The Johns Hopkins University database "Online Mendelian Inheritance in Man" provides an extensive listing for AML1, the vertebrate homolog of Lozenge and Runt.

Invertebrate Runt homologs

The central domain of Drosophila protein Lozenge contains a region homologous to AML1 (acute myeloid leukemia 1) and to Runt. This region includes the dimerization and DNA binding domain, as well as a putative ATP-binding site which is completely conserved between proteins of this group. There is a region of conserved amino acids of unknown function (VWRPY) at the C-terminus. In the homologous domain, LZ sequence has 71% identity to AML1 and 69% homology to Runt. The LZ sequence also possesses an alanine-rich stretch and a glutamine-rich region in the C-terminal portion of the molecule (Daga, 1996).

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-β (TGF-β) signal mediators. However, the involvement of RUNX in TGF-β signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-β signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed a downstream target of rnt-1. These data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans (Ji, 2004).

C. elegans mab-2 mutants have defects in the development of a male-specific sense organ because of a failure in the proliferation of the stem cell-like lateral hypodermal (seam) cells. mab-2 encodes RNT-1, the only C. elegans member of the Runx family of transcriptional regulators, that is postulated to act both as an oncogene and a tumour suppressor in mammalian cells. Importantly, rnt-1 is found to be a rate-limiting regulator of seam cell proliferation in C. elegans; overexpression of rnt-1 at particular developmental stages is capable of driving extra cell divisions, leading to seam cell hyperplasia. Loss of rnt-1 is correlated with upregulation of cki-1, a CDK inhibitor. Deregulated expression of Runx genes in humans is associated with various cancers, particularly leukaemias, suggesting a conserved role for Runx genes in controlling cell proliferation during development, especially in stem cell lineages. C. elegans is therefore an important model system for studying the biology, and oncogenic potential, of Runx genes (Nimmo, 2005).

The C. elegans CBFß homologue, BRO-1 (Drosophila homologs Brother and Big brother), was investigated. bro-1 mutants have a similar male-specific sensory ray loss phenotype to rnt-1 (the C. elegans homologue of the mammalian CBFß-interacting Runx factors), caused by failed cell divisions in the seam lineages. These studies indicate that BRO-1 and RNT-1 form a cell proliferation-promoting complex, and that BRO-1 increases both the affinity and specificity of RNT-1-DNA interactions. Overexpression of bro-1, like rnt-1, leads to an expansion of seam cell number and co-overexpression of bro-1 and rnt-1 results in massive seam cell hyperplasia. BRO-1 appears to act independently of RNT-1 in certain situations. These studies provide new insights into the function and regulation of this important cancer-associated DNA-binding complex in stem cells and support the view that Runx/CBFß factors have oncogenic potential (Kagoshima, 2007).

SpRunt-1 of sea urchin was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. This target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. Transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1 (Coffman, 1996).

There is an ongoing discussion as to whether segmentation in different phyla has a common origin sharing a common genetic program. However, before comparing segmentation between phyla, it is necessary to identify the ancestral condition within each phylum. Even within the arthropods it is not clear which parts of the genetic network leading to segmentation are conserved in all groups. In this paper, the expression of three segmentation genes of the pair-rule class is examined in the spider Cupiennius salei. Spiders are representatives of the Chelicerata, a monophyletic basic arthropod group. During spider embryogenesis, segments are sequentially added at the posterior end of the embryo, which resembles the formation of the abdominal segments in short-germ insect embryos. In spider embryos, the orthologues for the Drosophila primary pair-rule genes hairy, even-skipped, and runt are expressed in stripes in the growth zone, where the segments are forming, suggesting a role for these genes in chelicerate segmentation. These data imply that the involvement of hairy, even-skipped, and runt in arthropod segmentation is an ancestral character for arthropods and is not restricted to a particular group of insects (Damen, 2000).

A remarkable feature of the Cs-Hairy sequence is the change in the conserved carboxyl-terminal tetrapeptide WRPW found in the Hairy family of basic helix-loop-helix transcription factors, which include the Hairy, Deadpan, and Enhancer of split proteins. The WRPW tetrapeptide is changed to WRPF in Cs-H. The WRPW motif is required for interaction with the corepressor Groucho and for transcriptional repression. It is not known whether this 1-aa change in the tetrapeptide affects a putative interaction of Cs-H with Groucho. Runt domain proteins contain a very similar carboxyl-terminal motif, WRPY, which also is required for Groucho-dependent repression in Drosophila (Damen, 2000).

Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle, Dermestes maculatus

A set of pair-rule segmentation genes (PRGs) promote the formation of alternate body segments in Drosophila melanogaster While Drosophila embryos are long-germ, with segments specified more-or-less simultaneously, most insects add segments sequentially as the germband elongates. The hide beetle, Dermestes maculatus, represents an intermediate between short- and long-germ development, ideal for comparative study of PRGs. This study shows that eight of nine Drosophila PRG-orthologs are expressed in stripes in Dermestes. Functional results parse these genes into three groups: Dmac-eve, -odd, and -run play roles in both germband elongation and PR-patterning. Dmac-slp and -prd function exclusively as complementary, classic PRGs, supporting functional decoupling of elongation and segment formation. Orthologs of ftz, ftz-f1, h, and opa show more variable function in Dermestes and other species. While extensive cell death generally prefigured Dermestes PRG RNAi cuticle defects, an organized region with high mitotic activity near the margin of the segment addition zone likely contributes to truncation of eve(RNAi) embryos. These results suggest general conservation of clock-like regulation of PR-stripe addition in sequentially-segmenting species while highlighting regulatory re-wiring involving a subset of PRG-orthologs (Xiang, 2017).

Fish Runt homologs

RUNX1/AML1/CBFA2 is essential for definitive hematopoiesis, and chromosomal translocations affecting RUNX1 are frequently involved in human leukemias. Consequently, the normal function of RUNX1 and its involvement in leukemogenesis remain subject to intensive research. To further elucidate the role of RUNX1 in hematopoiesis, the zebrafish ortholog (runx1) was cloned and its function was analyzed. Zebrafish runx1 is expressed in hematopoietic and neuronal cells during early embryogenesis. runx1 expression in the lateral plate mesoderm co-localizes with the hematopoietic transcription factor scl, and expression of runx1 is markedly reduced in the zebrafish mutants spadetail and cloche. Transient expression of runx1 in cloche embryos results in partial rescue of the hematopoietic defect. Depletion of Runx1 with antisense morpholino oligonucleotides abrogates the development of both blood and vessels, as demonstrated by loss of circulation, incomplete development of vasculature and the accumulation of immature hematopoietic precursors. The block in definitive hematopoiesis is similar to that observed in Runx1 knockout mice, implying that zebrafish Runx1 has a function equivalent to that in mammals. These data suggest that zebrafish Runx1 functions in both blood and vessel development at the hemangioblast level, and contributes to both primitive and definitive hematopoiesis. Depletion of Runx1 also causes aberrant axonogenesis and abnormal distribution of Rohon-Beard cells, providing the first functional evidence of a role for vertebrate Runx1 in neuropoiesis (Kalev-Zylinska, 2002).

To provide a base for examining the role of Runx1 in leukemogenesis, the effects of transient expression of a human RUNX1-CBF2T1 transgene [product of the t(8;21) translocation in acute myeloid leukemia] in zebrafish embryos was examined. Expression of RUNX1-CBF2T1 causes disruption of normal hematopoiesis, aberrant circulation, internal hemorrhages and cellular dysplasia. These defects reproduce those observed in Runx1-depleted zebrafish embryos and RUNX1-CBF2T1 knock-in mice. The phenotype obtained with transient expression of RUNX1-CBF2T1 validates the zebrafish as a model system to study t(8;21)-mediated leukemogenesis (Kalev-Zylinska, 2002).

Xenopus Runt homologs

The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. Xaml, a Xenopus AML1 homolog, has been isolated in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventral blood island in a pattern that anticipates that of globin, which is expressed later. Analysis of globin and Xaml expression in explants, in embryos with perturbed dorsal ventral patterning, and by lineage tracing indicates that the formation of the ventral blood island is more complex than previously thought and involves contributions from both dorsal and ventral tissues. A truncated Xaml protein interferes with primitive hematopoiesis. Based on these results, it is proposed that Runt domain proteins function in the specification of hematopoietic stem cells in vertebrate embryos (Tracey, 1998).

Mammalian Runt homologs

cDNAs representing the alpha subunit of polyomavirus enhancer binding protein 2 (PEBP2; also called PEA2) are highly homologous to runt, with an N-proximal 128-amino acid region showing 66% identity. The Runt homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion beta subunit. The major species of PEBP2 alpha mRNA is expressed in tested T-cell lines but not in tested B-cell lines. Evidence indicates that PEBP2 functions as a transcriptional activator and is involved in regulation of T-cell-specific gene expression (Ogawa, 1993).

