Oncogene (2004) 23, 4211–4219
& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00
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Structure and regulated expression of mammalian RUNX genes
Ditsa Levanon1 and Yoram Groner*,1
1
Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel
The RUNX are key regulators of lineage-specific gene
expression in major developmental pathways. The expression of RUNX genes is tightly regulated, leading to a
highly specific spatio/temporal expression pattern and to
distinct phenotypes of gene knockouts. This review
highlights the extensive structural similarities between
the three mammalian RUNX genes and delineates how
regulation of their expression at the levels of transcription
and translation are orchestrated into the unique RUNX
expression pattern.
Oncogene (2004) 23, 4211–4219. doi:10.1038/sj.onc.1207670
Keywords: gene structure; promoter analysis; translation control; signaling pathways; tissue-specific expression
Introduction
Three highly conserved human and mouse runt domain
(RD) genes RUNX1, RUNX2 and RUNX3 (RUNX is
used when description applies to both human and
mouse, whereas Runx is used when findings involve only
the mouse genes) were identified and localized on human
chromosomes 21q22.12, 6p21 and 1p36.1 and on mouse
chromosomes 16, 17 and 4, respectively (Bae et al., 1994,
1995; Levanon et al., 1994; Avraham et al., 1995; Calabi
et al., 1995; Zhang et al., 1997a). The RUNX are key
regulators of lineage-specific gene expression in major
developmental pathways. The genes arose early in
evolution and maintained extensive structural similarities in mammals (Coffman, 2003; Levanon et al.,
2003b; Rennert et al., 2003). Gene knockout (KO)
studies revealed that Runx1 is indispensable for
definitive hematopoiesis (Okuda et al., 1996; Wang
et al., 1996), and Runx2 is essential for osteogenesis
(Komori et al., 1997; Otto et al., 1997). More recently,
Runx3 was shown to play a role in neurogenesis and
thymopoiesis (Inoue et al., 2002; Levanon et al., 2002,
2003b; Taniuchi et al., 2002; Woolf et al., 2003) and in
the control of cell proliferation and apoptosis of gastric
epithelium (Li et al., 2002). All RUNX proteins bind to
the same DNA motif and activate or repress transcription of target genes through recruitment of common
transcriptional modulators. In spite of this, the RUNX
*Correspondence: Y Groner; E-mail: [email protected]
gene products have well-defined biological functions,
which are reflected in the phenotypes of the corresponding KO mice. This occurrence is orchestrated through a
tightly regulated spatio/temporal expression pattern of
the RUNX genes.
Here we review the current knowledge about the
structure and regulated expression of the three mammalian RUNX genes. Some aspects of these issues were
previously reviewed (Ducy, 2000; Levanon et al.,
2003a, b; Otto et al., 2003). Due to space limitations,
regulation of RUNX expression by alternative splicing
and by post-translational modifications is not covered.
Genomic organization and regulatory elements of the
RUNX genes
RD-containing genes are found in phylogenetically
diverse organisms (Ito and Bae, 1997; Bae and Lee,
2000; Kataoka et al., 2000; Eggers et al., 2002; KalevZylinska et al., 2002; Levanon et al., 2003b; Rennert
et al., 2003; Stricker et al., 2003) (Figure 1a). Analysis of
DNA and protein sequence identified a nematode RD
gene as the most primitive orthologue, suggesting that
RUNX may have arisen in bilaterian metazoans
(Rennert et al., 2003). However, a final conclusion
should wait until the complete annotated genomic
sequence of the lower species Cnidarians becomes
available. Lower organisms contain a single RUNX
gene (Figure 1a), but climbing the phylogenetic ladder
to arthropods and vertebrates is accompanied by
multiplicity of RUNX genes that occurred through gene
duplication (Rennert et al., 2003).
The three mammalian RUNX genes maintained
extensive structural similarities (Ghozi et al., 1996;
Geoffroy et al., 1998; Xiao et al., 1998; Bangsow et al.,
2001; Levanon et al., 2001b; Rennert et al., 2003), which
extend beyond the RUNX locus itself to include the
neighboring paralogous genes CLIC and DSCR (Levanon et al., 2001b, 2003a; Eggers et al., 2002) (Figure 1b).
