Mammalian Gene Family Research Articles

Phosphorylation of Coronin 1B by Protein Kinase C Regulates Interaction with Arp2/3 and Cell Motility

Coronins are a conserved family of WD repeat-containing, actin-binding proteins that regulate cell motility in a variety of model organisms. Our results show that Coronin 1B is a ubiquitously expressed member of the mammalian Coronin gene family that co-localizes with the Arp2/3 complex at the leading edge of fibroblasts, and co-immunoprecipitates with this complex. Pharmacological experiments show that the interaction between Coronin 1B and the Arp2/3 complex is regulated by protein kinase C (PKC) phosphorylation. Coronin 1B is phosphorylated by PKC both in vitro and in vivo. Using tryptic peptide mapping and mutagenesis, we have identified serine 2 (Ser-2) on Coronin 1B as the major residue phosphorylated by PKC in vivo. Rat2 fibroblasts expressing the Coronin 1B S2A mutant show enhanced ruffling in response to phorbol 12-myristate 13-acetate (PMA) and increased speed in single cell tracking assays. Cells expressing the Coronin 1B S2D mutant have attenuated PMA-induced ruffling and slower cell speed. Expression of the S2A mutant partially protects cells from the inhibitory effects of PMA on cell speed, whereas expression of the S2D mutant renders cells hypersensitive to its effects. These data demonstrate that Coronin 1B regulates leading edge dynamics and cell motility in fibroblasts, and that its ability to control motility and interactions with the Arp2/3 complex are regulated by PKC phosphorylation at Ser-2. Furthermore, Coronin 1B phosphorylation is responsible for a significant fraction of the effects of PMA on fibroblast motility.

Na+/H+ exchangers: physiology and link to hypertension and organ ischemia

Na/H exchangers (NHEs) are ubiquitous proteins with a very wide array of physiological functions, and they are summarized in this paper in view of the most recent advances. Hypertension and organ ischemia are two disease states of paramount importance in which NHEs have been implicated. The involvement of NHEs in the pathophysiology of these disorders is incompletely understood. This paper reviews the principal findings and current hypotheses linking NHE dysfunction to hypertension and ischemia. With the advent of large-scale sequencing projects and powerful in-silico analyses, we have come to know what is most likely the entire mammalian NHE gene family. Recent advances have detailed the roles of NHE proteins, exploring new functions such as anchoring, scaffolding and pH regulation of intracellular compartments. Studies of NHEs in disease models, even though not conclusive to date, have contributed new evidence on the interplay of ion transporters and the delicate ion balances that may become disrupted. This paper provides the interested reader with a concise overview of NHE physiology, and aims to address the implication of NHEs in the pathophysiology of hypertension and organ ischemia in light of the most recent literature.

The Expression of Pannexin Family of Gap Junction Genes in the Mammalian Gonad

Gap junctions between germ line and somatic cells have been observed in animals ranging from worms to humans, and are presumed to be a crucial mechanism of regulation of gonad development and germ cell differentiation. In addition to connexins, a novel mammalian gene family of gap junctions, pannexins, has been recently identified (Baranova et al., Genomics 2004; 83: 706), whose expression pattern in the gonad has not been investigated in detail. Gap junctions in the fruit fly, Drosophila melanogaster, are only comprised of innexins, which share homology to pannexins and not connexins.

Comment on "Nervy Links Protein Kinase A to Plexin-Mediated Semaphorin Repulsion"

Terman and Kolodkin ([ 1 ][1]) recently suggested that Nervy, a Drosophila homolog of the mammalian myeloid translocation gene (MTG) family, functions at the plasma membrane as a protein kinase (PKA) anchoring protein (AKAP). Their findings were surprising because mammalian MTGs localize to nuclei

Cross-species analysis of the mammalian β-defensin gene family: presence of syntenic gene clusters and preferential expression in the male reproductive tract

Mammalian beta-defensins are an important family of innate host defense peptides with pleiotropic activities. As a first step to study the evolutionary relationship and biological role of the beta-defensin family, we identified their complete repertoires in the human, chimpanzee, mouse, rat, and dog following systemic, genome-wide computational searches. Although most beta-defensin genes are composed of two exons separated by an intron of variable length, some contain an additional one or two exons encoding an internal pro-sequence, a segment of carboxy-terminal mature sequences or untranslated regions. Alternatively, spliced isoforms have also been found with several beta-defensins. Furthermore, all beta-defensin genes are densely clustered in four to five syntenic chromosomal regions, with each cluster spanning <1.2 Mb across the five species. Phylogenetic analysis indicated that, although the majority of beta-defensins are evolutionarily conserved across species, subgroups of gene lineages exist that are specific in certain species, implying that some beta-defensins originated after divergence of these mammals from each other, while most others arose before the last common ancestor of mammals. Surprisingly, RT-PCR revealed that all but one rat beta-defensin transcript are preferentially expressed in the male reproductive tract, particularly in epididymis and testis, except that Defb4, a human beta-defensin-2 ortholog, is more restricted to the respiratory and upper gastrointestinal tracts. Moreover, most beta-defensins expressed in the reproductive tract are developmentally regulated, with enhanced expression during sexual maturation. Existence of such a vast array of beta-defensins in the male reproductive tract suggests that these genes may play a dual role in both fertility and host defense.

