The restriction (R)-point decision is fundamental to normal differentiation and the G(1)-S transition, and the decision-making machinery is perturbed in nearly all cancer cells. The mechanisms underlying the cellular context-dependent R-point decision remain poorly understood. We found that the R-point was dysregulated in Runx3(-/-) mouse embryonic fibroblasts (MEFs), which formed tumors in nude mice. Ectopic expression of Runx3 restored the R-point and abolished the tumorigenicity of Runx3-/-MEFs and K-Ras-activated Runx3(-/-) MEFs (Runx3(-/-); K-Ras(G12D/+)). During the R-point, Runx3 transiently formed a complex with pRb and Brd2 and induced Cdkn1a (p21(Waf1/Cip1/Sdi1); p21), a key regulator of the R-point transition. Cyclin D-CDK4/6 promoted dissociation of the pRb-Runx3-Brd2 complex, thus turning off p21 expression. However, cells harboring oncogenic K-Ras maintained the pRb-Runx3-Brd2 complex and p21 expression even after introduction of Cyclin D1. Thus, Runx3 plays a critical role in R-point regulation and defense against cellular transformation.

The tumor-suppressor RUNX3 has a critical role in a lineage determination, cell cycle arrest and apoptosis. Lozenge (Lz), a Drosophila homolog of mammalian RUNX family members, has integral roles in these processes and specifically in eye cell fate determination. To elucidate the genetic modifiers of Lz/RUNX3, we performed a large-scale functional screen in a fly mutant library. The screen revealed genetic interactions between the Lz, Rac and Hippo pathways. Analysis of interactions among these genes revealed that the defective phenotype resulting from activation of Yki, an end point effector of the Hippo pathway, was suppressed by Lz and enhanced by Rac-Trio. Molecular biological analysis using mammalian homologs reveled that LATS1/2-mediated YAP phosphorylation-facilitated dissociation of the YAP-TEAD4 complex and association of the YAP-RUNX3 complex. When cells were stimulated to proliferate, activated RAC-TRIO signaling inhibited LATS1/2-mediated YAP phosphorylation; consequently, YAP dissociated from RUNX3 and associated with TEAD, thereby replacing the YAP-RUNX3 complex with YAP-TEAD. RUNX3 contributed to both association and dissociation of YAP-TEAD complex, most likely through the formation of the YAP-TEAD-RUNX3 ternary complex. Ectopic expression of RUNX3 in MKN28 gastric cancer cells reduced tumorigenicity, and the tumor-suppressive activity of RUNX3 was associated with its ability to interact with YAP. These results identify a novel regulatory mechanism, mediated by the Hippo and RAC-TRIO pathways, that changes the binding partner of YAP.

The multifunctional enzyme transglutaminase 2 (TG2) primarily catalyzes cross-linking reactions of proteins via (γ-glutamyl) lysine bonds. Several recent findings indicate that altered regulation of intracellular TG2 levels affects renal cancer. Elevated TG2 expression is observed in renal cancer. However, the molecular mechanism underlying TG2 degradation is not completely understood. Carboxyl-terminus of Hsp70-interacting protein (CHIP) functions as an ubiquitin E3 ligase. Previous studies reveal that CHIP deficiency mice displayed a reduced life span with accelerated aging in kidney tissues. Here we show that CHIP promotes polyubiquitination of TG2 and its subsequent proteasomal degradation. In addition, TG2 upregulation contributes to enhanced kidney tumorigenesis. Furthermore, CHIP-mediated TG2 downregulation is critical for the suppression of kidney tumor growth and angiogenesis. Notably, our findings are further supported by decreased CHIP expression in human renal cancer tissues and renal cancer cells. The present work reveals that CHIP-mediated TG2 ubiquitination and proteasomal degradation represent a novel regulatory mechanism that controls intracellular TG2 levels. Alterations in this pathway result in TG2 hyperexpression and consequently contribute to renal cancer.

Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins.