Of the mammalian genes, RUNX3 is the smallest and
contains the fewest number of exons, all of which are
highly conserved among the three RUNX genes (Bangsow et al., 2001) (Figure 1b). The structural similarities
between the mammalian RUNX include genomic
elements that are involved in expression regulation.
Each of the three genes is transcriptionally regulated by
two distantly located promoter regions, P1 (distal) and
P2 (proximal) (Ghozi et al., 1996; Stewart et al., 1997;
Structure and expression of RUNX
D Levanon and Y Groner
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Figure 1 RUNX gene phylogeny, gene structure and elements involved in expression regulation. (a) Phylogenetic illustration of
RUNX genes showing the gene number and promoter usage in different animals. The three lower nondesignated branches in the
phylogenetic tree represent the animal groups (from bottom up) sponges, cnidarians (e.g. jellyfish) and acoelomates (e.g. flatworms).
The more primitive animals contain one gene regulated by the P2 promoter (Rennert et al., 2003). (b) Genomic organization of the
human RUNX genes (common exons are shown in similar color). Chromosomal localizations and conserved neighboring genes (CLIC
and DSCR) are depicted. The two promoters P1 and P2 and initiator ATGs are indicated. 50 UTRs are in yellow and orange and
30 UTRs are in blue. The proviral insertions in the CD2-MYC lymphomas are indicated by the name of the derived cell line (T6i, T47i,
T1i) along with the chromosomal insertion sites (til-1, Dsi1) (Cameron et al., 2003). (c) Scheme depicting the common structure of
mammalian P1-50 UTR. It contains four exons; the middle two are alternatively spliced and designated as exon/introns. RR marks the
two perfectly conserved RUNX-binding sites, underlined in the conserved 18 bp sequence 50 -CAACCACAGAACCACAAG-30 .
(d) Scheme depicting the common structure of the mammalian P2-50 UTR. It spans one exon, which terminates with an in-exon splice
site (AG) and is preceded by the intronic branch point signal CTRAY. The P2-50 UTRs contain IRES and are surrounded by an
exceptionally large CpG island marked by the gray cloud
Fujiwara et al., 1999; Drissi et al., 2000; Bangsow et al.,
2001; Rini and Calabi, 2001; Telfer and Rothenberg,
2001; Xiao et al., 2001). The P2 promoter is nested
within a particularly large CpG island (Bangsow et al.,
2001; Levanon et al., 2001b) (Figure 1d), which is also
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conserved in Fugu (Eggers et al., 2002). No such CpGrich region is found in the P1 promoter. Interestingly
however, a highly conserved CpG island is located at the
30 -end within the last coding exon of the RUNX genes
(Bangsow et al., 2001; Levanon et al., 2001b). These
Structure and expression of RUNX
D Levanon and Y Groner
4213
highly conserved CpG islands at both ends of RUNX
might be involved in tissue-specific expression of the
genes (Ehrlich, 2003), albeit the role of CpG islands in
regulation of tissue-specific gene expression is still
controversial.
The CpG island of RUNX3 P2 promoter is hypermethylated in human and mouse gastric cancer cell lines
and in primary human tumors (Guo et al., 2002; Li et al.,
2002; Waki et al., 2003). This may suggest that a tumorsuppressor function of RUNX3 is involved in the
etiology of stomach cancer (Guo et al., 2002; Li et al.,
2002; Waki et al., 2003). Alternatively, it may reflect the
increased activity of the DNA methyltransferase 1
(DNMT1) known to hypermethylate multiple CpG
islands in gastric cancers (Etoh et al., 2004). The P1
promoter of the three RUNX genes could be activated
upon integration of murine leukemia virus (Figure 1b),
leading to enhanced expression and subsequently to the
development of T-cell lymphoma in CD2-MYC transgenic mice (Stewart et al., 1997, 2002; Cameron et al.,
2003). These findings suggest that RUNX3 could act
both as a dominant oncogene in T-cell lymphoma, and
as a tumor suppressor in stomach cancer. RUNX1 and
RUNX3 expression is also affected by the Epstein–Barr
virus (EBV) transcription factor EBNA-2. Following
infection of B cells by EBV, expression of RUNX1
mRNA is reduced, whereas expression of RUNX3
mRNA is strikingly elevated, indicating that B-cell
transformation by EBV is associated with upregulation
of RUNX3 (Levanon et al., 1994; Spender et al., 2002,
and our unpublished data).
