Critical roles for c-Myb in hematopoietic progenitor cells

Seminars in Immunology 20 (2008) 247–256

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Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim

Review

Critical roles for c-Myb in hematopoietic progenitor cells Kylie T. Greig a,b , Sebastian Carotta a , Stephen L. Nutt a,∗ a b

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia The Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia

a r t i c l e Keywords: c-Myb Transcription factor Hematopoiesis p300 Differentiation

i n f o

a b s t r a c t While it has long been known that the transcription factor c-Myb is an essential regulator of hematopoiesis, its precise molecular targets have remained elusive. Cell line studies suggest that c-Myb promotes proliferation and at the same time inhibits differentiation, however the early lethality of c-Myb deficient embryos precluded analysis of its role in adult hematopoiesis. Here we review insights derived from recently developed mouse models of c-Myb deficiency that are viable as adults. These studies reveal a complex array of functions for c-Myb in multiple hematopoietic cell types that will redefine our understanding of this crucial transcription factor. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction c-Myb was identified as the cellular homologue of v-Myb, an oncogene found in two avian retroviruses that induce leukemia (reviewed in [1]). In the AMV retrovirus, v-Myb induces myeloblastic leukemia, while a fusion between v-Myb and v-ets in the E26 retrovirus causes a mixed myeloid/erythroid leukemia. Following the molecular cloning of c-Myb, numerous studies performed in cell lines implicated this gene in the proliferation, survival and differentiation of hematopoietic cells (reviewed in [2]). The first observation that c-Myb also plays a physiological role came from studies of mice that lack c-Myb, which die embryonically with failures of erythroid and myeloid development [3]. While this demonstrated that c-Myb is essential for normal hematopoiesis, the embryonic lethality of these mice precluded further analysis of the function of c-Myb during differentiation. This review will focus on studies using recently developed viable alleles of c-Myb to gain fresh insights into the physiological function of c-Myb in the hematopoietic system. c-Myb has also been shown to play a role

Abbreviations: BCR, B cell receptor; BFU-E, blast forming unit-erythroid; CFU-E, colony forming unit-erythroid; DBD, DNA-binding domain; DN, double negative; DP, double positive; Epo, erythropoietin; ES cell, embryonic stem cell; HAT, histone acetyltransferase; HDAC, histone deacetylase; HSC, hematopoietic stem cell; Ig, immunoglobulin; MEP, megakaryocyte erythroid progenitor; Lin, lineage; LSK, lin− c-kit+ Sca-1+ ; LZ, leucine zipper; NRD, negative regulatory domain; SCF, stem cell factor; SP, single positive; TAD, transactivation domain; TCR, T cell receptor; Tpo, thrombopoietin. ∗ Corresponding author. Tel.: +61 3 9345 2483; fax: +61 3 9347 0852. E-mail addresses: [email protected] (K.T. Greig), [email protected] (S. Carotta), [email protected] (S.L. Nutt). 1044-5323/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2008.05.003

in non-hematopoietic cells; these studies are the subject of other reviews [4] and are not addressed here.

2. c-Myb structure/function c-Myb is a 75-kDa transcription factor [5]. The protein comprises three functional domains: an amino-terminal DNA-binding domain (DBD); a central transactivation domain (TAD) and a carboxy-terminal negative regulatory domain (NRD) (reviewed in [2]) (Fig. 1). The NRD includes a leucine zipper (LZ) motif that is postulated to mediate homodimer formation [6], however there is no evidence that this occurs in vivo. Alternative splicing of cMyb mRNA results in an additional exon between exons 9 and 10, generating an 89-kDa product [7]. The p89 form of c-Myb displays higher transactivation activity [8], however little is known about its functional relevance. c-Myb binds to the consensus sequence PyAACG/TG in vitro [9] and activates transcription via interactions with coactivator proteins, particularly CBP/p300 [10,11]. CBP and p300 are highly related proteins that function as coactivators for many transcription factors. CBP/p300 enhance transcription by providing a bridge between c-Myb and the basal transcription machinery. In addition, they possess histone acetyltransferase activity, enabling them to influence the chromatin context at promoters [12]. c-Myb participates by binding to histone H3 and positioning it for acetylation [13]. As well as modifying histones, CBP/p300 can also acetylate c-Myb, increasing its affinity both for DNA [14] and for CBP [15]. While the complex of c-Myb with CBP/p300 promotes transcriptional activity, recent experiments have shown that c-Myb also interacts with a histone deacetylase complex to dampen promoter

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Fig. 1. (A) Genomic structure of murine c-Myb, showing regions of conservation between the murine and human c-Myb genes; exons are shaded in blue, and conserved non-coding regions are shaded in pink. Note the highly conserved regions in intron 1 and in the 3 UTR. Sequence analysis was performed using the mVista program [86]. (B) Schematic of the murine c-Myb gene, showing the effects of the various c-Myb alleles at the DNA level. Diagram not to scale. (C) c-Myb protein structure, showing the effects of the various c-Myb alleles at the protein level. R1, R2, R3, regions of the DNA-binding domain; TAD, transcription activation domain; NRD, negative regulatory domain; LZ, leucine zipper domain.

activity [16]. Further experiments are required to investigate what regulates the balance of HAT and HDAC recruitment by c-Myb, as this may have important implications for local chromatin structure and promoter activity.

present it is unclear to what extent miR-150 is involved in downregulation of c-Myb during development. It will also be fascinating to examine what regulates miR-150 expression itself.

