid lineage (North et al., 2004), and Runx1 is a key regulator of hematopoiesis. In addition, Runx1 activities are commonly perturbed during leukemogenesis (Friedman, 1999). This review focuses on the role of Runx1 in the formation and proliferative expansion versus quiescence of embryonic and adult hematopoietic stem cells (HSC) and adult lineage-specific progenitors. Aspects of Runx1 gene and protein regulation and functional interaction with other pathways relevant to these topics will also be discussed.

Definitive HSC Formation and Lineage Maturation Requires Runx1

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited

Runx1(−/−) mice develop primitive but not definitive hematopoietic cells (Okuda et al., 1996; Wang et al., 1996). In the aorta/gonad/mesonephros (AGM) region of the developing mouse embryo, Runx1 expression is found both in the ventral aspect of the dorsal aorta and in c-kit+CD34+ hematopoietic cells emerging into the lumen at 10.5 dpc (North et al., 1999). Selection of Runx1-expressing cells identifies the HSC population in this region of the embryo, and deletion of Runx1 from the vascular endothelium prevents HSC emergence (North et al., 1999, 2002; Yokomizo et al., 2001; Chen et al., 2009).

Notch signaling cooperates with Runx1 to regulate HSC emergence. Induction of exogenous Notch-intracellular domain (NICD) from the heat shock promoter in irradiated adult zebrafish leads to fourfold induction of Runx1 and threefold increase in blood precursors, and anti-sense Runx1 inhibits NICD-mediated expansion of zebrafish embryo AGM-region HSC (Burns et al., 2005). In addition, Runx1 rescues hematopoiesis in Notch1−/− para-aortic splanchnopleural cells (Nakagawa et al., 2006). Notably, adult HSC apparently do not require Notch signaling, as they reconstitute hematopoiesis normally in the presence of dominant-negative Mastermind-like1 or in the absence of RBPJ (Maillard et al., 2008), although Notch ligand expands human NOD/SCID repopulating hematopoietic stem cells in a dose-dependent manner (Delaney et al., 2005).

The effect of Runx1 deletion on adult HSC activity has been investigated. One study found expansion of lin-Sca-1+c-kit+ (LSK) cells and reduced marrow competitive repopulation (Growney et al., 2005), whereas another confirmed the increase in LSK cells but found an increase in quiescent side population cells and in the fraction of c-kit+ cells in G0 in the absence of Runx1 and increased competitive repopulation (Ichikawa et al., 2008). Exogenous expression of the full-length AML1b isoform of Runx1 reduces murine HSC competitive repopulation, whereas expression of the alternatively spliced AML1a, lacking 238 C-terminal residues, has the opposite effect (Tsuzuki et al., 2007). If the shorter isoform acts in a dominant-negative manner, then it may act similarly to Runx1 deletion and thus confirm the idea that absence of Runx1 activity leads to HSC quiescence and increased stem cell activity, with exogenous Runx1 driving HSC proliferation and thereby inducing differentiation to a less pluripotent state.

Adult mice lacking Runx1 have markedly reduced numbers of common lymphoid progenitors, pro-B, pre-B, and mature B cells; they also harbor T-cell developmental abnormalities, thrombocytopenia, increased granulocyte–monocyte progenitors, and expansion of mature granulocytic and monocytic myeloid cells (Ichikawa et al., 2004; Growney et al., 2005). Runx1 activates several lineage-specific genes, including those encoding myeloperoxidase, neutrophil elastase, and the M-CSF receptor in immature myeloid cells, T cell receptor subunits, and the megakaryocytic glycoprotein αIIb promoter (Redondo et al., 1992; Suzow and Friedman, 1993; Nuchprayoon et al., 1994; Zhang et al., 1994; Elagib et al., 2003). In addition, Runx1 silences the CD4 enhancer in T-lineage cells (Taniuchi et al., 2002).

Regulation of the Runx1 Locus

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited

The Runx1 gene contains two promoters, with P1 located 160 kb upstream of P2 in the human gene; P2 is contained within an evolutionarily conserved CpG island (Ghozi et al., 1996; Levanon et al., 2001). Use of alternative promoters leads to a change in the N-terminal Runx1 residues, potentially leading to differential gene regulation. The Runx1 P1 promoter contains two Runx-binding sites; these enable repression by Runx3 in B-lineage cells but potentially mediate auto-activation in other contexts (Spender et al., 2005). The first intron of the Runx1 gene contains, at +23 kb, an enhancer regulated by GATA-2, Ets, and SCL (Nottingham et al., 2007; Landry et al., 2008), and BMP4 activity also modulates Runx1 transcription (Pimanda et al., 2007).