Transcription factor PEBP2/CBF consists of a DNA binding alpha subunit, and a regulatory beta subunit. The alpha subunit has an evolutionarily conserved 128-amino acid region termed "Runt domain" that is responsible for both DNA binding and heterodimerization with the beta subunit. The Runt domain in all mammalian submembers of the alpha subunit contains two conserved cysteine residues, and its DNA binding activity undergoes redox regulation. To investigate the mechanism of this redox regulation, site-directed mutagenesis of the two conserved cysteines in the Runt domain of the mouse PEBP2alphaA homolog was performed. Substitution of Cys-115 with serine results in a partially impaired DNA binding, which remains highly sensitive to a thiol-oxidizing reagent, diamide. Conversely, the corresponding substitution of Cys-124 causes an increased DNA binding concomitant with an increased resistance to diamide. In contrast, substitution of either cysteine to aspartate is destructive to DNA binding to marked extents. These results have revealed that both Cys-115 and Cys-124 are responsible for the redox regulation in their own ways with low and high oxidizabilities, respectively. Two cellular thiol-reactive proteins, thioredoxin and Ref-1, work effectively and synergistically for activation of the Runt domain. Interestingly, the beta subunit further enhances the activation by these proteins and reciprocally prevents the oxidative inactivation by diamide. These findings collectively suggest the possibility that the Runt domain's function in vivo could be dynamically regulated by the redox mechanism with Trx, Ref-1, and the beta subunit as key modulators (Akamatsu, 1997).

Mutation and alternative splicing of mammalian Runt homologs

The gene AML1/PEBP2 alphaB encodes the alpha subunit of transcription factor PEBP2/CBF and is essential for the establishment of fetal liver hematopoiesis. Rearrangements of AML1 are frequently associated with several types of human leukemia. Three types of AML1 cDNA isoforms have been described to date; they have been designated AML1a, AML1b, and AML1c. All of these isoforms encode the conserved-Runt domain, which harbors the DNA binding and heterodimerization activities. A new isoform of the AML1 transcript, termed AML1 deltaN, has been discovered in which exon 1 is directly connected to exon 4 by alternative splicing. The AML1 deltaN transcript is detected in various hematopoietic cell lines of lymphoid to myeloid cell origin. The protein product of AML1 deltaN lacks the N-terminal region of AML1, including half of the Runt domain, and neither binds to DNA nor heterodimerizes with the beta subunit. However, AML1 deltaN is found to interfere with the transactivation activity of PEBP2, and the molecular region responsible for this activity was identified. Stable expression of AML1 deltaN in 32Dcl3 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor. These results suggest that AML1 deltaN acts as a modulator of AML1 function and serves as a useful tool to dissect the functional domains in the C-terminal region of AML1 (Zhang, 1997).

Mammalian Runt homologs are called alternative osteoblast-specific transcription factor (OSF), core binding factor (CBF) or polyoma enhancer-binding protein (PEBP). A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. Cbfa1/Osf2 is expressed from day 10.5 of fetal development in developing limbs. Cbfa1 is first detected in the region surrounding cartilaginous condensation and in limb tendons from 13.5 days after fertilization. To elucidate the function of Cbfa1, mice were generated with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 die just after birth without breathing. Examination of their skeletal systems shows a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appear at the perichondrial region, neither vascular nor mesenchymal cell invasion is observed in the cartilage. Therefore, both intramembranous and endochondral ossification are completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice; hence Cbfa1 plays an essential role in osteogenesis (Komori, 1997).

AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein that cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree (Biggs, 2006).

Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA binding of Runx2

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Hand1 and Hand2 are expressed by a subset of neural crest cells in the anterior branchial arches and are involved in craniofacial development. However, the precise mechanisms by which Hand proteins mediate biological actions and regulate downstream target genes in branchial arches is largely unknown. This study reports that Hand2 negatively regulates intramembranous ossification of the mandible by directly inhibiting the transcription factor Runx2, a master regulator of osteoblast differentiation. Hand proteins physically interact with Runx2, suppressing its DNA binding and transcriptional activity. This interaction is mediated by the N-terminal domain of the Hand protein and requires neither dimerization with other bHLH proteins nor DNA binding. Partial colocalization of Hand2 and Runx2 was observed in the mandibular primordium of the branchial arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice display insufficient mineralization and ectopic bone formation in the mandible due to accelerated osteoblast differentiation, which is associated with the upregulation and ectopic expression of Runx2 in the mandibular arch. This study shows that Hand2 acts as a novel inhibitor of the Runx2-DNA interaction and thereby regulates osteoblast differentiation in branchial arch development (Funato, 2009).

Runt homologs and muscle, bone, blood, liver and immune system development

The osteoblast is the bone-forming cell. Cells of mesenchymal origin, once terminally differentiated, produce most of the proteins present in the bone extracellular matrix (ECM), and also control the mineralization of the ECM. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. An osteoblast-specific cis-acting element, termed OSE2, is present in the Osteocalcin promoter. Osf2/Cbfa1 is a Runt domain protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation (Ducy, 1997).