4. Regulation of c-Myb protein activity 3. c-Myb expression during hematopoiesis Studies in hematopoietic cell lines suggest that c-Myb expression is largely restricted to progenitor cells, with expression being downregulated as cells differentiate [17,18]. Such downregulation is significant, as overexpression of c-Myb inhibits the differentiation of erythroid and myeloid cell lines [19,20]. While c-Myb is generally not expressed in terminally differentiated cells, it can be upregulated in mature B and T cells upon activation [21]. Compared with these cell line studies, surprisingly little is known about cMyb expression in vivo, particularly in hematopoietic progenitor cells where c-Myb appears to function. Development of reagents to track c-Myb expression such as a gfp reporter mouse would help to address this issue. Such reagents would also aid experiments examining how the c-Myb gene is regulated. Various factors including NF␬B [22], WT-1 [23] and Ets-1 [24] have been suggested to regulate c-Myb transcription, while other studies conclude that c-Myb is primarily regulated by an attenuation block in the first intron [25]. More recent work has uncovered a micro-RNA, miR150, that binds to conserved sites in the 3 UTR of c-Myb and results in decreased protein expression [26]. In the lymphoid lineages miR-150 shows the opposite expression pattern to c-Myb, being expressed in mature B and T cells but not in their progenitors. At

c-Myb protein activity is also tightly regulated. Posttranslational modifications that generally inhibit c-Myb activity include phosphorylation [27], ubiquitination [28] and sumoylation [29,30]. Many of these modifications depend on the NRD and are lost in v-myb, contributing to the dysregulated activity of this protein. While clearly important, post-translational modifications of c-Myb have not been directly linked to signalling from any particular stimulus in vivo. For instance, p42MAPK [27] and casein kinase II [31] can phosphorylate c-Myb in vitro, but whether this occurs in vivo and the effect this might have on c-Myb activity are unclear. Wnt signalling can also lead to phosphorylation and degradation of c-Myb [32], but again the cellular contexts in which this occurs have not been determined. In contrast, the serine/threonine kinase Pim-1 stimulates c-Myb activity [33], possibly through direct phosphorylation [34]. Parallels between the loss of Pim-1 and loss of c-Myb activity (see below) suggest that this interaction may be biologically relevant, however it is unknown whether cytokines that induce Pim-1 also affect c-Myb activity. Collectively, these studies demonstrate that c-Myb activity is controlled on a number of levels. This is perhaps not surprising, given that overexpression of c-Myb inhibits differentiation and dys-

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Fig. 2. Diagram of hematopoiesis, illustrating the multiple defects observed in mouse models of c-Myb deficiency. (↓) A decrease in the frequency of the designated cell type/maturation stage in the absence of c-Myb. (↑) Cell populations whose frequency is increased by the loss of c-Myb function. Tick, cell types that are independent of c-Myb. Whether c-Myb plays a role in multipotent progenitors, including MEPs is unclear. Abbreviations: LMPP, lymphoid-primed multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte–macrophage progenitor; CLP/ELP, common lymphoid progenitor/early lymphoid progenitor; Meg, megakaryocyte.

regulated activity of v-myb contributes to cancer. While regulation of c-Myb activity is significant, many questions remain. A plethora of biochemical studies have been performed on c-Myb, uncovering many other interacting partners (reviewed in [35]), however most of these studies have been performed in vitro with overexpressed proteins, making their functional relevance unclear. The difficulties in studying c-Myb activity are partly due to the short half-life of c-Myb [36], but perhaps a more pressing problem is the lack of adequate assays for c-Myb activity. c-Myb activity is typically assessed by activation of artificial promoters in luciferase assays, a system that is unlikely to closely reflect subtle changes in c-Myb activity that may occur in vivo. 5. The function of c-Myb during early hematopoiesis Studies of the physiological role of c-Myb have utilised a large variety of mutant alleles (Figs. 1 and 2, Table 1). The earliest of these was a null allele (c-Myb− ) that established c-Myb as an essential gene for definitive hematopoiesis [3]. More recently, other alleles have been developed that are viable as adults, enabling the precise roles of c-Myb in hematopoiesis to be analysed. These include three independently derived conditional alleles of c-Myb (c-Mybex3–6 , cMybex6 , c-Mybex2 ) that have been used to examine the effects of c-Myb deficiency in the B and T cell lineages [37–42]. A c-Myb allele that results in reduced expression (c-MybKD ) was inadvertently created during the development of the c-Mybex3–6 conditional allele [38]. Reduced c-Myb expression is also observed in mice that carry a miR-150 transgene, while mice deficient for miR-150 display increased c-Myb expression [26]. Other alleles result in decreased c-Myb activity. Fusion of the c-Myb DBD to the engrailed repressor created a dominant interfering allele that has been expressed as a transgene in T cells (MEnT) [43]. Three hypomorphic alleles have been isolated in forward genetic screens; these contain point mutations in the DBD (c-MybPlt3 ), TAD (c-MybM303V ) and LZ (c-MybPlt4 ) domains that lead to reduced c-Myb activity [44,45]. In the case