Along with differential promoter usage, alternative splicing predicts multiple Runx1 isoforms (Levanon et al., 2001). The P1 promoter encodes AML1c, and the P2 promoter encodes AML1b; in AML1b, 5 unique residues replace 32 N-terminal AML1c amino acids. The shorter AML1a isoform is also encoded from the P2 promoter (Miyoshi et al., 1995). In differentiating murine embryonic stem cells (ESC), AML1c has a later onset of expression than AML1b (Fujita et al., 2001). Similarly, in cultured human ESC, embryoid bodies or primitive, mixed colony forming units (CFU-P) express AML1b but not AML1c, whereas definitive colonies (CFU-D) express both isoforms (Zambidis et al., 2005). Yet, mice with greatly diminished P2 promoter activity due to neomycin-cassette insertion in one Runx1 allele and absence of the second allele fail to develop definitive hematopoiesis, suggesting that P1 activity (AML1c) is sufficient for primitive hematopoiesis whereas P2 activity (AML1b) is required for definitive, adult hematopoiesis (Pozner et al., 2007). Further analysis of isoform expression patterns in yolk sac, fetal liver, and AGM hemogenic endothelium and HSC may help clarify their respective roles during embryogenesis.

Runx1 Regulates the Cell Cycle

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited

The first indication that Runx1 has the capacity to directly regulate cell proliferation came from the finding that CBFβ-SMMHC, a dominant-inhibitory myeloid oncoprotein, slows G1 to S cell cycle progression in growth factor-dependent Ba/F3 cells, associated with hypo-phosphorylation of the Rb protein (Cao et al., 1997). This initial study was extended by showing that deletion of 10 amino acids located at the N-terminus of the CBFβ domain within CBFβ-SMMHC and required for Runx1 interaction obviates cell cycle inhibition (Cao et al., 1998). CBFβ-SMMHC also markedly slows proliferation of primary murine or human marrow myeloid progenitors, again dependent upon interaction with Runx1, but does not reduce the number or size of erythroid colonies, which lack Runx1 (D'Costa et al., 2005). Similarly, RUNX1-ETO, another dominant-inhibitory myeloid oncoprotein, or KRAB-RUNX1-ER, an estrogen-regulated fusion protein containing the RUNX1 DNA-binding domain linked to the KRAB repression domain, each slow cell cycle progression in hematopoietic cells (Lou et al., 2000; Burel et al., 2001), and mutation of the DNA-binding domain in RUNX1-ETO prevents cell cycle inhibition (Kummalue et al., 2002).

Conversely, exogenous RUNX1 or RUNX1-ER stimulates 32Dcl3, Ba/F3, and lineage-negative murine marrow cell G1 to S cell cycle progression (Lou et al., 2000; Strom et al., 2000; D'Costa et al., 2005). RUNX1 lacking its C-terminal repression domain, residues 419–480, retains the ability to stimulate Ba/F3 proliferation, whereas further deletion of its trans-activation domain, residues 321–418, generates a dominant-inhibitory protein that interferes with G1 to S cell cycle progression (Bernardin and Friedman, 2002). Activation of cdk4 and cyclin D3 transcription and repression of the p21 promoter likely contribute to stimulation of proliferation by Runx1 (Lou et al., 2000; Lutterbach et al., 2000; Bernardin-Fried et al., 2004). In addition, cyclin D directly interacts with RUNX1 to reduce its trans-activation potency (Peterson et al., 2005), potentially providing a feedback mechanism to limit RUNX1-mediated proliferation.

Regulation of cell proliferation by Runx proteins represents an evolutionarily conserved activity. Stimulation of HSC expansion in zebrafish by Runx1 was discussed above. In the sea urchin S. purpuratus, depletion of the Runx ortholog SpRunt-1 reduces blastocyst cell proliferation and inhibits expression of cyclinD, wnt6, and wnt8 RNAs, whose promoters bind SpRunt-1 in a chromatin immunoprecipitation assay; moreover GSK-3 inhibition, mimicking Wnt signaling, leads to increased expression of SpRunt-1 protein (Robertson et al., 2008).