The P1- and P2-derived primary transcripts are
processed into a diverse repertoire of alternatively
spliced mRNA isoforms that are differentially expressed
in various cell types and at different developmental
stages. Considering that the P1 and P2 promoters
generate different 50 -untranslated regions (50 UTRs)
and thus distinct N-terminal coding regions, the
repertoire of RUNX gene products is even larger
(Figure 1b). The combination of alternative promoters
and elaborate splicing alternatives lead to production of
a large number of protein isoforms with a variety of
biological functions (Ogawa et al., 1993; Bae et al., 1994;
Miyoshi et al., 1995; Tanaka et al., 1995b; Levanon
et al., 1996, 2001b; Mundlos et al., 1997; Stewart et al.,
1997; Zhang et al., 1997b; Ben Aziz-Aloya et al., 1998;
Thirunavukkarasu et al., 1998; Bangsow et al., 2001).
The significance of the various isoforms to RUNX
biology remains to be elucidated.
Tissue-specific transcription control of RUNX genes
The P1 and P2 genomic regions of the three RUNX
genes were evaluated for promoter features. Interestingly, both P1 and P2 contain several dispersed RUNXbinding sites, raising the possibility of cross-regulations
between the RUNX genes (Ghozi et al., 1996; Ducy et al.,
1999; Drissi et al., 2000, 2002b; Bangsow et al., 2001;
Otto et al., 2003). Significantly, in all the three human
and mouse genes, two RUNX sites are identically
located, at the beginning of the P1 50 UTR within a
perfectly conserved sequence of 18 bp (Drissi et al., 2000;
Bangsow et al., 2001) (Figure 1c). Several in vitro studies
have attributed either positive or negative transcriptional regulatory role to these sites through binding of
RUNX proteins (Ducy et al., 1999; Drissi et al., 2000;
Alliston et al., 2001). However, the in vivo significance of
these sites remains to be elucidated. The observations
that in the various Runx KO mice expression of the
other non-targeted Runx genes is not affected (Stricker
et al., 2002; Yamashiro et al., 2002) may argue that these
RUNX-binding sites do not always function in crossregulation.
Functional analyses using transfection assays have
demonstrated that the P1 and P2 regions of the three
genes possess promoter activity (Ghozi et al., 1996;
Fujiwara et al., 1999; Drissi et al., 2000; Bangsow et al.,
2001; Rini and Calabi, 2001; Xiao et al., 2001; Zambotti
et al., 2002). In most cases, the activity was cell-type
specific, with higher promoter–reporter readouts in cells
that normally express the genes. However, detailed
information about the positive and negative elements
that regulate tissue-specific expression of RUNX genes in
vivo is still missing.
In vitro studies by several groups evaluated the
skeletal specific activity of RUNX2 P1 promoter. The
region spanning nucleotides 92 to 78, which is
entirely conserved in humans, rat and mouse, was
identified as a vitamin D response element (VDRE).
This element binds the VDR/RXR heterodimer and
thereby mediates the direct suppressive effect of 1,25(OH)2-Vitamin D3 on transcription of RUNX2 (Drissi
et al., 2002a). This region also harbors binding sites for
AP1 and RUNX that mediate the response to selective
estrogen receptor modulators (SERMS) (Tou et al.,
2001). However, the in vivo significance of this finding is
not clear because responsiveness to estrogen was not
demonstrated. Another highly conserved RUNX2 P1
region at 415 to 375 contains binding sites for NF1
and AP1 and acts as an osteoblast-specific enhancer
(Zambotti et al., 2002). The highly conserved RUNX
binding sites between 89 and þ 46 are involved in
regulation of RUNX2 expression (Ducy et al., 1999;
Drissi et al., 2000; Alliston et al., 2001) (Figure 1c and
see below).