of the c-MybM303V allele, this is at least partly due to decreased interaction with p300. A similar effect is seen in mice homozygous for point mutations in the p300 KIX domain (p300KIX ) that mediates its interaction with the TAD of c-Myb. Surprisingly, analogous mutations in the KIX domain of CBP do not cause the same phenotypic effects, confirming the importance of the c-Myb–p300 but not the c-Myb–CBP interaction for hematopoiesis [46]. This section will describe how these various mouse models have been used to illuminate the role of c-Myb in normal hematopoiesis. 5.1. Hematopoietic stem cells Despite the multilineage defects observed in c-Myb deficient embryos, definitive hematopoietic stem cells (HSCs) are generated in the absence of c-Myb. The c-Myb−/− fetal liver does contain some cells with a hematopoietic progenitor phenotype, albeit at a reduced number [47]. Progenitor cells are also generated by in vitro differentiation of c-Myb−/− embryonic stem (ES) cells [48] and cMyb−/− ES cells give rise to T cell progenitors in Rag1−/− chimeras [49]. These studies suggest that the failure of erythroid and myeloid development in c-Myb−/− embryos is not due to a lack of definitive HSCs, but rather due to a defect in expansion and/or differentiation of these cells. By contrast, mice that have decreased c-Myb expression or activity show increased numbers of hematopoietic progenitor cells. Embryos with a knockdown allele of c-Myb display an increased frequency of progenitor cells in their fetal liver [38]. Similarly, c-MybM303V/M303V and c-MybPlt4/Plt4 mice show increased numbers of lin− c-kit+ Sca-1+ (LSK) cells within their bone marrow, and MybPlt4/Plt4 bone marrow generates elevated numbers of blast colonies in vitro [44,45]. While these studies suggest that the progenitor compartment is expanded when c-Myb activity is reduced, it is unclear whether this is due to an increase in long-term HSCs or an increase in short-term progenitor cells. Assays to quantify longterm HSCs rely on both HSC activity and subsequent differentiation,

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Table 1 Phenotypes described for the various mutant alleles of mouse c-Myb Mouse model

Allele

Effect on c-Myb

Phenotype of homozygous mice

References

Null

c-Myb−

Neomycin cassette in exon 6 introduces a stop codon

[3]

Knockdown

c-Mybkd

Neomycin cassette in intron 6 reduces protein expression

Point mutation in DBD

c-MybPlt3

Hypomorphic allele, unknown mechanism

Point mutation in TAD

c-MybM303V

Hypomorphic allele, at least partly due to decreased interaction with p300

Point mutation in LZ

c-MybPlt4

Hypomorphic allele, unknown mechanism

Conditional knockout

c-Mybex3–6

Deletion of exons 3–6 generates a protein lacking the DBD

Embryonic lethal by E15.5; drastically reduced numbers of erythroid and myeloid cells, megakaryocytes present but reduced in number Reduced viability; anemic; increased proportion of immature cells; block at the DN2 to DN3 transition; reduced number of B cells Increased numbers of megakaryocytes and platelets; reduced numbers of B and T cells; anemic Increased numbers of megakaryocytes and platelets; mildly anemic; partial block at DN3 to DN4 transition; partial block at pro-B to pre-B transition; increased number of LSK cells Increased numbers of megakaryocytes and platelets; reduced numbers of B and T cells; anemic GATA-Cre (germline deletion): indistinguishable from c-Myb−/− mice CD4-Cre (deletion in DP cells): decreased CD4/CD8 ratio in the thymus CD2-Cre (deletion in DN2 cells): block at the DN3 to DN4 transition cwLck-Cre (deletion in DN2 cells): partial block at the DN3 to DN4 transition CD4-Cre (deletion in DP cells): number of DP cells modestly reduced, decreased CD4/CD8 ratio in the thymus Lck-Cre (deletion in DP cells): reduced number of DP and CD4 SP cells cwLck-Cre (deletion in DN2 cells): reduced number of DN, DP and SP cells, reduced number of ␥␦ T cells, block at the DN3 to DN4 transition CD19-Cre (deletion in pro-B cells): partial block at the pro-B to pre-B transition Partial block at the pro-B to pre-B transition; partial block at the DN3 to DN4 transition; decreased number of B1 cells Increased number of B1a cells Partial block at the DN3 to DN4 transition; reduced number of DP thymocytes Increased number of platelets; reduced numbers of B and T cells; anemic

Conditional knockout

c-Mybex6

c-Mybex2

Deletion of exon 6 disrupts the DBD and introduces a stop codon

Deletion of exon 2 generates a frameshift, leading to a drastically truncated protein

Ectopic expression of miR-150

miR-150 tg

Decreased c-Myb protein expression

Deletion of miR-150 Dominant interfering c-Myb in T cells

miR-150− MEnT

Disrupted p300 interaction

p300KIX

Increased c-Myb protein expression Dominant interfering protein; most likely interferes with all Myb proteins Mutations in the KIX domain of p300 disrupt its interaction with c-Myb