Background: Runt-related transcription factor 2 (RUNX2) is a transcription factor that is closely related to bone formation, and prostate cancer (CaP) is the most common cancer to metastasize to bone. The present study investigated the expression levels of RUNX2 in human prostate tissue, and the correlation between RUNX2 levels and the clinicopathological characteristics of CaP. Methods: A case–control study was conducted including 114 cases of newly diagnosed CaP and 114 age-matched BPH patients as controls. RUNX2 expression was estimated using real-time PCR and immunohistochemical staining. Results: The mRNA expression of RUNX2 did not differ between CaP tissues and non-cancer BPH controls ( P =0.825). However, RUNX2 expression was significantly decreased in patients with elevated PSA levels (⩾20 ng ml −1 ), a Gleason score ⩾8 and metastatic disease compared to those with low PSA, low Gleason score and non-metastatic disease ( P =0.023, 0.005 and 0.014, respectively). Immunohistochemical analysis showed that 65.2% of the patients with positive RUNX2 nuclear staining had metastatic disease, which was present in only 25.9% of those with negative staining ( P =0.010). Conclusions: RUNX2 mRNA expression was negatively correlated with CaP aggressiveness. Moreover, the nuclear location of RUNX2 may be a prognostic marker of metastasis in CaP.

Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3−/− mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3−/− bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3−/− epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3+/− mice (∼18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3+/− mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.

We reported earlier that RUNX3 is expressed in human and mouse gastrointestinal tract (GIT) epithelium and that it functions as a tumor suppressor in gastric and colorectal tissues. However, there have been conflicting reports describing the absence of Runx3 in GIT epithelial cells. A part of the controversy may be derived from the use of a specific antibody by other groups (referred to as G-poly). Here, we show further evidence to support our earlier observations and provide a possible explanation for this apparent controversy. We generated multiple anti-RUNX3 monoclonal antibodies and found that RUNX3 antibodies recognizing the RUNX3 N-terminal region (residues 1–234) react with RUNX3 in gastric epithelial cells, whereas those recognizing the C-terminal region (beyond residue 234) did not. G-poly primarily recognizes the region beyond 234 and hence, is unable to detect Runx3 in this tissue.

The RUNX family members play pivotal roles in normal development and neoplasia. RUNX1 and RUNX2 are essential for hematopoiesis and osteogenesis, respectively, while RUNX3 is involved in neurogenesis, thymopoiesis and functions as a tumor suppressor. Inappropriate levels of RUNX activity are associated with leukemia, autoimmune disease, cleidocranial dysplasia, craniosynostosis and various solid tumors. Therefore, RUNX activity must be tightly regulated to prevent tumorigenesis and maintain normal cell differentiation. Recent work indicates that RUNX activity is controlled by various extracellular signaling pathways, and that phosphorylation, acetylation and ubiquitination are important post-translational modifications of RUNX that affect its stability and activity. Defining the precise roles, these modifications that play in the regulation of RUNX function may reveal not only how the RUNX proteins are regulated but also how they are assembled into other regulatory machineries.

Two major isoforms of the Runx2 gene are expressed by alternative promoter usage: Runx2 type I (Runx2-I) is derived from the proximal promoter (P2), and Runx2 type II (Runx2-II) is produced by the distal promoter (P1). Our previous results indicate that Dlx5 mediates BMP-2-induced Runx2 expression and osteoblast differentiation (Lee, M.-H., Kim, Y-J., Kim, H-J., Park, H-D., Kang, A-R., Kyung, H.-M., Sung, J-H., Wozney, J. M., Kim, H-J., and Ryoo, H-M. (2003) J. Biol. Chem. 278, 34387-34394). However, little is known of the molecular mechanisms by which Dlx5 up-regulates Runx2 expression in BMP-2 signaling. Here, Runx2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and Runx2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and Runx2-I and Msx2 in the sutural mesenchyme. Furthermore, Runx2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas Runx2 P2 promoter activity was not. Runx2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between -756 and -342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the Runx2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the Runx2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.

RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor beta1 (TGF-beta1). It is known that TGF-beta1 induces cell growth arrest by activating CDKN1A (p21(WAF1)(/Cip1)), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-beta-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-beta-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.

The Runt domain transcription factors (RUNXs) play essential roles in normal development and neoplasias. Genetic analyses of animals and humans have revealed the involvement of RUNX1 in hematopoiesis and leukemia, RUNX2 in osteogenesis and cleidocranial dysplasia, and RUNX3 in the development of T-cells and dorsal root ganglion neurons and in the genesis of gastric cancer. Here we report that RUNX3 is a target of the acetyltransferase activity of p300. The p300-dependent acetylation of three lysine residues protects RUNX3 from ubiquitin ligase Smurf-mediated degradation. The extent of the acetylation is up-regulated by the transforming growth factor-beta signaling pathway and down-regulated by histone deacetylase activities. Our findings demonstrate that the level of RUNX3 protein is controlled by the competitive acetylation and deacetylation of the three lysine residues, revealing a new mechanism for the posttranslational regulation of RUNX3 expression.