A runtB chicken homolog has been found to be 84% identical to the mouse PEBP2alphaB2 isoform. RuntB mRNA levels increase dramatically with the transition from stage 0 (dedifferentiated) to stages I and II (hypertrophic chondrocytes). Runt polypeptides are present in chondrocytes both in vivo and in vitro. These results suggest that runt plays a role in chondrogenic differentiation (Castagnola, 1996).

Organization of the transforming growth factor-beta (TGF-beta) type I receptor (TRI) promoter predicts constitutive transcription, although its activity increases with differentiation status in cultured osteoblasts. Several sequences in the rat TRI promoter comprise cis-acting elements for CBFa (AML/PEBP2alpha) transcription factors. A principal osteoblast-derived nuclear factor that binds to these sites is identifed as CBFa1 (AML-3/PEBP2alphaA). Rat CBFa1 levels parallel expression of the osteoblast phenotype and increase under conditions that promote mineralized bone nodule formation in vitro. Fusion of CBFa binding sequence from the TRI promoter to enhancer-free transfection vector increases reporter gene expression in cells that possess abundant CBFa1, and overexpression of CBFa increases the activity of transfected native TRI promoter/reporter plasmid. Consequently, phenotype-restricted use of cis-acting elements for CBFa transcription factors can contribute to the high levels of TRI that parallel osteoblast differentiation and to the potent effects of TGF-beta on osteoblast function (Ji, 1998).

The Pebpb2/Cbfb gene encodes the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF. Although PEBP2beta/CBFbeta was detected in various tissues to various degrees, interesting features of expression are observed in the skeletal myogenic cells. PEBP2beta/CBFbeta is found mainly to occur as cytoplasmic staining: the intensity of this staining increases, depending on the differentiation stage of the cells. In the undifferentiated myoblasts, PEBP2beta/CBFbeta is undetectable, whereas moderate levels of PEBP2beta/ CBFbeta are detected in the elongated and aligned myocytes. PEBP2beta/CBFbeta appears to accumulate further when the cells fuse to each other to become multinucleated myotubes. Once the muscle fibers are established, PEBP2beta/CBFbeta is relocated onto or around the Z-lines. PEBP2beta/CBFbeta is also detected in the cytoplasm of cardiac myocytes and in the smooth muscle cells of the digestive tract. In all the above, the skeletal myotubes are the only case that show both nuclear and cytoplasmic staining of PEBP2beta/CBFbeta. Thus, the differentiation dependent pattern of PEBP2beta/CBFbeta expression occurs in muscle development, and PEBP2beta/CBFbeta is a cytoplasmic as well as nuclear protein in vivo (Chiba, 1998).

The Pebpb2 gene encodes a non-DNA binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF), and is rearranged in inversion of chromosome 16 associated with human acute myeloid leukemia. To investigate its physiological function, Pebpb2 was mutated by a targeting strategy to generate a null mutant. The homozygous mutation in mice proves lethal in embryos at approximately embryonic day 12.5, apparently due to massive hemorrhaging in the central nervous system. In addition, definitive hematopoiesis in the liver is severely impaired. The observed phenotype is indistinguishable from that reported for homozygous disruption of AML1, which encodes a DNA binding subunit of PEBP2/CBF. Thus, the results indicate that the two subunits function together as a heterodimeric PEBP2/CBF in vivo and that PEBP2/CBF plays an essential role in the development of definitive hematopoiesis (Niki, 1997).

Cbfa2 (AML1) encodes the DNA-binding subunit of a transcription factor in the small family of core-binding factors (CBFs). Cbfa2 is required for the differentiation of all definitive hematopoietic cells, but not for primitive erythropoiesis. Cbfa2 is expressed in definitive hematopoietic progenitor cells, and in endothelial cells in sites from which these hematopoietic cells are thought to emerge. Endothelial cells expressing Cbfa2 are in the yolk sac, the vitelline and umbilical arteries, and in the ventral aspect of the dorsal aorta in the aorta/genital ridge/mesonephros (AGM) region. Endothelial cells lining the dorsal aspect of the aorta, and elsewhere in the embryo, do not express Cbfa2. Cbfa2 appears to be required for maintenance of Cbfa2 expression in the endothelium, and for the formation of intra-aortic hematopoietic clusters from the endothelium (North, 1999).

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life (called hereafter bone formation) are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation transgenic mice that only postnatally overexpressed Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts were generated. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself; has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts are less active than wild-type osteoblasts. Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally (Ducy, 1999).