Wnt signaling activates cyclin D expression, suggesting potential functional cooperation with Runx1 in regulating stem cell proliferation. In fact, expression of activated β-catenin leads to HSC proliferation and differentiation, with resultant stem cell exhaustion (Reya et al., 2003; Kirstetter et al., 2006; Scheller et al., 2006), mimicking the effect of full-length Runx1 over-expression (Tsuzuki et al., 2007). On the other hand, osteoblast-specific expression of Dickkopf1, a Wnt inhibitor, leads to reduced p21 and increased HSC proliferation with reduced long-term engraftment potential (Fleming et al., 2008), indicating that Wnt signals maintain stem cells when expressed specifically in the adult osteocyte niche. Deletion of β-catenin and γ-catenin in adult mice does not compromise HSC potential, a result which still allows a role for non-canonical Wnt signals in adult HSC homeostasis, whereas Vav-Cre-mediated β-catenin excision reduces adult HSC engraftment potential, perhaps indicative of a role for Wnt in developmental HSC expansion (Zhao et al., 2007; Jeannet et al., 2008; Koch et al., 2008).

In C. elegans, the Runx ortholog RNT-1 stimulates seam cell proliferation, with rnt-1 mutants have reduced numbers of seam cells and animals expressing exogenous RNT-1 having an expansion of seam cells (Kagoshima et al., 2005; Nimmo et al., 2005). Seam cells have stem-cell properties, carrying out symmetrical and assymetrical divisions to produce several types of differentiated progeny. Loss of RNT-1-induced expression of CKI, a cyclin-dependent kinase inhibitor, potentially accounts for reduced seam cell numbers in rnt-1 mutant animals (Nimmo et al., 2005). Moreover, mutation of bro-1, encoding the CBFβ homolog BRO-1, similarly reduces seam cell proliferation, over-expression of BRO-1 expands seam cells, and simultaneous over-expression of BRO-1 and RNT-1 induces massive seam cell expansion, exceeding wild-type numbers by three- to fourfold (Kagoshima et al., 2007). These analyses have added significance due to the ability to study proliferative versus differentiation decisions at the single cell level during C. elegans development.

The Cell Cycle and Signaling Pathways Regulate Runx1

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited

Regulation of proliferation by Runx1 inspired the idea that Runx1 in turn might be regulated during the cell cycle. Indeed, endogenous or exogenous Runx1 protein levels increase as IL3-dependent Ba/F3 cells progress from G1 to S to G2/M, whereas Runx1 RNA levels remain constant (Bernardin-Fried et al., 2004). RUNX1 contains three serine residues that match the cyclin-dependent kinase (Cdk) consensus, (S/T)PX(R/K), S48, S303, and S424 (Fig. 1). Cdk phosphorylation of S303 induces RUNX1 degradation during G2/M, mediated by the anaphase-promoting complex (Biggs et al., 2005, 2006; Wang et al., 2007). Cdk also phosphorylates S48 and S424 in hematopoietic cells, and mutation of these serine residues to the phospho-mimetic aspartic acid increases trans-activation potency (Zhang et al., 2008). Although change to aspartic acid did not lead to increased affinity for the p300 co-activator, RUNX1(S424D) has reduced affinity for HDAC1 and HDAC3 (Guo and Friedman, 2008), co-repressors previously shown to bind RUNX1 in the vicinity of S424 (Reed-Inderbitzin et al., 2006). Moreover, mutation of S48, S303, and S424 to alanine weakens trans-activation and reduces the ability of RUNX1 to stimulate proliferation (Zhang et al., 2008). Thus, RUNX1 phosphorylation by Cdk strengthens its trans-activation potency by weakening its interaction with HDACs, allowing stimulation of proliferation. Analogously, ERK-mediated phosphorylation of RUNX1 S276/S293 or arginine methylation of R206 and R210 inhibits mSin3A co-repressor interaction, and HIPK2-mediated phosphorylation of RUNX1 S276/T300/S303 enhances p300 co-activator interaction (Fig. 1; Imai et al., 2004; Aikawa et al., 2006; Wee et al., 2008; Zhao et al., 2008). Cytokine-mediated ERK signaling also induces cyclin D1 transcription (Lavoie et al., 1996). In addition, BMP/TGFβ signals activate SMAD proteins, which directly interact with Runx1 to modulate its activity (Hanai et al., 1999).

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Figure 1. Diagram of the AML1c isoform of Runx1, showing location of the Runt DNA-binding domain (DBD), the sites of interaction with the C/EBPα and Ets family transcription factors (Sun et al., 1995; Zhang et al., 1996; Mao et al., 1999), and Cdk and ERK phosphorylation sites. Also depicted are the locations of Runx1 activation domains and repression domains (AD, RD) along with the co-activators or co-repressors mediating their activities, including Groucho, YAP, an undefined N-terminal activating factor (Imai et al., 1998; Yagi et al., 1999; Liu et al., 2006), and additional mediators cited in the text.