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 downstream target of rnt-1. Our data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans.

RUNX family transcription factors are integral components of TGF-β signaling pathways and have been implicated in cell cycle regulation, differentiation, apoptosis, and malignant transformation. It was noted previously that allele loss and loss of expression of RUNX3 are causally involved in gastric carcinogenesis. Our results demonstrate that RUNX3 is inactivated by aberrant DNA methylation in approximately 19% of lung cancer cell lines and 24% of primary lung cancer specimens. RUNX3 methylation is tumor-specific, since it is not observed in surrounding normal lung tissues. Our results suggest that loss of RUNX3 expression by DNA hypermethylation is frequently associated with the evolution of lung cancer.

Dorsal root ganglion (DRG) neurons project their axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality (Neuron 30 (2001), 707; Cell 101 (2000), 485; Neuron 31 (2001), 59; J. Comp. Neurol. 380 (1997), 215; Sensory Neurons, Oxford Univ. Press, New York, 1992, p. 131). Expression of runt-related Runx/AML genes (Mech. Dev. 109 (2001), 413) on subtypes of DRG neurons suggests their involvement in lamina-specific afferent differentiation and maturation. Here we show that Runx3−/− mice display severe limb ataxia and abnormal posture and that most of them die shortly after birth. They show that proprioceptive afferent axons fail to reach the ventral horn and have a smaller dorsal funiculus in their spinal cords. Despite the strong resemblance of this phenotype to that of knockout mice deficient in neurotrophin-3 (NT-3) (Cell 77 (1994), 503; Nature 369 (1994), 658) and its receptor, trkC, (Nature 368 (1994), 249), which show proprioceptive afferent loss through selective neuronal cell death, Runx3−/− mice maintain normal number of TrkC/trkC positive DRG neurons throughout development. Our results suggest that Runx3 controls the target-specific axon pathfinding of trkC-expressing DRG neurons in the spinal cord.