The role of the AML1 transcription factor in the development of hematopoiesis in the paraaortic splanchnopleural (P-Sp) and the aorta-gonad-mesonephros (AGM) regions of mouse embryos was examined. The activity of colony-forming units of colonies from the P-Sp/AGM region is reduced severalfold by heterozygous disruption of the AML1 gene, indicating that AML1 functions in a dosage-dependent manner to generate hematopoietic progenitors. In addition, no hematopoietic progenitor activity is detected in the P-Sp/AGM region of embryos with an AML1 null mutation. Similar results were obtained when a dispersed culture was first prepared from the P-Sp/AGM region before assay of the activity of the colony-forming units. In a culture of cells with the AML1(+/+) genotype, both hematopoietic and endothelial-like cell types emerge, but in a culture of cells with the AML1(-/-) genotype, only endothelial-like cells emerge. Interestingly, introduction of AML1 cDNA into the P-Sp/AGM culture with the AML1(-/-) genotype partially restores the production of hematopoietic cells. This restoration is observed for cultures prepared from 9.5-day postcoitum (dpc) embryos but not for cultures prepared from 11.5-dpc embryos. Therefore, the population of endothelial-like cells capable of growing in the AML1(-/-) culture would appear to contain inert but nonetheless competent hematogenic precursor cells up until at least the 9.5-dpc period. All these results support the notion that the AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic P-Sp/AGM region (Mukouyama, 2000).

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells reveal that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediate repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors is not observed in BSP promoters in which Cbfa sites are deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts is similarly downregulated by forced expression of Cbfa factors. These data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influences repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieves suppression of the BSP promoter. These results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development (Javed, 2001).

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis during development, but little is known about the factors that control its expression. The first example of tissue specific loss of VEGF expression as a result of targeting a single gene, Cbfa1/Runx2, is provided. During endochondral bone formation, invasion of blood vessels into cartilage is associated with upregulation of VEGF in hypertrophic chondrocytes and increased expression of VEGF receptors in the perichondrium. This upregulation is lacking in Cbfa1 deficient mice, and cartilage angiogenesis does not occur. Finally, over-expression of Cbfa1 in fibroblasts induces an increase in their VEGF mRNA level and protein production by stimulating VEGF transcription. The results demonstrate that Cbfa1 is a necessary component of a tissue specific genetic program that regulates VEGF during endochondral bone formation (Zelzer, 2001).

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, chicken Runx2 was cloned and overexpressed in chick embryos using a retroviral system. Infected chick wings show multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation is not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, transgenic mice were generated that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, in situ hybridization was performed with Runx2- and Runx3-specific probes. Both genes are coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs (Stricker, 2002).

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4+CD8+), which are selected to become either CD4+CD8-helper cells or CD4-CD8+ cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4-CD8+ thymocytes. Binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4-CD8- thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes (Taniuchi, 2002).

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, a novel function of the Stat1 transcription factor is demonstrated in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-ß. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. This study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling (Kim, 2003).

Differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed in Runx2-deficient (Runx2–/–) mice, terminal differentiation of chondrocytes does occur, indicating that additional factors are involved in chondrocyte maturation. The involvement of Runx3 in chondrocyte differentiation was investigated by generating Runx2-and-Runx3-deficient (Runx2–/–3–/–) mice. Chondrocyte differentiation is inhibited depending on the dosages of Runx2 and Runx3, and Runx2–/–3–/– mice show a complete absence of chondrocyte maturation. Further, the length of the limbs is reduced depending on the dosages of Runx2 and Runx3, due to reduced and disorganized chondrocyte proliferation and reduced cell size in the diaphyses. Runx2–/–3–/– mice do not express Ihh, which regulates chondrocyte proliferation and maturation. Adenoviral introduction of Runx2 in Runx2–/– chondrocyte cultures strongly induces Ihh expression. Moreover, Runx2 directly binds to the promoter region of the Ihh gene and strongly induces expression of the reporter gene driven by the Ihh promoter. These findings demonstrate that Runx2 and Runx3 are essential for chondrocyte maturation and that Runx2 regulates limb growth by organizing chondrocyte maturation and proliferation through the induction of Ihh expression (Yoshida, 2004).

Runx2 is necessary and sufficient for osteoblast differentiation, yet its expression precedes the appearance of osteoblasts by 4 days. Twist proteins transiently inhibit Runx2 function during skeletogenesis. Twist-1 and -2 are expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurs only after their expression decreases. Double heterozygotes for Twist-1 and Runx2 deletion have none of the skull abnormalities observed in Runx2+/- mice; a Twist-2 null background rescues the clavicle phenotype of Runx2+/- mice, and Twist-1 or -2 deficiency leads to premature osteoblast differentiation. Furthermore, Twist-1 overexpression inhibits osteoblast differentiation without affecting Runx2 expression. Twist proteins' antiosteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runx2 DNA binding domain to inhibit its function. In vivo mutagenesis confirms the antiosteogenic function of the Twist box. Thus, relief of inhibition by Twist proteins is a mandatory event precluding osteoblast differentiation (Bialek, 2004).