[44]

[45]

[44]

[38] [41] [39] [40] [40]

[37] [37]

[42] [26]

[26] [43,70] [46]

K.T. Greig et al. / Seminars in Immunology 20 (2008) 247–256

Conditional knockout

[38]

K.T. Greig et al. / Seminars in Immunology 20 (2008) 247–256

both of which are abnormal in c-Myb deficient mice. Characterization of progenitor cells by flow cytometry is also problematic as a key marker of these cells, c-kit, appears to be regulated by c-Myb in at least some of these populations (unpublished data and [50]). The LSK compartment in c-MybM303V/M303V mice contains a higher proportion of cycling cells [45], which would be consistent with an expansion of progenitor cells rather than long-term HSCs. 5.2. Myeloid cells Many of the early studies of c-Myb concentrated on its role in myeloid development. This focus is understandable, given that vmyb was identified due to its involvement in myeloid leukemia. Consistent with this role, overexpression of c-Myb was found to inhibit myeloid differentiation [19] and many of the canonical c-Myb target genes were identified in myeloid cells (Table 2). In contrast, defects in myeloid development are notably absent amongst the hematopoietic defects found in mice with reduced c-Myb activity (Fig. 1). Mice with hypomorphic alleles of c-Myb lack eosinophils, yet have normal numbers of other myeloid cells including monocytes, macrophages and neutrophils [44,45]. Careful analysis of myeloid progenitors in c-MybPlt4/Plt4 mice failed to show any major defects in their frequency or growth factor responsiveness [51], suggesting that the differentiation and proliferation of myeloid cells is not overly sensitive to the level of c-Myb activity. These studies demand a reassessment of the role of c-Myb in myelopoiesis. It is possible that studies of v-myb and overexpressed c-Myb reveal roles that are not displayed in vivo. The fact that c-Myb−/− embryos fail to develop myeloid cells [3] implies that c-Myb is important for myeloid development, but it is also possible that myeloid cells do not develop in these embryos because of defects in hematopoietic progenitor cells. c-Myb−/− ES cells do not appear to contribute to myeloid development in wild type or Rag−/− chimeras [47,49], however these studies are difficult to interpret as they depend on c-Myb−/− HSC activity and the marker they used to detect c-Myb−/− cells (Ly9) is not expressed by all myeloid cells [52]. It is interesting to consider whether the discrepancies between c-Myb−/− embryos and c-Myb deficient adult mice – both in the stem cell and myeloid compartments – may reflect differences between adult and fetal hematopoiesis. A similar situation is observed in mouse models of PU.1 and AML-1 deficiency, both of which demonstrate a lack of myeloid development in the fetal liver but not in the neonate or adult [53–55]. Alternatively, the discrepancies may relate to the level of c-Myb activity in each mouse model; perhaps the only viable hypomorphic alleles that can be isolated are those that retain sufficient c-Myb activity to support myelopoiesis. A third possibility is that the closely related gene B-Myb can compensate for c-Myb in myeloid, but not stem cell, development. B-Myb is expressed in a large range of cell types, including myeloid cells, and in at least some instances can activate the same promoters as c-Myb [56]. Studies involving conditional deletion of B-Myb and/or c-Myb in myeloid cells would provide further insights into the potential roles of these proteins in myeloid development. 5.3. Erythroid cells Erythropoiesis occurs in two distinct waves during development. Primitive hematopoiesis occurs in the yolk sac and produces large nucleated erythroid cells that synthesize embryonic globins. Definitive erythropoiesis is later established in the aorta-gonad-mesonephros region, fetal liver and subsequently in the bone marrow. During both primitive and definitive hematopoiesis, the erythroid lineage arises from the bipotential megakaryocyte–erythroid progenitor (MEP) [57]. The earliest com-

