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PubMed1996

Comparison of the human genomic structure of the Runt domain-encoding PEBP2/CBFalpha gene family.

We cloned the human cDNA corresponding to the cDNA (PEBP2alphaB-451) encoding the mouse polyomavirus enhancer-binding protein 2alphaB-451, representing a major splice variant from acute myeloid leukemia gene 1 (AML1). Genomic DNA clones of AML1 were also isolated and the exon/intron structure was determined. Furthermore, we determined and compared the genomic structures of three mammalian Runt domain-containing genes, PEBP2alphaA,AML/PEBE2alphaB and PEBP2alphaC.

Acute Disease↗
PubMed2006

Graded activity of transcription factor Runx3 specifies the laminar termination pattern of sensory axons in the developing spinal cord.

Different functional classes of dorsal root ganglion sensory neurons project their axons to distinct target zones within the developing spinal cord. To explore the mechanisms that link sensory neuron subtype identity and axonal projection pattern, we analyzed the roles of Runx and ETS transcription factors in the laminar targeting of sensory afferents. Gain- and loss-of-function studies in chick embryos reveal that the status of Runx3 expression is a major determinant of the dorso-ventral position of termination of proprioceptive and cutaneous sensory axons. In addition, the level of expression and/or activity of Runx3 in individual proprioceptive sensory neurons appears to specify whether their axons terminate in intermediate or ventral regions. Our findings suggest that the selectivity of Runx3 expression, and its level of activity, control sensory afferent targeting in the developing spinal cord.

Animals↗
PubMed2006

A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification.

Subpopulations of sensory neurons in the dorsal root ganglion (DRG) can be characterized on the basis of sensory modalities that convey distinct peripheral stimuli, but the molecular mechanisms that underlie sensory neuronal diversification remain unclear. Here, we have used genetic manipulations in the mouse embryo to examine how Runx transcription factor signaling controls the acquisition of distinct DRG neuronal subtype identities. Runx3 acts to diversify an Ngn1-independent neuronal cohort by promoting the differentiation of proprioceptive sensory neurons through erosion of TrkB expression in prospective TrkC+ sensory neurons. In contrast, Runx1 controls neuronal diversification within Ngn1-dependent TrkA+ neurons by repression of neuropeptide CGRP expression and controlling the fine pattern of laminar termination in the dorsal spinal cord. Together, our findings suggest that Runx transcription factor signaling plays a key role in sensory neuron diversification.

Animals↗
PubMed2002

The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.

The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

Acute Disease↗
PubMed2002

Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators.

OBJECTIVE: The AML/RUNX family of transcription factors plays important roles in hematopoiesis, neurogenesis, bone development, and segmentation in vertebrate embryos. The aim of this study was to isolate runt-related genes in a genetically and embryologically exploitable system, the zebrafish, and characterize their function during hematopoietic development. MATERIALS AND METHODS: Two runt-related genes were isolated by degenerate PCR and standard library screening, and a radiation hybrid panel, T51 RH, was used to resolve their chromosomal localization. In situ hybridization demonstrated their expression whereas their transcriptional activity was assessed using an AML1-responsive reporter gene in the MLA 144 T-cell line. RESULTS: We isolated the zebrafish runxa and runxb cDNAs, which encode proteins highly homologous to the human and murine Runx1 (AML1) and Runx3 (AML2) proteins. In contrast to a recent report, we detected runxa expression in both hematopoietic and neural tissues of the developing zebrafish. runxa transcripts first appear during segmentation in bilateral mesodermal cells that coexpress one of the earliest blood and endothelial cell markers, scl/tal-1. By 24 hours postfertilization (hpf), runxa transcripts are seen in the ventral wall of the dorsal aorta. Hematopoietic runxa expression is lost in cloche mutants, which are defective in blood and endothelial cell formation. runxb transcripts are seen in nonhematopoietic domains. Both Runxa and Runxb transactivate an AML1-responsive human promoter in hematopoietic cells. Genomic localization studies demonstrate that runxa is located on linkage group 1 (LG1), and the runxb gene is located on LG13. CONCLUSIONS: Our gene expression analysis strongly suggests that both the functional and spatial aorta-gonad-mesonephros (AGM) region has been conserved throughout evolution. Our runxa spatiotemporal expression data shed light on the role of vertebrate Runx1/AML1 in primitive vs definitive hematopoietic development.