Across vertebrates, there are three principal skeletal tissues: bone, persistent cartilage, and replacement cartilage. Although each tissue has a different evolutionary history and functional morphology, they also share many features. For example, they function as structural supports, they are comprised of cells embedded in collagen-rich extracellular matrix, and they derive from a common embryonic stem cell, the osteochondroprogenitor. Occasionally, homologous skeletal elements can change tissue type through phylogeny. Together, these observations raise the possibility that skeletal tissue identity is determined by a shared set of genes. Misexpression of either Sox9 or Runx2 can substitute bone with replacement cartilage or can convert persistent cartilage into replacement cartilage and vice versa. The data also suggest that these transcription factors function in a molecular hierarchy in which chondrogenic factors dominate. A binary molecular code is proposed that determines whether skeletal tissues form as bone, persistent cartilage, or replacement cartilage. Finally, these data provide insights into the roles that master regulatory genes play during evolutionary change of the vertebrate skeleton (Eames, 2004).

To elucidate the roles of molecular determinants in dictating skeletal histogenesis, Sox9 and Runx2 functions were analyzed in the cranial skeleton, where the development of replacement cartilage, bone, and persistent cartilage is spatially distinct. Differential expression of Sox9 and Runx2 correlates with three skeletal fates as cranial neural crest cells initiated histogenesis. Sox9 and Runx2 were confirmed as functional regulators of cranial skeletogenesis by viral misexpression. Sox9 misexpression produces ectopic cartilage, while Runx2 misexpression results in ectopic bone. Closer histological and molecular analyses of Sox9- or Runx2-treated embryos revealed transformations of skeletal tissue fate, the most striking of which was the appearance of replacement cartilage within bone as a result of Sox9 misexpression. This conclusion was further supported by comparing the experimental phenomenon with secondary cartilage, a tissue in normal, uninfected embryos, which results from a conversion of cells from osteogenic to chondrogenic fates. In addition, Sox9 misexpression transforms replacement cartilage to persistent cartilage, whereas ectopic Runx2 expression converts persistent cartilage to replacement cartilage. From both the restricted pattern of these skeletal tissue transformations and expression data in uninfected embryos, it is proposed that chondrogenic determinants are dominant to osteogenic ones in skeletogenic cell fate decisions. These findings have direct implications for analyzing evolutionary phenomena in molecular terms (Eames, 2004).

Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. This study analyzes the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system leads to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induces runx1 gene expression and increases multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase is followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo (Burns, 2005).

The adult stem cell niche has been characterized in the mouse bone marrow and consists of an endosteal (quiescent) and vascular (proliferative) compartment. Under steady-state conditions, it is thought that most HSCs reside in the G0 phase of the cell cycle in close contact with stromal cells, including osteoblasts. The balance between quiescent and cycling stem cells appears to rely on the amount of soluble cytokines, which result in HSCs relocating from the osteoblastic to the vascular niche. This mobilization of stem cells into peripheral circulation may be necessary for reconstituting the HSC pool. Many signaling pathways are thought to contribute to stem cell self-renewal in the marrow niche including Notch, Wnt, Hedgehog, and factors that negatively regulate the cell cycle, such as Tie2/Angiopoietin-1. Cooperation of such pathways is thought to maintain stem cell homeostasis in vivo (Burns, 2005).

Several studies have hypothesized that Notch affects HSCs, although direct proof of the activity and the downstream targets have remained to be elucidated. In murine cell culture, constitutive Notch1 expression in HSC/progenitor cells establishes immortalized cell lines able to generate progeny with either lymphoid or myeloid characteristics. Retroviral Notch1 activation in recombination activating gene-1 (RAG-1)-deficient mouse stem cells results in an increase in HSC self-renewal and favors lymphoid over myeloid differentiation (Burns, 2005).

The studies presented here differ from others in that a brief pulse of Notch activity was administered and the cells were able to terminally differentiate. Other experiments with retroviruses and conditional alleles permanently express NICD and thus alter the normal maturation of cells. For instance, in adult assays an increase in the lymphoid cell fate was not concomitant with a decrease in the myeloid lineage, as previously seen. Based on these results, it is proposed that activated Notch expands the stem and progenitor cell compartment by either influencing undifferentiated cells to adopt a HSC fate or by causing a G0 HSC population to up-regulate runx1-dependent gene expression (Burns, 2005).