In this issue, Y. Groner and colleagues discuss the possible involvement of the transcription factor Runx3 in stomach cancer (Concept, pp.560–564). The fundamental point of the article is that, although both of our groups observed the same neurological and T-cell phenotypes in Runx3 knockout (KO) mice, the C57BL6 strain that we used (type I KO; Li et al., 2002) developed gastric abnormalities, whereas the ICR strain used by Groner (type II KO; Levanon et al., 2002) did not. Although careful analysis to find reasons for the differences in these studies is necessary, we feel that important points were not made in this Concept and need to be addressed here. We acknowledged the differences between the two strains in one of our subsequent papers (Inoue et al., 2002), but as the ICR strain is outbred, we did not further analyse its phenotype. However, we have shown that Runx3 is involved in the transforming growth factor-β (TGF-β) signalling pathway, and it is well known that responses to TGF-β vary in different strains of mice (Kallapur et al., 1999), which could explain the difference in the gastric phenotypes of the type I and type II KO mice. The other main query raised over the use of the C57BL6 mice was that they are more susceptible to Helicobacter felis infection. However, we examined the stom-ach epithelium of Runx3−/− C57BL6 mice before the mice drank milk, ruling out the possibility of Helicobacter involvement in the phenotype we described. Groner's group was unable to detect the Runx3 protein in the gastric epithelium of mouse embryos and therefore question whether the gastric abnormalities seen in the type I KO mice are due to a lack of Runx3. On the basis of their results, they conclude that Runx3 is not expressed in mouse stomach epithelial cells at any time during their life cycle. This is the most direct contradiction between the two groups and therefore merits careful investigation. Genes involved in development and differentiation, such as the Runx genes, change their expression patterns during development. We have shown that Runx3 is expressed in the glandular stomach epithelial cells of 10-week-old mice and also in the embryonic epithelial cells, albeit at much lower levels (Li et al., 2002). Thus, the levels at this early stage might have been too low to be detected in Groner's study. Importantly, specific antibodies were used for detection in Groner's studies, whereas we tested for the presence of Runx3 RNA. The titre of the antibody might not have been high enough to detect such low levels of protein and, in addition, the Runx3 protein could be more labile in stomach than in other tissues. As the main issue here is whether Runx3 is expressed in stomach epithelial cells, we suggest that they analyse adult mouse stomach. Conversely, we agree with the finding of Groner's group that Runx3 is expressed in mesenchymal tissues of mouse embryo stomach. However, we reported that this Runx3 expression in mesenchyme is low compared with that in epithelial cells. Therefore, there seems to be a marked change in the relative expression levels of Runx3 in epithelial cells and mesenchymal cells from embryo to adult. As doubts have been cast on the expression of Runx3 in the stomach, it is interesting to consider the roles of Runx3 from an evolutionary perspective. Runx3 is thought to be the most ancient form of the three mammalian Runx genes and is involved in the neurogenesis of the monosynaptic reflex arc. But it is also known that Caenorhabditis elegans and sea urchins contain only one Runx gene and, in these animals, this is expressed in the intestine and foregut, respectively (Nam et al., 2002; Robertson et al., 2002). Thus, Runx3 might have had an important role in controlling growth and differentiation of gut epithelial cells throughout evolution. Several crucial observations that were made in the original paper describing the type I KO mice have not been mentioned in the Concept. For example, the growth of tumours in nude mice, induced by a human gastric cancer cell line that does not express RUNX3, was strongly inhibited by exogenous expression of RUNX3. This observation suggests that RUNX3 has a tumour-suppressive effect. Although rare, we also found a loss-of-function mutation in RUNX3, termed RUNX3(R122C), in one gastric carcinoma patient. RUNX3(R122C) did not have the tumour-suppressive effect mentioned above. Finally, although cell lines isolated from the gastric epithelia of p53−/− Runx3+/+ mouse embryos did not induce tumours in nude mice, those from p53−/− Runx−/− mice induced adenocarcinoma (Li et al., 2002). These data alone are sufficient to suggest strongly that there is a causal relationship between the loss of expression of RUNX3 and gastric cancer. The construction of the target vector used to generate the type I KO mice is also cited as a possible source of the discrepancy between the gastric phenotypes. However, we feel that the method was not sufficiently clear in our original paper and readers may have interpreted that LacZ was directly fused at the SmaI site in exon 4 of Runx3 (designated exon 3 in the original paper). This would eliminate only a small part of the carboxy-terminal end of the Runt domain and the resulting protein product might still interact with polyomavirus enhancer-binding protein 2β (PEBP2-β)/core-binding factor β (CBF-β). In fact, although the DNA was cleaved at the SmaI site, the 3′ end was digested and a KpnI site was inserted. Therefore, the final construct is lacking 24 amino acids from the C terminus of exon 4 and has only the 12 remaining amino acids fused in-frame to LacZ; this is unlikely to bind to PEBP2-β/CBF-β. Another point raised in the Concept is that, in type I KO mice, the inserted phosphoglycerate kinase (PGK)–neo gene could also drive the expression of Clic4, 50 kb downstream, which has been shown to abrogate apoptosis. The PGK promoter can activate the expression of neighbouring genes when they are clustered and of distantly located genes when the promoter is inserted in the locus control region (Scacheri et al., 2001). Neither of these applies to our targeted locus. Furthermore, the finding by Scacheri and colleagues that a gene located 2 kb from the PGK promoter was not affected, suggests that a gene 50 kb away would also not be affected. Runx3 has two promoters, P1 and P2, and the latter is silenced by hypermethylation in human gastric tumours. However, Groner and colleagues suggest that Runx3 expression could then be driven by P1. We have previously performed RT–PCR (PCR after reverse transcription) with two primer sets, Ps-N for P2-specific messenger RNA and Ps-C for common mRNA, and did not observe any pro-duct in either of the reactions in P2-methylated cell lines (Li et al., 2002). So, in the case of gastric cancer cell lines, a promoter switch of Runx3 from P2 to P1 was not observed. Finally, the Concept highlights the fact that Runx1 is expressed in stomach epithelium, with which we agree. A potential regulatory role of Runx1 in the stomach will be an interesting subject for future study.

Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling induces the expression of Runx2, a key transcription factor in osteoblast differentiation, but little is known about the molecular signaling mechanisms that mediate this. Here we examined the role of the protein kinase C (PKC) pathway in regulating Runx2 gene expression and its transactivation function. Treatment with FGF2 or FGF4, or transfection with a vector expressing a mutant FGFR2 that is constitutively activated in the absence of ligand, strongly stimulates Runx2 expression. Electrophoretic mobility shift assays also showed that FGF2 treatment increases the specific binding of Runx2 to the cognate response element in the osteocalcin gene promoter. Blocking PKC completely inhibited FGF2-induced Runx2 expression, whereas mitogen-activate protein kinase inhibitors had no effect. The FGF/FGFR-stimulated 6xOSE2 promoter activity was also blocked by inhibiting PKC, as was the FGF2 stimulation of the DNA-binding activity of Runx2. Experiments with PKC isoform-specific inhibitors and dominant negative isoforms of PKC indicate that PKCS is one of key isoforms involved in the FGF2-stimulated Runx2 expression. In addition, experiments with Runx2-knockout cells showed that, although the PKC pathway largely regulates FGF2-stimulated Runx2 activity by upregulating Runx2 expression, it also modifies Runx2 protein post-translationally and thereby increases its transcriptional activity. Thus, we show for the first time that FGF/FGFR signaling stimulates the DNA-binding and transcriptional activities of Runx2 as well as its expression, and these are largely regulated by the PKC pathway.

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. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that 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.

The Caenorhabditis elegans run gene encodes a Runt domain factor. Runx1, Runx2, and Runx3 are the three known mammalian homologs of run. Runx1, which plays an essential role in hematopoiesis, has been identified at the breakpoint of chromosome translocations that are responsible for human leukemia. Runx2 plays an essential role in osteogenesis, and inactivation of one allele of Runx2 is responsible for the human disease cleidocranial dysplasia. To understand the role of run in C. elegans, we used transgenic run::GFP reporter constructs and a double-stranded RNA-mediated interference method. The expression of run was detected as early as the bean stage exclusively in the nuclei of seam hypodermal cells and lasted until the L3 stage. At the larval stage, expression of run was additionally detected in intestinal cells. The regulatory elements responsible for the postembryonic hypodermal seam cells and intestinal cells were separately located within a 7.2-kb-long intron region. This is the first report demonstrating that an intron region is essential for stage-specific and cell type-specific expression of a C. elegans gene. RNA interference analysis targeting the run gene resulted in an early larva-lethal phenotype, with apparent malformation of the hypodermis and intestine. These results suggest that run is involved in the development of a functional hypodermis and gut in C. elegans. The highly conserved role of the Runt domain transcription factor in gut development during evolution from nematodes to mammals is discussed.

PEBP2 beta/Cbf beta is the beta subunit of PEBP2/Cbf, which has been demonstrated to have important biological activities in hematopoiesis and osteogenesis. However, PEBP2 beta is ubiquitously expressed, suggesting that PEBP2 has other additionally important physiological activities. In an effort to elucidate other possible functions for PEBP2, we have isolated a novel gene that encodes a PEBP2 beta-interacting protein from a mouse cDNA library. We have called this gene Crl-1 for charged amino acid rich leucine zipper-1 (Crl-1) because it is rich in charged amino acids and contains a putative leucine zipper region. Expression studies in a 17.5 days post-coitum mouse embryo demonstrated Crl-1 expression mainly in the olfactory bulb and cerebral cortex. Post-natally, Crl-1 expression was additionally observed in the cerebellar cortex with strong expression in the hippocampus. These findings show that this novel PEBP2 beta-interacting protein is expressed mainly in subsets of neuronal cells, suggesting that Crl-1 plays some role in the developing mouse brain.

BACKGROUND:When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor-beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein-2 (BMP-2) not only blocks myogenic differentiation but also induces osteoblastic differentiation. However, the molecular mechanisms governing the ability of TGF-beta and BMP to induce ligand-specific responses and inhibit myogenic differentiation are not known. The objective of our studies was to elucidate the molecular mechanisms that block myoblastic differentiation and induce osteoblastic differentiation in C2C12 cells. METHODS:Induction of RUNX2/PEBP2alphaA/Cbfa1 by TGF-beta and BMP was examined by electrophoretic mobility shift assay (EMSA) and Northern blot analysis. C2C12 cells stably expressing RUNX2 or Smad, or both, were established, and the role of these genes in the process of osteoblastic differentiation was analyzed by examining the expression of osteoblast-specific markers. RESULTS:Treatment of C2C12 with TGF-beta and BMP-induced RUNX2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis. Cooperation between RUNX2 and BMP-activated Smad induced osteoblastic differentiation. CONCLUSIONS:Both TGF-beta and BMP activate transcription of RUNX2, which is sufficient to inhibit myogenesis. To induce osteogenesis, BMP-induced RUNX2 must cooperate with BMP-activated Smads.