Tissue-specific activity of one construct spanning 3 kb
of Runx2 P1 promoter was evaluated in vivo using
transgenic mice (Lengner et al., 2002). Expression was
detected in a subset of committed chondrocyte progenitors, which are involved in formation of the axial
skeleton, but not in those of the appendicular limbs.
Expression was not observed after birth, indicating that
this promoter region lacks sequences conferring expression in hypertrophic chondrocytes and osteoblasts.
Expression of Runx2 in mice deficient for the homeobox
transcription factor Bapx1 is specifically downregulated
in the developing axial skeleton (Tribioli and Lufkin,
1999). Given the role of Bapx1 in the early prechondrogenic stages of axial skeleton development, it is
tempting to speculate that Bapx1 regulates transcription
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Structure and expression of RUNX
D Levanon and Y Groner
4214
of Runx2 during axial skeleton development. Finally,
expression of Runx2 is also affected by changes in the
expression of several other transcription factors including Hoxa-2, Msx2, Twist and PPARg2 (Kanzler et al.,
1998; Lecka-Czernik et al., 1999; Satokata et al., 2000;
Yousfi et al., 2002). Future research might prove
whether any of these transcription factors directly
affects the promoters/enhancers of RUNX2.
Translation control of RUNX expression
As noted earlier, transcription of the RUNX genes is
regulated at the transcriptional level by two promoters
that give rise to mRNAs with distinct 50 UTRs
(Figure 1b). The two 50 UTRs (designated P1-50 UTR
and P2-50 UTR) differ in size and structure, and each has
characteristic features that are conserved between
paralogues, even though the DNA sequences are not
conserved (Bangsow et al., 2001; Levanon et al., 2003b).
The P1 50 UTR is usually shorter than the P2 50 UTR and
contains four exons. The initiator ATG and the highly
conserved P1 N-terminal peptide (MASy) are encoded
in the fourth exon (Figure 1c). The P2-50 UTR spans
only one exon that harbors a highly conserved in-exon
splice site and directs translation of the conserved P2 Nterminal peptide (MRIPV) (Figure 1d).
The two 50 UTRs play an important role in translation
regulation of RUNX expression. In RUNX1, the P150 UTR is 452 bp long and directs efficient translation in
vitro. On the other hand, the P2-50 UTR is exceptionally
long (1631 bp) and has structural characteristics that
largely impede its in vitro translation activity. Functional analysis revealed that the P1-50 UTR directs capdependent translation control, whereas the P2-50 UTR
regulates translation by an internal ribosomal entry site
(IRES) mechanism (Pozner et al., 2000). Recent studies
revealed that in Runx2 both the P1 and P2 50 UTRs are
able to mediate IRES-dependent translation control in
vitro (Xiao et al., 2003). It would be interesting to
determine whether the CpG-rich P2 50 UTR of RUNX3
also possesses IRES elements and functions in translation regulation of RUNX3.
What could be the biological significance of capversus IRES-dependent translation control of RUNX?
Initially discovered in picornaviruses, IRES elements
were since found in many cellular mRNAs, including
several regulatory proteins whose expression is tightly
regulated (Vagner et al., 2001). It is believed that IREScontaining mRNAs are translated at times when capdependent translation is physiologically impaired: during mitosis, differentiation or stress conditions. Thus,
the existence of both cap- and IRES-dependent translation control in RUNX mRNAs adds an additional level
at which expression of these genes is regulated. Moreover, as the P1- and P2-50 UTR-containing mRNAs
encode RUNX proteins that differ at their N termini,
the two translation control mechanisms can provide the
flexibility needed for production of the relevant type of
protein isoform in the appropriate amount at the proper
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time and in the right cell type. For example, RUNX1 P250 UTR mediates translation through IRES-dependent
control during megakaryocytic differentiation in K562
cells at a time window when only the P2 promoter is
transcriptionally active (Pozner et al., 2000), highlighting the fine-tuned regulation and the coupling between
transcriptional regulation and translation control. The
IRES elements of Runx2 50 UTR1 and 50 UTR2 endued
increased translation under genotoxic stress induced by
mitomycin C and during osteoblastic maturation,
further emphasizing the importance of translation
control in RUNX expression (Sudhakar et al., 2001;
Xiao et al., 2003).