Amino Acid Sequence↗
PubMed1999

Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone.

PTH is a major regulator of calcium homeostasis by mobilizing calcium through bone resorption. We show that the expression of collagenase-3 (MMP-13), a member of the family of matrix metalloproteinases, required for the cleavage of collagens in the bone, is increased upon PTH injection in mice. A cis-acting element in the collagenase-3 promoter was identified which, together with AP-1, is required for induction by PTH. This element contains CCACA motifs which are required for binding of the 65 kDa osteoblast-specific splice variant of Cbfal. Introduction of mutations in this binding site that interfere with protein interaction also eliminates PTH inducibility and transactivation by Cbfa/ Runt proteins. While DNA binding activity of AP-1 is increased upon PTH treatment, high basal level of Cbfa/Runt binding activity is detectable in untreated cells which is not further increased by PTH, suggesting that AP-1 and Cbfal contribute to transcriptional activation through different mechanisms. In agreement with the critical role of both proteins defined in tissue culture cells, expression of collagenase-3 is reduced in mice lacking c-fos and is completely absent in cbfa1-/-embryos. These data provide the first evidence for a critical role of Cbfal, a major regulator of bone development, in PTH-dependent processes such as bone resorption.

Animals↗
PubMed2005

Transcriptional cross-regulation of RUNX1 by RUNX3 in human B cells.

RUNX transcription factors are important in development and in numerous types of human cancer. They act as either transcriptional activators or repressors and can be proto-oncogenes or tumour suppressors. Understanding their regulation and interaction may explain how RUNX factors contribute to such different and often opposing biological processes. We show that RUNX3 regulates RUNX1 expression, contributing to the mutually exclusive expression of RUNX3 and RUNX1 in human B lymphoid cell lines. RUNX3 repressed the RUNX1 P1 promoter by binding specifically to conserved RUNX sites near the transcription start of the promoter. siRNA inhibition of RUNX3 in lymphoblastoid cells resulted in increased RUNX1 expression, indicating that continuous expression of physiological levels of RUNX3 is required to maintain repression. Furthermore, expression of RUNX3 was required for efficient proliferation of B cells immortalized by Epstein-Barr virus. Cross-regulation between different RUNX family members is therefore a means of controlling RUNX protein expression and must now be considered in the interpretation of pathological changes due to loss of RUNX3 tumour suppressor function or following gene duplication or translocation events.

B-Lymphocytes↗
PubMed2001

Functional conservation of hematopoietic factors in Drosophila and vertebrates.

The GATA, Friend of GATA, and Runt homology domain protein families function during hematopoiesis to promote progenitor cell development and regulate lineage commitment and differentiation. The hematopoietic functions of these factors have been remarkably conserved across taxonomic groups, ranging from flies to humans. Furthermore, aspects of hematopoiesis and hemocyte function appear to be conserved. Thus, comparative studies using Drosophila and vertebrate models should enhance our understanding of blood cell development.

Animals↗
PubMed2003

Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis.

The RUNX transcription factors are important regulators of lineage-specific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, the in vitro cytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3-/-;Runx1+/-), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8+ T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors of CD4 expression during T cell lineage decisions.

Animals↗
PubMed2005

The vitamin D receptor, Runx2, and the Notch signaling pathway cooperate in the transcriptional regulation of osteopontin.