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mitted erythroid progenitors are the slowly proliferating burst forming unit-erythroid (BFU-E) cells. These cells further differentiate into rapidly dividing colony forming unit-erythroid (CFU-E) cells. The differentiation of CFU-E progenitors requires 3–5 cell divisions and results in decreased cell size, chromatin condensation and hemoglobinization, leading to the enucleation and expulsion of other organelles. While primitive erythrocytes do not express c-Myb, expression is detected at the onset of definitive erythropoiesis [58]. In accordance with this expression pattern, primitive erythropoiesis is unaffected in c-Myb−/− embryos, which maintain normal hematocrit levels until E13.5. Definitive erythropoiesis, however, is profoundly impaired in c-Myb−/− embryos, which die by E15.5 with severe anemia [3]. This failure to generate definitive erythrocytes in vivo is not due to abnormalities in the fetal liver environment, as in vitro differentiated c-Myb−/− ES cells fail to generate detectable BFU-E colonies and produce a reduced number of CFU-E colonies [48]. The anemia of c-Myb−/− embryos is also recapitulated in other c-Myb deficient mice, as well as in p300KIX/KIX mice (Table 1). Studies in cell lines demonstrate that c-Myb is required for expansion of erythroid progenitors but must be downregulated to permit differentiation into mature erythrocytes. Consistent with this, experiments performed on cultured pro-erythroblasts (corresponding to late BFU-Es [59]) reveal that c-Myb is expressed in proliferating cells and is rapidly downregulated upon erythropoietin (Epo)-induced differentiation [60,61]. The switch from c-Myb-controlled erythroblast proliferation to Epo-induced erythrocyte differentiation is mediated by the transcription factor GATA-1. At the onset of terminal differentiation, cytoplasmic GATA1 translocates into the nucleus and represses c-Myb expression by binding to GATA-1 binding sites within the c-Myb promoter thus allowing further differentiation [60]. Insights have been made into how c-Myb promotes the proliferation of erythroid progenitors. Erythroid proliferation depends on the combined action of Epo and stem cell factor (SCF), the ligand of c-kit, while differentiation requires Epo alone. Similar to c-Myb deficient mice, strains harbouring mutations in the genes encoding c-kit (Kit) and SCF (Kitl) exhibit prenatal lethality due to severe anemia, while primitive yolk sac derived erythropoiesis is normal [62]. The similarity between these phenotypes in the erythroid compartment makes c-kit an attractive candidate to at least partially mediate the function of c-Myb in erythroid progenitors. Indeed, c-Myb was shown to bind to the Kit promoter and positively regulate its expression [63] (Table 2). Furthermore, inducible deletion of c-Myb in erythroblasts results in decreased Kit expression [64], which may contribute to the defective proliferation of erythroid progenitors observed in c-Myb deficient mice. c-Myb not only regulates the proliferation of committed erythroid progenitors but also is important for the commitment of immature progenitors towards the erythroid lineage. Two recent studies using mice with reduced c-Myb expression showed that low levels of c-Myb lead to the accumulation of immature hematopoietic progenitors which express GATA2, PU.1 and Fli1, factors that are normally downregulated to allow erythroid commitment [38,64]. Whether this impaired erythroid commitment directly results in MEPs defaulting to the megakaryocytic pathway remains to be determined (see below). 5.4. Megakaryocytes The early development of megakaryocytes from the bipotential MEP remains poorly understood. MEPs give rise to the highly proliferative BFU–megakaryocyte and the more mature smaller CFU–megakaryocyte, both of which express the progenitor marker CD34 (reviewed in [65]). During development the diploid

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Table 2 A partial list of reported c-Myb target genes c-Myb binds to endogenous promoter?

Effect of inducible c-Myb expression in vitro?

Altered expression in mouse models of c-Myb deficiency?

References

Adenosine deaminase (Ada)

Yes: T and myeloid cell lines

?

[84,87]

Bcl2 (Bcl2)

Yes: splenic T cells

Normal in DP thymocytes

[37,40,72,88–91]

Carbonic anhydrase 1 (Car1)

Yes: erythroid cell line

?

[92]

c-Kit (Kit)

?

↓ on CFU-E cells

[50,63,64,93]

c-myc (Myc)

Yes: T and myeloid cell lines

?

[84,91,94–96]

CD4 antigen (Cd4)

?

↓ when dominant interfering c-Myb is induced (T cell line—MERT) ↓ when c-Myb expression is reduced (myeloid–erythroid cell line—siRNA) ↑ when c-Myb is induced (myeloid cell line—myb-ER) ↓ when dominant interfering c-Myb is induced (erythroid cell line—MERT) ↓ when c-Myb is withdrawn (myeloid cells—myb-ER) ↑ when c-Myb is induced; direct effect (myeloid cell line—myb-ER) ↓ when c-Myb expression is reduced (myeloid–erythroid cell line—siRNA) ?

[41,75,97]

Cyclin B1 (Ccnb1)

No: myeloid–erythroid cell line

Normal on DP thymocytes and CD4 SP cells ?

[98]

Neutrophil elastase 2 (Ela2)

No: T and myeloid cell lines

?

[84,99,100]

Gata-3 (Gata3)

Yes: thymocytes

↓ in DP and CD4 SP thymocytes

[41]

H2A histone Z (H2afz)

Yes: thymocytes

↓ in DN and DP thymocytes

[39]

Methionine adenyl-transferase II, alpha (Mat2a)

Yes: T and myeloid cell lines

?

[84,101,102]

Mim-1 (Lect2) Pre-T cell antigen receptor alpha (Ptcra) Recombination activating gene 2 (Rag2)

? No: T and myeloid cell lines Yes: thymocytes, T and B cell lines Yes: thymocytes ?

? Normal in DN thymocytes Normal in DN thymocytes

[103,104] [37,69,84] [37,79,105,106]

Decreased number of ␥␦ T cells Decreased number of ␥␦ T cells

[74,107,108] [73]

TCR delta chain (Tcrd) TCR gamma chain (Tcrd)

↑ when c-Myb is induced; direct effect (myeloid–erythroid cell line—myb-ER) ↓ when c-Myb expression is reduced (myeloid cell line, thymocytes and BM CD34+ cells—siRNA) No effect when c-Myb expression is reduced (myeloid–erythroid cell line—siRNA) ↓ when dominant interfering c-Myb is induced (T cell line—MERT) ↓ when dominant interfering c-Myb is induced (T cell line—MERT) No effect when c-Myb expression is reduced (myeloid–erythroid cell line—siRNA) ? ? ? ? ?

c-Myb has been demonstrated to bind to the promoters of each of these genes and can activate transcription in reporter assays; additional evidence that these genes are targets of c-Myb is listed. An additional 29 target genes were identified by subtraction cloning; 24 showed decreased expression in CD4 T cells expressing dominant interfering c-Myb and binding of c-Myb to the endogenous promoter could be demonstrated for 12 target genes in a progenitor cell line [85]. MERT, inducible dominant interfering c-Myb.