Signaling pathways regulating expression of RUNX genes
A number of investigators have studied the signaling
pathways that regulate RUNX expression in response to
external stimuli. The current wisdom is that RUNX not
only functions as part of the TGF-b/BMP signaling
pathway (Ito and Miyazono, 2003), but that the genes
are also regulated by components of this pathway. For
example, TGF-b induces transcription of RUNX2 in the
pluripotent mesenchymal precursor cell line C2C12 (Lee
et al., 1999, 2000, 2002), but downregulates Runx2 in
primary osteoblasts and in the rat osteosarcoma cell line
ROS 17/2.8 (Li et al., 1998; Alliston et al., 2001). These
data correspond with reports that TGF-b stimulates and
inhibits early and terminal osteoblast differentiation,
respectively (Bonewald and Dallas, 1994; Centrella et al.,
1994). Inhibition by TGF-b may be associated with
repression of the Runx2 P1 promoter via the RUNXbinding sites located in the highly conserved 89 to
þ 46 region mentioned earlier (Alliston et al., 2001)
(Figure 1c). This observation raises an interesting
possibility that the P1 promoter region bearing the
two RUNX-binding sites within the highly conserved
18 bp sequence serves as a TGF-b responsive element in
all the three RUNX genes, through a RUNX autoregulatory loop.
Induction of osteoblastic differentiation by BMP2 in
the C2C12 cell line involves increased expression of
Runx2 mRNA preceded by upregulation of Smad5,
JunB, activation of p38MAPK (Lee et al., 2000, 2002)
and induction of Dlx5 (Lee et al., 2003). The promoter
region, which confers response to BMP, has not been
characterized. In transfection assays, a 50 P1 promoter
region spanning up to 3 kb did not respond to BMP
treatment (Banerjee et al., 2001; Xiao et al., 2001).
Unlike RUNX2, much less is known about the
involvement of TGF-b signaling in the expression of
the two other RUNX genes. It was demonstrated that
TGF-b induces the expression of Runx3, which then
activates the Ig a promoter in the B cell line m 1.29 (Shi
and Stavnezer, 1998) and that Runx1 expression is
upregulated in Smad5-deficient mice (Liu et al., 2003).
Runx3 functions as a component of the TGF-b signaling
cascade in dendritic cells and mediates their TGF-bdependent development and function in vivo (Fainaru
Structure and expression of RUNX
D Levanon and Y Groner
4215
et al., 2004). TGF-b regulates the development of
epithelium by inhibiting proliferation and inducing
differentiation, and mice deficient for TGF-b1 display
enhanced epithelial proliferation, resulting in hyperplasia (Crawford et al., 1998). Runx1 is expressed in the
epithelia of several developing organs including those
affected in TGF-b1 KO mice (Levanon et al., 2001a;
Yamashiro et al., 2002, and see Table 1). Whether
expression of Runx1 in these epithelia is regulated by
TGF-b is an interesting open question.
Expression of RUNX genes is also affected by the
FGF signaling pathway. In various cell lines, the
response to FGF varies depending on the cell type.
For example, FGF-dependent induction of Runx2 was
observed in the mesenchymal pluripotent cell line
C3H10T1/2 (Zhou et al., 2000), whereas repression
was observed in the rat osteosarcoma cell line ROS 17/
2.8 (Tsuji and Noda, 2001). In the developing teeth,
Runx2 is expressed in the dental mesenchyme and is
regulated by FGF. Implantation experiments using
beads soaked in FGF indicated that the mesenchymal
expression of Runx2 is induced by FGF from the
surrounding epithelium (D’Souza et al., 1999).