Osteopontin (OPN), a glycosylated phosphoprotein that binds calcium, is present in bone extracellular matrix and has been reported to modulate both mineralization and bone resorption. Targeted disruption in mice of the vitamin D receptor (VDR) or Runx2 results in marked inhibition of OPN expression in osteoblasts. In this study, we addressed possible cross-talk between VDR and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) or Runx2 stimulated OPN transcription (mouse OPN promoter -777/+79) 2-3-fold. However, coexpression of Runx2 and VDR in COS-7 cells and treatment with 1,25(OH)(2)D(3) resulted in an 8-fold induction of OPN transcription, indicating for the first time functional cooperation between Runx2 and VDR in the regulation of OPN transcription. In ROS 17/2.8 and MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO, which acts as a repressor of Runx2, significantly inhibited 1,25(OH)(2)D(3) induction of OPN transcription, OPN mRNA, and protein expression. Both a Runx2 site (-136/-130) and the vitamin D response element (-757/-743) in the OPN promoter are needed for cooperative activation. Chromatin immunoprecipitation analyses showed that 1,25(OH)(2)D(3) can enhance VDR and Runx2 recruitment on the OPN promoter, further indicating cooperation between these two factors in the regulation of OPN. In osteoblastic cells, Hes-1, a downstream factor of the Notch signaling pathway, was found to enhance basal and 1,25(OH)(2)D(3)-induced OPN transcription. This enhancement was inhibited by AML-1/ETO, an inhibitor of Runx2. Immunoprecipitation assays indicated that Hes-1 and Runx2 interact and that 1,25(OH)(2)D(3) can enhance this interaction. Taken together, these findings define novel mechanisms involving the intersection of three pathways, Runx2, 1,25(OH)(2)D(3), and Notch signaling, that play a major role in the regulation of OPN in osteoblastic cells and therefore in the process of bone remodeling.

Animals↗
PubMed2004

Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog.

The 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. We investigated the involvement of Runx3 in chondrocyte differentiation by generating Runx2-and-Runx3-deficient (Runx2(-/-)3(-/-)) mice. We found that chondrocyte differentiation was inhibited depending on the dosages of Runx2 and Runx3, and Runx2(-/-)3(-/-) mice showed a complete absence of chondrocyte maturation. Further, the length of the limbs was 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 did not express Ihh, which regulates chondrocyte proliferation and maturation. Adenoviral introduction of Runx2 in Runx2(-/-) chondrocyte cultures strongly induced Ihh expression. Moreover, Runx2 directly bound to the promoter region of the Ihh gene and strongly induced 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.

Animals↗
PubMed2002

Expression of transcription factor AML-2 (RUNX3, CBF(alpha)-3) is induced by Epstein-Barr virus EBNA-2 and correlates with the B-cell activation phenotype.

To identify cell proteins regulated by the Epstein-Barr virus (EBV) transcription factor EBNA-2, we analyzed a cell line with conditional EBNA-2 activity by using microarray expression profiling. This led to the identification of two novel target genes induced by EBNA-2. The first of these, interleukin-16, is an immunomodulatory cytokine involved in the regulation of CD4 T cells. The second, AML-2, is a member of the Runt domain family of transcription factors. Quiescent B cells initially expressed AML-1 but, 48 h after virus infection, the levels of AML-1 decreased dramatically, whereas the amount of AML-2 protein increased. Analysis of a panel of B-cell lines indicated that AML-2 expression is normally predominant in EBV latency III, whereas AML-1 is associated with EBV latency I or EBV-negative cells. The AML genes are the first example of cell transcription factors whose expression correlates with the latency I/III phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The capacity of polyomavirus enhancer binding protein 2alphaB (AML1/Cbfa2) to stimulate polyomavirus DNA replication is related to its affinity for the nuclear matrix.

The nuclear matrix is thought to play an important role in the DNA replication of eukaryotic cells, although direct evidence for such a role is still lacking. A nuclear matrix-associated transcription factor, polyomavirus (Py) enhancer binding protein 2alphaB1 (PEBP2alphaB1) (AML1/Cbfa2), was found to stimulate Py replication through its cognate binding site. The minimal replication activation domain (RAD) was identified between amino acid (aa) 302 and aa 371 by using a fusion protein containing the GAL4 DNA binding domain (GAL4-RAD). In addition, the region showed affinity for the nuclear matrix and, on the basis of competition studies, binding activity for one or more proteins involved in the initiation of Py DNA replication. A leukemogenic chimeric protein, AML1/ETO(MTG8), which does not contain this region of PEBP2alphaB1/AML1, was also localized in the nuclear matrix fraction and competed for nuclear matrix association with PEBP2alphaB1 and GAL4-RAD. Moreover, AML1/ETO inhibited Py DNA replication stimulated by PEBP2alphaB1 and GAL4-RAD. The inhibition was specific for replication mediated by PEBP2alphaB1 and GAL4-RAD, and proportional to the degree of loss of these activators from the nuclear matrix, suggesting a requirement for nuclear matrix targeting in the stimulation of Py DNA replication by RAD. These results are the first to suggest a molecular link between the initiation of DNA replication and the nuclear matrix compartment.