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Target gene

K.T. Greig et al. / Seminars in Immunology 20 (2008) 247–256

megakaryocyte progenitors undergo repeated rounds of DNA replication without cell division, resulting in a nuclear DNA ploidy that ranges from 2N to 128N. This process is accompanied by increases in cell size and protein levels, and eventually leads to the development of long cytoplasmic extensions that shed platelets into the circulation. In contrast to its positive role in regulating erythropoiesis, c-Myb negatively influences megakaryopoiesis, as c-Myb deficient mice and p300KIX/KIX mice exhibit significant thrombocytosis (Table 1). Interestingly, the overproduction of megakaryocytes is independent of thrombopoietin (Tpo), the major physiological regulator of megakaryopoiesis, as c-MybPlt4/Plt4 and c-MybPlt3/Plt3 mice show a similar increase in platelet count when deficient for the Tpo receptor, c-Mpl [44,51]. c-Myb appears to inhibit megakaryocyte differentiation very early in development as c-Myb hypomorphic mice display increased numbers of megakaryocyte progenitors, and the lineage potential of early progenitor cells is skewed towards megakaryocyte development [45,51]. The megakaryocytes that develop in c-Myb deficient and p300KIX/KIX mice display a reduction in modal ploidy [46,51,66], however it is unclear whether this is due to defects in maturation or reflects alterations in the cell cycle. As megakaryocytes and erythroid cells arise from a common progenitor, the above data may indicate that c-Myb is a key regulator of the erythrocyte/megakaryocyte lineage choice. c-Myb is expressed in MEPs and disruption of the c-Myb–p300 interaction leads to reduced erythroid differentiation accompanied by increased megakaryocyte differentiation ([66] and (Table 1)). The molecular mechanism by which c-Myb regulates MEP differentiation is unknown, as is how c-Myb interacts with the known mediators of erythro-myeloid lineage specification such as GATA1/2, Scl and PU.1. In order to understand the role of c-Myb in megakaryocytic commitment it will be important to determine at which megakaryocytic developmental stages c-Myb is expressed, and with which factors c-Myb interacts. 5.5. Early lymphopoiesis The early lethality of c-Myb−/− embryos initially obscured the role of c-Myb in B and T cell development. The first evidence that c-Myb is required for lymphopoiesis came from chimeric mice in which c-Myb−/− ES cells failed to contribute to either mature B or T cell development [47,49]. Subsequently, new alleles of c-Myb have demonstrated a critical role for c-Myb in multiple stages of lymphocyte development. 5.5.1. T cells Early T cell progenitors in the thymus lack expression of the coreceptors CD4 and CD8 and are described as double negative (DN). The DN compartment can be subdivided into four stages: DN1, DN2, DN3 and DN4. Rearrangement of the Tcrb locus is completed within the DN3 stage and the resulting pre-T cell receptor (TCR) provides signals for proliferation, survival and maturation to the DN4 and subsequently the CD4+ CD8+ double positive (DP) stage of development. DP thymocytes undergo rearrangement of the Tcra locus, followed by positive and negative selection and lineage commitment to become either CD4 or CD8 single positive (SP) thymocytes (reviewed in [67]). c-Myb shows highest expression in DN and DP thymocytes, with expression declining at the SP stage [37]. This downregulation appears critical in allowing differentiation to proceed, as overexpression of c-Myb is associated with T cell acute lymphoblastic leukemia in humans [68]. Mice deficient for c-Myb display decreased numbers of thymocytes and defects at multiple stages of T cell development (Table 1). In particular, the transition from the DN3 stage to the DN4 stage is