These findings that the stem cell markers runx1, scl, and lmo2 are transcriptionally increased in response to NICD indicates that stem and progenitor cells are expanded in the adult marrow, possibly by increasing stem cell self-renewal. Recently, a conditional allele of runx1 was generated in the mouse to study the loss of Runx1 function during adult hematopoiesis. In transplantation studies, Runx1-excised marrow cells show a reduced competitive repopulating ability in long-term engraftment assays, demonstrating that Runx1 is essential for normal stem cell function. The NICD-induced expansion of HSCs in the AGM is dependent on Runx1. The proximal and distal promoters of the human runx1 gene were examined, no DNA-binding sites for RBPjkappa, the primary Notch pathway mediator that physically interacts with DNA to modulate target gene transcription, were found. It is still possible that Notch directly regulates runx1 transcription through alternative binding sites, although it may indirectly activate runx1 expression. In either case, the Notch-Runx pathway is likely operative in both the AGM and adult marrow and may lead to the activation of downstream targets critical for stem cell homeostasis (Burns, 2005).

Notch signaling has been extensively linked to the process of both normal and aberrant stem cell self-renewal. The human Notch1 receptor, TAN-1, was first identified as a partner gene in a (7;9) chromosomal translocation found in <1% of all T-cell acute lymphoblastic leukemias (T-ALL) . Recently, >50% of all human T-ALLs were shown to have activating mutations in the notch1 gene. These data emphasize how dysregulation of the Notch signaling pathway can result in uncontrolled self-renewal that ultimately produces malignancy (Burns, 2005).

Transplantation of HSCs has been successful in the treatment of malignancies and other diseases, such as aplastic and sickle-cell anemia. After irradiation or chemotherapy is given to patients, restoration of normal hematopoiesis is critical to prevent infection and bleeding. This study has shown that a pulse of Notch activity expands stem cell number in the adult marrow without permanently altering blood lineage homeostasis. This finding has obvious therapeutic implications. Small molecule agonists that induce Notch signaling could be used to pharmacologically expand stem cell numbers and blood progenitors. For instance, embryonic cord blood stem cells are often insufficient for adult stem cell transplants. Notch activators may be used to increase mobilization of HSCs for transplantation, similar to the clinical activity of G-CSF in peripheral stem cell harvests. These data provide rationale for future clinical work to focus on methods that manipulate the Notch signaling pathway to amplify blood stem cells, and thus multilineage hematopoiesis (Burns, 2005).

Disruptions in the use of skeletal muscle leads to muscle atrophy. After short periods of disuse, muscle atrophy is reversible, and even after prolonged periods of inactivity, myofiber degeneration is uncommon. The pathways that regulate atrophy, initiated either by peripheral nerve damage, immobilization, aging, catabolic steroids, or cancer cachexia, however, are poorly understood. Runx1 (AML1) has critical roles in hematopoiesis and leukemogenesis, is poorly expressed in innervated muscle, but strongly induced in muscle shortly after denervation. To determine the function of Runx1 in skeletal muscle, mice were generated in which Runx1 was selectively inactivated in muscle. Runx1 is required to sustain muscle by preventing denervated myofibers from undergoing myofibrillar disorganization and autophagy, structural defects found in a variety of congenital myopathies. Only 29 genes, encoding ion channels, signaling molecules, and muscle structural proteins, depend upon Runx1 expression, suggesting that their misregulation causes the dramatic muscle wasting. These findings demonstrate an unexpected role for electrical activity in regulating muscle wasting, and indicate that muscle disuse induces compensatory mechanisms that limit myofiber atrophy. Moreover, these results suggest that reduced muscle activity could cause or contribute to congenital myopathies if Runx1 or its target genes were compromised (Wang, 2005).

The genes that are misregulated in runx1 mutant muscle provide clues to the causes for the profound structural changes. Notably, expression of myosin heavy-chain IIA (myh2) is not maintained (sixfold decrease) and embryonic myosin heavy chain (myh3) fails to be induced (19-fold decrease) following denervation of runx1 mutant muscle. This reduction in myosin expression may therefore explain the absence of thick filaments and distinct A- and I-bands in runx1 mutant denervated muscle. Two keratin genes (Krt1-18, Krt2-8), which encode subunits of a heterodimer, fail to be appropriately induced (five- and ninefold decrease, respectively) in denervated runx1 mutant muscle. As these keratins are thought to link Z-discs and M-lines with costameres, plasma membrane structures proposed to anchor myofibrils, a reduction in Krt1-18 and Krt2-8 expression may account for the fragmentation and misalignment of Z-discs. Moreover, these findings raise the possibility that a failure to induce myosin and keratin expression not only leads to myofibrillar disorganization, but by leading to structural perturbations, may also trigger a stress response that stimulates autophagy. Alternatively, muscle wasting may occur independently from the myofibrillar defects. Since myofiber size can be regulated by signaling proteins, such as IGF-1, the inappropriate expression of genes encoding secreted signaling molecules may contribute to wasting of runx1 mutant denervated muscle. The failure to induce osteopontin and thrombospondin I (31-fold and fourfold decrease, respectively) raises the possibility that their induction may be required to counter-balance the loss of innervation-dependent growth signals and promote muscle growth/maintenance. In addition, overexpression of orosomucoid 2 and lipocalin 2 (seven- and fivefold increase, respectively) in runx1 mutant denervated muscle is consistent with the possibility that their anomalous expression promotes muscle wasting. Further, increased expression of genes that regulate metabolism, such as resistin-like alpha or cytosolic acyl-CoA-thioesterase (10-fold and fivefold increase, respectively) may stimulate autophagy and contribute to muscle wasting. Given the small number of Runx1 target genes, further studies of these genes should lead to a detailed understanding of the mechanisms that regulate skeletal muscle wasting and may allow for a rational strategy to control autophagy in diseased muscle (Wang, 2005).