Animals↗
PubMed2005

Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth.

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.

Animals↗
PubMed2005

Increased dosage of the RUNX1/AML1 gene: a third mode of RUNX leukemia?

RUNX1/AML1, located on chromosome 21, is a key factor in the generation and maintenance of hematopoietic stem cells and the gene most frequently implicated in human leukemias. Chromosome translocations and point mutations are well-documented genetic alterations in RUNX leukemia (also known as CBF leukemia). In addition, overdosage or overexpression of RUNX1 is suspected to be a third mode of RUNX1 involvement in leukemogenesis. The possibility that this mode might underlie Down syndrome-related leukemias caused by trisomy of chromosome 21 is discussed.

Animals↗
PubMed2004

Localization of the domains in Runx transcription factors required for the repression of CD4 in thymocytes.

The runt family transcription factors Runx1 and Runx3 are expressed in developing murine thymocytes. We show that enforced expression of full-length Runx1 in CD4(-)CD8(-) thymocytes results in a profound suppression of immature CD4/CD8 double-positive thymocytes and mature CD4 single-positive thymocytes compared with controls. This effect arises from Runx1- or Runx3-mediated repression of CD4 expression, and is independent of positively selecting signals. Runx1 is able to repress CD4 in CD4/CD8 double-positive thymocytes, but not in mature splenic T cells. Runx-mediated CD4 repression is independent of association with the corepressors Groucho/TLE or Sin3. Two domains are required for complete Runx-mediated CD4 repression. These are contained within Runx1 aa 212-262 and 263-360. The latter region contains the nuclear matrix targeting sequence, which is highly conserved among runt family transcription factors across species. The presence of the nuclear matrix targeting sequence is required for Runx-mediated CD4 repression, suggesting that Runx transcription factors are stabilized on the CD4 silencer via association with the nuclear matrix.

Amino Acid Sequence↗
PubMed2002

Pathways in blood and vessel development revealed through zebrafish genetics.

Studies in zebrafish have potential to contribute to understanding of the vertebrate hematopoietic and vasculogenic systems. Our research has examined the roles of several molecules in pathways that lead to the development of blood and vessels in zebrafish, and has provided insights into the regulation of these processes. Gdf6a/radar, a member of the bone morphogenetic protein (BMP) family, is expressed in the zebrafish hypochord and primitive gut endoderm; structures that flank the developing dorsal aorta and posterior cardinal vein. This pattern of expression positions Gdf6a/radar as a candidate regulator of vasculogenesis. Support for such a role has come from experiments where Gdf6a/radar function was depleted with antisense morpholino oligonucleotides. This resulted in vascular leakiness, suggesting that Gdf6a/radar is involved in maintenance of vascular integrity. The transcription factor Runx1 is known to play a critical role in mammalian definitive hematopoiesis. When Runx1 expression domains and function were analyzed in zebrafish, the importance of this gene in definitive hematopoiesis was confirmed. However there was also evidence for a wider role, including involvement in vascular development and neuropoiesis. This work has laid the foundation for an ethylnitrosourea (ENU) mutagenesis screen based on runx1 whole-mount in situ hybridzation, that aims to identify genes operative in the runx1 pathway. An additional member of the Runx family, Runx3, is also involved in developmental hematopoiesis, with a function distinct from that of Runx1. We hypothesize that Runx1 and Runx3 form a continuum of transcriptional control within the hematopoietic system. An added attraction of zebrafish is that models of human disease can be generated, and we have shown that this system has potential for the study of Runx1-mediated leukemogenesis.

Animals↗