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RUNX2 levels vary during the cell cycle in osteoblasts, reaching its peak in G1, and RUNX2 slows proliferation in this lineage dependent upon integrity of its DNA-binding and C-terminal activation/repression domains (Galindo et al., 2005; Teplyuk et al., 2008). RUNX2 may, in part, regulate proliferation via control of rRNA transcription (Young et al., 2007). Interestingly, Cdk phosphorylation of RUNX2 S451, the homolog of RUNX1 S424, stimulates endothelial cell proliferation (Wee et al., 2002; Qiao et al., 2006). The Wnt effector LEF-1/TCF cooperates with RUNX1 to activate the T cell receptor α enhancer and also cooperates with RUNX2 to stimulate FGF18 transcription during early bone formation and directly interacts with the RUNX2 DNA-binding domain to repress osteocalcin transcription during bone maturation (Mayall et al., 1997; Kahler and Westendorf, 2003; Reinhold and Naski, 2007). In addition, RUNX3 binds the β-catenin/TCF4 complex to attenuate Wnt signaling in intestinal epithelial cells (Ito et al., 2008). Thus, Wnt signals cooperate or interfere with RUNX activities dependent upon cellular, gene, and developmental context.

Summary and Considerations for Future Investigations

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited

Emergence of adult HSC requires Runx1, with Polycomb complexes suppressing Runx1 transcription in totipotent stem cells (Lee et al., 2006). During development, Notch signals mediate Runx1 induction in cooperation with SCL/GATA/Ets factors, and Wnt signals potentially cooperate with Runx1 to facilitate adult HSC expansion via cooperative induction of cyclin D, cdk4, and additional cell cycle regulators (Fig. 2). In adult marrow, Wnt, Runx1 and additional signals cooperate to mediate HSC quiescence versus proliferation, allowing HSC to enter the cell cycle as needed to maintain a stem cell pool and to support hematopoiesis throughout life. Thus, within HSC Runx1 integrates external cues for developmental and proliferative decisions. In addition, cytokine signals may cooperate with Runx1 to regulate the rate of proliferation of lineage-specific progenitors in response to environmental demands.

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Figure 2. Model for control of hematopoietic stem and progenitor cell proliferation by Runx1 and cooperating signals. Notch ligands induce Runx1 expression, facilitiating HSC formation. Wnt ligands stimulate cyclin D and c-Myc expression to favor cell proliferation, cyclin D induces Cdk-mediated phosphorylation of Runx1, and Runx1 cooperatives with cyclin and c-Myc to stimulate G1 entry from G0 and G1 to S cell cycle progression. Cytokine signals activate ERK, thereby inducing cyclin D expression and stimulating Runx1 activity via direct modification. Wnt and Notch signals may also contribute to stem and progenitor cell expansion indirectly, by inhibiting their further maturation.

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Runx1 regulates cell cycle transitions dependent upon functional/physical interactions with additional proteins, including HDACs, mSin3A, p300, SMAD proteins, and LEF-1/TCF. These interactions in turn depend upon external cues and direct Runx1 modifications. Epigenetic/transcriptional control, RNA processing, and regulation of protein stability also likely influence Runx1 activities in stem and progenitor cells. Future investigations will identify the direct links between various signaling pathways, Runx1 isoform expression and modification, and Runx1-target gene induction or repression that mediate hematopoietic stem and progenitor cell development, proliferation versus quiescence, and lineage-specific maturation.

These future studies will facilitate efforts to develop and expand adult HSC for clinical applications, and will also provide insights into mechanisms underlying transformation by CBF oncoproteins, which inhibit RUNX1 activities (Friedman, 1999), or by RUNX1 over-expression, which has been observed in a subset of acute lymphoblastic leukemias (Niini et al., 2000; Mikhail et al., 2002; Penther et al., 2002). CBF oncoprotein require cooperation with additional mutations that favor proliferation to induce acute leukemia (Yang et al., 2002; Bernardin et al., 2002), whereas RUNX1 over-expression may itself provide a proliferative signal to malignant lymphoblasts.

Literature Cited

  1. Top of page
  2. Abstract
  3. Definitive HSC Formation and Lineage Maturation Requires Runx1
  4. Regulation of the Runx1 Locus
  5. Runx1 Regulates the Cell Cycle
  6. The Cell Cycle and Signaling Pathways Regulate Runx1
  7. Summary and Considerations for Future Investigations
  8. Acknowledgements
  9. Literature Cited
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