MINT, the Msx2 interacting nuclear matrix target, enhances Runx2-dependent activation of the osteocalcin fibroblast growth factor response element

Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/Cbfa1. To better understand Msx2 regulation of the OCFRE, functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation, were studied. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naive CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses (Sierra, 2004).

Hedgehog (Hh)-Patched1 (Ptch1) signaling plays essential roles in various developmental processes, but little is known about its role in postnatal homeostasis. This study demonstrate regulation of postnatal bone homeostasis by Hh-Ptch1 signaling. Ptch1-deficient (Ptch1+/-) mice and patients with nevoid basal cell carcinoma syndrome show high bone mass in adults. In culture, Ptch1+/- cells showed accelerated osteoblast differentiation, enhanced responsiveness to the runt-related transcription factor 2 (Runx2), and reduced generation of the repressor form of Gli3 (Gli3rep). Gli3rep inhibited DNA binding by Runx2 in vitro, suggesting a mechanism that could contribute to the bone phenotypes seen in the Ptch1 heterozygotes. Moreover, systemic administration of the Hh signaling inhibitor cyclopamine decreased bone mass in adult mice. These data provide evidence that Hh-Ptch1 signaling plays a crucial role in postnatal bone homeostasis and point to Hh-Ptch1 signaling as a potential molecular target for the treatment of osteoporosis (Ohba, 2008).

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1f/f mice and α1(II) Runx1f/f mice, respectively] were generated to circumvent the embryonic lethality of Runx1-deficient mice. These mice were mated with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and α1(II) DKO mice, respectively]. Prx1 Runx1f/f mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, α1(II) Runx1f/f mice and α1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of α1(II) collagen (Col2a1 -- Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of α1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6 (Kimura, 2010).

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7; see Drosophila Kismet) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 (see Drosophila Runt) occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation (Hsu, 2020).

Runt homologs and neural development

The RUNX transcription factors are important regulators of linage-specific gene expression in major developmental pathways. Runx3 is highly expressed in developing cranial and dorsal root ganglia (DRGs). Within the DRGs, Runx3 is specifically expressed in a subset of neurons, the tyrosine kinase receptor C (TrkC) proprioceptive neurons. Runx3-deficient mice develop severe limb ataxia due to disruption of monosynaptic connectivity between intra spinal afferents and motoneurons. The underlying cause of the defect is a loss of DRG proprioceptive neurons, reflected by a decreased number of TrkC-, parvalbumin- and ß-galactosidase-positive cells. Thus, Runx3 is a neurogenic TrkC neuron-specific transcription factor. In its absence, TrkC neurons in the DRG do not survive long enough to extend their axons toward target cells, resulting in lack of connectivity and ataxia. The data provide new genetic insights into the neurogenesis of DRGs and may help elucidate the molecular mechanisms underlying somatosensory-related ataxia in humans (Levanon, 2002).

Runt homologs and tooth development

The transcription factor Osf2/Cbfa1 is a key regulator of osteogenic differentiation while BSP, a major non-collagenous protein, is a marker of osteoblastic differentiation. To determine the relationship between Osf2/Cbfa1 and the formation of mineralized tissues in tooth development the temporal expression of Osf2/Cbfa1 and BSP mRNA were studied using in situ hybridization. These studies show that Osf2/Cbfa1 is expressed early in mesenchymal and epithelial tissues destined to form the mineralized tissues of the tooth and periodontal tissues, whereas BSP provides a specific marker for the differentiated cells in each of these tissues. Expression of Osf2/Cbfa1, but not BSP, is observed in the periodontal ligament indicating that expression of Osf2/Cbfa1 is not restricted to mineralizing tissues (Jiang, 1999).

Association of Runt homologs with a beta subunit and with the nuclear matrix

Continued: Runt Evolutionary homologs part 2/2

runt : Biological Overview | Regulation | Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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