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partially blocked by several mutant alleles of c-Myb. This defect is apparently not due to differences in pre-TCR expression [37], despite the gene encoding the pre-TCR (Ptcra) being described as a c-Myb target [69] (Table 2). c-Myb deficient DN3 thymocytes are also inefficient at completing V(D)J rearrangement at the Tcrb locus, even though they show normal expression of Rag1 and Rag2 [37], however it is not clear that this is the sole reason for the DN3 arrest. While DN3 cells show normal survival and proliferation [37], it is possible that there is a defect in survival or proliferation of DN4 cells. Examination of mice that express a dominant interfering form of c-Myb in T cells suggests that c-Myb is required for thymic proliferation following ␤-selection [70], with the caveat that this protein likely interferes with the related protein B-Myb as well. That the kinase reported to regulate c-Myb activity, Pim-1, has also been implicated in proliferative signalling during ␤-selection is intriguing [71]. Pim1 transgenic mice show an increased number of DN4 cells, and expression of the Pim1 transgene rescues the DN3 block in Rag1−/− thymocytes except in the context of the dominant interfering c-Myb allele [70]. In addition to the block at the DN3 to DN4 transition, c-Myb deficient thymocytes show a decreased percentage of DP cells. This defect has been related to increased apoptosis of DP cells [37,40,72], however while some ascribe this to decreased Bcl2 expression [72], others fail to find any difference in expression of Bcl2 or other anti-apoptotic genes [37,40]. Moreover, the defect cannot be rescued by a Bcl2 transgene, suggesting that decreased survival is not the sole reason for the phenotype (Bender TP, unpublished data). Decreased Tcra rearrangement has also been proposed to contribute [37], suggesting that as for DN cells, c-Myb plays a complex role in DP differentiation. Moreover, the defective Tcrb and Tcra recombination, coupled with reports that c-Myb regulates Tcrd and Tcrg expression [73,74] (Table 2), suggests a generalised role for c-Myb in regulating TCR rearrangement. c-Myb also plays a role in commitment to the CD4 lineage. c-Myb mRNA levels decline more rapidly in cells undergoing commitment to the CD8 lineage than the CD4 lineage [41], and c-Myb deficient mice show a more severe decrease in CD4 than CD8 SP thymocytes [37]. Moreover, this decrease in CD4 cells is not rescued by providing a productively rearranged TCR that is positively selected on MHCII [41]. Although there are some studies suggesting that c-Myb regulates Cd4 expression [75] (Table 2), this is not a consistent finding and is unlikely to be the reason for the defect observed. Instead, recent work suggests that this effect on CD4 cells is at least partly due to defects in Gata3 expression [41]. Gata-3 is a transcription factor required for CD4 lineage commitment [76]. c-Myb deficient DP cells show reduced levels of Gata3 expression, and c-Myb binds to the Gata3 promoter in vivo, suggesting that Gata3 is a direct target of c-Myb activity (Table 2). The authors of this work suggest a model by which c-Myb plays a role in upregulating Gata3 following TCR ligation and positive selection. This work provides a rare example where the activity of c-Myb can be linked to both an upstream regulatory pathway and a downstream effect on differentiation. This system may enable further studies of the signal transduction pathways that regulate c-Myb activity. 5.5.2. B cells The earliest B cell progenitors in the bone marrow are variously termed pre-pro-B, CLP-2 or Fraction A cells [77]. These cells differentiate to become pro-B cells, which upregulate expression of the transcription factor Pax5 and become committed to the B cell lineage. Committed B cells can be identified by their expression of Cd19, a Pax5 target gene (reviewed in [78]). Successful rearrangement of the Ig heavy chain (Igh) locus at the pro-B cell stage causes cells to proceed to the pre-B cell stage. Pre-B cells express the IgH on their cell surface in association with the surrogate light chains

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to form the pre-B cell receptor (BCR). Signals from the pre-BCR lead to proliferation and initiate recombination of the Ig light chain (Igl) locus. Productive Igl rearrangement results in expression of the BCR and progression to the immature B cell stage; these cells exit the bone marrow and complete their development in the periphery. c-Myb is expressed at the pro-B and pre-B cell stages of development, but is downregulated in immature and mature B cells [42], suggesting it may play a role early in B cell development. Mice with decreased c-Myb expression or activity exhibit profound reductions in their B cell compartment [38,42,44,45]. In most cases this is described as being a block in the transition from the pro-B cell stage to the pre-B cell stage, however data from our laboratory suggests that the defect is apparent even within the pro-B cell compartment, with a reduction in the number of committed CD19+ pro-B cells (KTG and Bender TP, unpublished data). The only conditional deletion of c-Myb reported in B cells to date used mice expressing Cre under the Cd19 promoter [42]. These mice express Cre efficiently from the late pro-B cell stage on, meaning that they could not be used to assess the requirement for c-Myb prior to the point of B cell commitment. Given that hypomorphic mutants of c-Myb do show a defect prior to this point (KTG, unpublished), it would be interesting to investigate the effect of deleting c-Myb at an earlier stage of B cell development. B cell development appears to be exquisitely sensitive to the level of c-Myb, with significant defects apparent in both heterozygous mice and when miR-150 is ectopically expressed [26]. At present there is little understanding of why B cell development is so dependent on c-Myb activity. c-Myb is reported to regulate Rag2 expression [79] (Table 2), however both Rag1 and Rag2 levels are normal in B cells from c-Myb hypomorphic mice (KTG, unpublished data). Moreover, decreased Rag activity would not explain the decrease in CD19+ pro-B cells. Others have suggested that c-Myb deficient pro-B cells show decreased survival when cultured [26]. Emerging data from our laboratory, and others, suggests that the reduced B lymphopoiesis in c-Myb deficient mice is related to a profound defect in IL-7 signalling (KTG and Bender TP, unpublished data). IL-7 is a critical cytokine for B cell development, acting on both pro- and pre-B cells, and IL-7 deficient mice display similar defects in the B cell lineage to c-Myb deficient mice [80,81]. It is interesting to note that Pim1 is upregulated by IL-7 signalling [82], and the level of Pim1 expression influences the ability of pro-B cells to respond to IL-7 [83]. As for T cells, there is no data demonstrating a link between c-Myb and Pim-1 in B cells, however there is enough circumstantial evidence to warrant further investigation of the relationship between these two proteins. In addition to conventional B2 cells, B1 cells also appear to be sensitive to the level of c-Myb expression. c-Myb deficient mice have decreased B1 cells in their peritoneum [42], while mice that lack miR-150 have a corresponding increase in B1a cells in both their spleen and peritoneal cavity [26]. Since loss of miR-150 does not lead to a detectable increase in c-Myb protein levels in B1a cells, the precise reason for the increased number of B1 cells is unclear. Moreover, it is interesting that loss of miR-150 has no effect on developing B cells, implying that miR-150 may predominantly play a role in fine-tuning c-Myb levels in mature cells, where miR150 is expressed and c-Myb can be induced upon activation.