Mice bearing a highly active mutant FGF receptor 1
(Fgfr1) exhibit an increase in Runx2 mRNA associated
Table 1 Summary of predominant Runx gene expression sitesa
Tissue/cell type
DRG
TrkA
TrkC
Runx1
Runx2
+
+
Hematopoiesis
AGM
+
Fetal liver
Fetal thymus
Thymus cortex
+
+
+
Thymus medulla
Spleen
+
Runx3 KO: impaired
development of
TrkC neurons
Runx1 KO:
Lack of definitive
hematopoiesis
+
+
+
Runx1 and Runx3
KO: impaired T-cell
development
+
+
Skeleton
Immature and
permanent cartilage
Prehypertrophic
cartilage
Hypertrophic
cartilage
Osteoblasts
+
+
Membranous bone
+b
+
Epidermal
appendages
Epithel
Mesenchyme
Runx3 KO phenotype
+
+
+
+
Runx2 KO: impaired
development of
osteoblasts
+
+c
+
a
References are indicated in the text. Details are also discussed in other
chapters of this issue. bLimited to sutures. cDetected only in dental
pappila
with enhanced osteoblast differentiation around the
cranial sutures (Zhou et al., 2000), providing an in vivo
indication that Runx2 expression in osteoblast differentiation is regulated by components of FGF signaling
pathway. In its capacity as an angiogenic growth factor,
FGF induces Runx2 and Runx1 mRNA and protein
expression in the murine endothelial cells MSS31
(Namba et al., 2000). Basic FGF induces expression of
Runx1 mRNA in the olfactory neuroblastoma cell line
JFEN (Nibu et al., 2000). Interestingly, Runx1 induction
in this cell line was associated with induction of tyrosine
kinase A (TrkA) expression (Nibu et al., 2000). This
Runx1-associated TrkA induction correlates with the
specific in vivo expression of Runx1 and TrkA in the
dorsal root ganglia nociceptive neurons (Levanon et al.,
2001a, 2002). Angiogenesis-associated expression of
Runx2 and Runx1 is also enhanced by IGF and VEGF,
respectively (Namba et al., 2000; Sun et al., 2001).
Expression of Runx2 is also downregulated by TNFa as
part of its inhibitory effect on osteoblast differentiation
(Gilbert et al., 2002).
The RUNX genes may also function as part of
the retinoic acid (RA) signaling pathway, as expression
of the three genes is upregulated by RA treatment
(Tanaka et al., 1995a; Le et al., 1999; Jimenez et al.,
2001). Identification of the RUNX promoter regions
involved in the induction by RA is still missing.
Expression of Runx2 is also regulated by members
of the hedgehog signaling pathway. Treatment of
primary chondrocytes or the pluripotent cell line
C3H10 T1/2 by sonic hedgehog resulted in activation
of the Runx2 P1 promoter and in increased levels of
Runx2 mRNA (Spinella-Jaegle et al., 2001; Takamoto
et al., 2003).
Recently, it was shown that the function of Drosophila runt domain gene Lozenge in fly hematopoiesis is
regulated by notch signaling pathway (Lebestky et al.,
2003). As the functional hierarchy of the hematopoietic
transcription factors GATA, FOG and RUNX is
conserved between Drosophila and mammals (Lebestky
et al., 2000; Fossett and Schulz, 2001; Evans et al.,
2003), the possibility arises that components of the
notch signaling pathway also regulate expression of
mammalian RUNX in the hematopoietic system. Consistent with this speculation, Runx1 and Runx3, as well
as notch signaling, are involved in regulating the
development and maturation of T cells (Deftos et al.,
2000; Levanon et al., 2001a; Taniuchi et al., 2002; Woolf
et al., 2003).
Tissue-specific expression pattern of RUNX genes
The tightly regulated gene expression of RUNX is
translated into a highly distinct tissue-specific expression
of the three family members (Table 1). Few exceptional
cases of either spatial or temporal overlapping expression have also been described. Both Runx1 and Runx3
are highly expressed in cranial and dorsal root ganglia
(DRG), but in different neuronal populations. Runx1
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D Levanon and Y Groner
4216
expression starts at embryonic day (E) 12.5 in the
small-diameter nociceptive TrkA neurons, while
Runx3 expression starts earlier at E10.5 and is confined
to the large-diameter proprioceptive TrkC neurons
(Simeone et al., 1995; Levanon et al., 2001a, 2002).
While information about the role of Runx1 in the
DRG is still missing, the expression pattern of
Runx3 in the DRG correlates well with the phenotypes of Runx3 KO mice (Inoue et al., 2002; Levanon
et al., 2002).
In mouse, embryo expression of Runx1 is first
detected in definitive hematopoietic stem cells (HSC)
and in endothelial cells at HSC emergence sites, that is,
yolk sac, vitelline and umbilical arteries and aortagonad-mesonephros (AGM) as well as in the liver
(North et al., 1999, 2002; Cai et al., 2000; Mukouyama
et al., 2000). These observations correlate well with the
earlier findings that homozygous disruption of Runx1
results in a complete absence of fetal liver hematopoiesis
and death of embryos between E11.5 and E12.5 from
hemorrhages in the central nervous system (Okuda et al.,
1996; Wang et al., 1996). In adult hematopoietic system,
Runx1 is highly expressed in several cell lineages
including myeloid, B- and T-lymphoid cells (Lorsbach
et al., 2003), and Runx3 is expressed in mature dendritic
cells (Fainaru et al., 2004) and T cells (Taniuchi et al.,
2002; Woolf et al., 2003).
All the three Runx proteins are expressed in the
thymus (Satake et al., 1995; Levanon et al., 1996, 2001a;
Komori et al., 1997; Hayashi et al., 2000, 2001; Taniuchi
et al., 2002; Woolf et al., 2003). In the early developing
thymus, expression pattern of Runx1 and Runx3 is very
similar and appears to overlap at the cellular level
(Levanon et al., 2001a). Later, at E18, when thymus’
regional demarcation of cortex and medulla becomes
clear, expression of Runx1 is mainly in the cortex,
whereas Runx3 is in both the cortex and medulla. In
adults, expression of Runx1 is predominantly in the
subcortical layer where immature thymocytes reside
(Levanon et al., 2001a; Woolf et al., 2003), whereas
Runx3 is found mainly in the medulla where CD8/CD4
single-positive T cells develop (Levanon et al., 2001a;
Taniuchi et al., 2002; Woolf et al., 2003). At the cellular
level, expression of Runx1 was detected in immature
double-negative thymocytes (Taniuchi et al., 2002)
and Runx3 in both double-negative cells and in
mature single-positive CD8 cells (Taniuchi et al., 2002;
Woolf et al., 2003). The expression pattern of
Runx1 and Runx3 in thymus corresponds to their in
vivo function as positive and negative regulators in
T-cell development during thymopoiesis (Taniuchi et al.,
2002; Woolf et al., 2003). Runx2 is also expressed
during early thymic development in double-negative
thymocytes and KO embryos display marked reduction in the overall number of thymocytes (Komori
et al., 1997; Vaillant et al., 2002). Consistent with
these findings, enforced expression of Runx2 in transgenic mice affects early T-cell development (Vaillant
et al., 2002).