6. The challenge ahead As outlined above, c-Myb plays many roles in hematopoiesis, both in progenitor cells and in differentiation towards multiple lineages. In many cases it is a challenge to determine whether mature cell defects in c-Myb deficient mice are due to effects of c-Myb on progenitor cells as opposed to lineage-specific functions.

It is also difficult to ascertain whether the phenotypes observed are due to effects on proliferation, differentiation or survival, as in many cases c-Myb has multiple effects, even within the same lineage. While some target genes of c-Myb have been identified (Table 2), these do not fully explain the defects observed. Moreover, many target genes have only been identified in cell lines and have not been adequately validated either in vitro or in vivo; in a recent study, very few reported c-Myb target genes were affected by siRNA-mediated knockdown of c-Myb expression [84]. Given the large variety of defects observed when c-Myb is deficient, there is unlikely to be a single target gene in each cell type that explains the phenotype. Global analyses of c-Myb suggest there are likely to be multiple target genes, and many of these may be cell-type specific [85]. The finding that c-Myb regulates expression of the histone variant H2A.Z suggests that c-Myb may subtly influence the transcription of many genes indirectly through impacts on chromatin [39]. It is likely that c-Myb functions with multiple other proteins in a complex regulatory network, however beyond the c-Myb–p300 interaction there is little data on protein partners of c-Myb in the various contexts where they operate. Work over many years has led to established systems for manipulating c-Myb activity in defined cell types and maturation stages, and well characterised cell based systems to assay proliferation, differentiation and survival are available. Combining these systems with recently developed tools such as ChIP-chip could provide a powerful approach to examining cMyb function in a sophisticated way. In addition, forward genetic screens have been immensely useful in identifying viable hypomorphic alleles of c-Myb; such screens could also be used to find genes that interact with c-Myb to generate the mutant phenotype. Acknowledgements The authors thank Timothy Bender, Douglas Hilton, Axel Kallies and Ian Majewski for critical comments on the manuscript. References [1] Wolff L. Myb-induced transformation. Crit Rev Oncog 1996;7:245–60. [2] Oh IH, Reddy EP. The myb gene family in cell growth, differentiation and apoptosis. Oncogene 1999;18:3017–33. [3] Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, et al. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 1991;65:677–89. [4] Ramsay RG. c-Myb a stem-progenitor cell regulator in multiple tissue compartments. Growth Factors 2005;23:253–61. [5] Weston K, Bishop JM. Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell 1989;58:85–93. [6] Nomura T, Sakai N, Sarai A, Sudo T, Kanei-Ishii C, Ramsay RG, et al. Negative autoregulation of c-Myb activity by homodimer formation through the leucine zipper. J Biol Chem 1993;268:21914–23. [7] Shen-Ong GL. Alternative internal splicing in c-myb RNAs occurs commonly in normal and tumor cells. EMBO J 1987;6:4035–9. [8] Woo CH, Sopchak L, Lipsick JS. Overexpression of an alternatively spliced form of c-Myb results in increases in transactivation and transforms avian myelomonoblasts. J Virol 1998;72:6813–21. [9] Weston K. Extension of the DNA binding consensus of the chicken c-Myb and v-Myb proteins. Nucleic Acids Res 1992;20:3043–9. [10] Dai P, Akimaru H, Tanaka Y, Hou DX, Yasukawa T, Kanei-Ishii C, et al. CBP as a transcriptional coactivator of c-Myb. Genes Dev 1996;10:528–40. [11] Oelgeschlager M, Janknecht R, Krieg J, Schreek S, Luscher B. Interaction of the co-activator CBP with Myb proteins: effects on Myb-specific transactivation and on the cooperativity with NF-M. EMBO J 1996;15:2771–80. [12] Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 1996;87:953–9. [13] Mo X, Kowenz-Leutz E, Laumonnier Y, Xu H, Leutz A. Histone H3 tail positioning and acetylation by the c-Myb but not the v-Myb DNA-binding SANT domain. Genes Dev 2005;19:2447–57. [14] Tomita A, Towatari M, Tsuzuki S, Hayakawa F, Kosugi H, Tamai K, et al. c-Myb acetylation at the carboxyl-terminal conserved domain by transcriptional coactivator p300. Oncogene 2000;19:444–51. [15] Sano Y, Ishii S. Increased affinity of c-Myb for CREB-binding protein (CBP) after CBP-induced acetylation. J Biol Chem 2001;276:3674–82.

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