The expression of the three Runx proteins in the
developing skeleton is intriguing. Although several
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groups carried out careful analyses, most of the
available information relates to Runx2. Collectively,
these analyses disclosed that all the three RUNX are
expressed in cartilage. However, only Runx1 is expressed during bone development in immature cartilage
and its expression in adults persists in permanent
cartilage (Simeone et al., 1995; Levanon et al., 2001a;
Lian et al., 2003). Runx2 and Runx3 are both expressed
in pre- and hypertrophic chondrocytes with Runx2
predominating in the latter (Inada et al., 1999; Kim
et al., 1999; Enomoto-Iwamoto et al., 2001; Levanon
et al., 2001a; Takeda et al., 2001; Choi et al., 2002;
Stricker et al., 2002; Komori, 2003). In osteoblasts,
Runx2 is the major form (Komori et al., 1997; Komori,
2003; Otto et al., 1997), Runx1 is expressed in early
osteoblasts (Lian et al., 2003) and Runx3 was not
detected (Stricker et al., 2002). In membranous bone of
the skull, expression of Runx2 is detected throughout
development (Park et al., 2001; Choi et al., 2002), while
Runx1 is expressed in mesenchymal condensates, in the
sutures and in immature cartilage (Yamashiro et al.,
2002; Lian et al., 2003). No detailed studies of Runx3
expression in membranous bone are available. The
biological function of Runx2 in bone development, as
reflected in the phenotype of Runx2 KO mice, correlates
well with the expression pattern of the gene (Komori
et al., 1997; Otto et al., 1997). No such information is
available about the role of Runx1 and Runx3 in the
developing skeleton. Detailed analysis of this aspect in
the recently derived CBF-b-deficient mice (Kundu et al.,
2002; Miller et al., 2002; Yoshida et al., 2002) may
provide information about the role of Runx1 in
osteogenesis.
Structures that arise through ectodermal invagination
such as salivary and mammary glands, and epidermal
appendages including whiskers and teeth, express both
Runx1 and Runx3, but again not in the same compartment. In whiskers and teeth, expression of Runx1 and
Runx3 is confined to the epithelium and mesenchymal
papilla, respectively (Levanon et al., 2001a; Yamashiro
et al., 2002). Expression of Runx2 is also found in the
mesenchymal papilla of developing teeth (D’Souza et al.,
1999) but is downregulated at the odontoblast stage,
whereas expression of Runx3 persists (Yamashiro et al.,
2002). Epidermal appendages develop through regulated
epithelial–mesenchymal cross-talk. Runx2 appears to
participate in these interactions; its mesenchymal
expression is induced by epithelial signals and is needed
for epithelial morphogenesis (D’Souza et al., 1999).
During mouse development, the expression distribution
of Runx1 and Runx3 into the epithelium and mesenchyme, respectively, is strictly maintained. Expression of
Runx1, however, is broader than that of Runx3, as
it is detected in several tissues where Runx3 is not
expressed, including the epithelia of palatal ridges,
bronchi, respiratory and olfactory mucosa, and in the
mucosa of the esophagus and stomach (Simeone et al.,
1995; Levanon et al., 2001a). Runx1 is also expressed
in mesenchyme of several other organs, as well as
in the heart and in the central nervous system (Levanon
et al., 2001a).
Structure and expression of RUNX
D Levanon and Y Groner
4217
Perspective
Expression of RUNX genes is tightly regulated at the
transcriptional and post-transcriptional levels. Consequently, the genes display distinct tissue- and developmental-specific expression. Details about the molecular
mechanisms that control the spatial and temporal
patterns of RUNX expression during growth, differentiation and development are still lacking. The genomic
architecture of the three mammalian genes is highly
similar, as are the basic regulatory elements. As the
RUNX family appeared early in evolution and the
mammalian genes maintain structural similarities, it is
conceivable that the control element enduing tissue- and
timing-specific expression evolved concomitantly with
the growing repertoire of RUNX-specific biological
tasks. Information about the expression patterns of
RUNX during development and about the signaling
pathways in which the genes participate will help to
identify the tissue- and stage-specific regulatory elements
and allow to better understand how these important
regulators are regulated.
Acknowledgements
We thank Ori Brenner, Joseph Lotem, Yael Bernstein, Varda
Negreanu and Amir Pozner for helpful discussions and
comments on the manuscript. The work was supported by
grants from the Commission of the EU, the Israel Science
Foundation, Minerva Foundation Germany and Shapell Family
Biomedical Research Foundation at the Weizmann Institute.
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