The Wnt–NLK Signaling Pathway Inhibits A-Myb Activity by Inhibiting the Association with Coactivator CBP and Methylating Histone H3

The c-myb proto-oncogene product (c-Myb) regulates proliferation and differentiation of hematopoietic cells. Recently we have shown that c-Myb is degraded in response to Wnt-1 stimulation via a pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK and HIPK2 bind directly to c-Myb and phosphorylate c-Myb at multiple sites, inducing its ubiquitination and proteasome-dependent degradation. The mammalian myb gene family contains two members in addition to c-myb, A-myb, and B-myb. Here, we report that the Wnt-NLK pathway also inhibits A-Myb activity, but by a different mechanism. As in the case of c-Myb, both NLK and HIPK2 bound directly to A-Myb and inhibited its activity. NLK phosphorylated A-Myb, but did not induce A-Myb degradation. Overexpression of NLK inhibited the association between A-Myb and the coactivator CBP, thus, blocking A-Myb-induced trans-activation. The kinase activity of NLK is required for the efficient inhibition of the association between A-Myb and CBP, although the kinase-negative form of NLK also partly inhibits the interaction between A-Myb and CBP. Furthermore, NLK induced the methylation of histone H3 at lysine-9 at A-Myb-bound promoter regions. Thus, the Wnt-NLK pathway inhibits the activity of each Myb family member by different mechanisms.

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.

Type II keratins precede type I keratins during early embryonic development

We and others have recently demonstrated that the keratin (K) gene family in mammals is even more complex than previously thought [Eur. J. Cell Biol. 83, 19–26]. To address the function of keratins during early development, precise information on their spatio-temporal expression is required. Here, we examined the expression of selected mouse keratins from pre-implantation to mid-gestational embryonic stages using RT-PCR and immunofluorescence. At E0.5, transcripts encoding K5, K6, K7, K8, K14, K15, K18, and K19 are apparently absent. We report on a post-transcriptional regulation of type I keratins, preventing filament formation in 8- to 16-cell stage embryos. In these embryos, mRNAs coding for K7, K8, K18, and K19 are present, but only K7 and K8 are translated into protein which is deposited in aggregates. Following the accumulation of K18 protein at E3.5, keratin filaments are formed. Delayed onset of type I keratin protein expression was additionally observed in later embryonic stages for K5 and K14. K5 protein expression starts in the forelimb surface ectoderm as early as E9.25, while the expression of its partner, K14, begins at E9.75. From E9.25 to E9.75, K5 forms atypical filaments with K18. Remarkably, in embryonic K5 −/− mice, K14 formed normal filaments until E12.5 despite the absence of its partner K5, due to the presence of K8. Following periderm formation, K14-containing filaments disappeared and K14 became localized in aggregates in basal keratinocytes. Despite the absence of a keratin cytoskeleton, there was no cytolysis. We suggest that the formation of the first embryonic cytoskeleton from soluble keratins is regulated by unknown mechanisms. Whether the premature expression of type II keratins relates to their proposed role in TNF- and Fas-mediated signalling is presently unknown.

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the alpha-globin gene family in mammals by isolating and characterizing alpha-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar alpha-globin family consists of at least four genes including a single adult-expressed gene (alpha), two embryonic/neonatally expressed genes (zeta and zeta'), and theta-globin, each orthologous to the respective alpha-, zeta-, and theta-globin genes of eutherian mammals. The results suggest that the theta-globin lineage arose by duplication of an ancestral adult alpha-globin gene and had already evolved an unusual promoter region, atypical of all known alpha-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that theta-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human theta-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three alpha-like and one beta-like globin genes linked in the order 5'-zeta-alpha-theta-omega-3' existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar zeta-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.

Regulation of odorant receptors: one allele at a time

The odorant receptors (ORs) make up the largest gene family in mammals. Each olfactory sensory neuron chooses just one OR from the more than 1000 possibilities encoded in the genome and transcribes it from just one allele. This process generates great neuronal diversity and forms the basis for the development and logic of the olfactory circuit between the nose and the brain. The mechanism behind this monoallelic regulation has been the subject of intense speculation and increasing experimental investigation, yet remains enigmatic. Recent genetic experiments have brought the outlines of the process into sharper relief, identifying a feedback mechanism in which the first odorant receptor expressed, generates a signal that stabilizes its choice, thus maintaining singular selection. In the absence of this signal, the olfactory neuron re-enters the selection process and switches to choose an alternate OR. Irreversible genetic changes in the nuclei of olfactory neurons do not accompany OR selection, which must therefore be initiated by an epigenetic process that may involve a stochastic mechanism.

Tertiary Windowing to Detect Positive Diversifying Selection

As a protein-encoding gene evolves, different selective pressures act on the gene temporally and spatially. An examination of the ratio of nonsynonymous-to-synonymous nucleotide substitution rate ratios (K(a)/K(s)) has proven to be a valuable method to examine selective pressures on protein encoding genes, including detecting positive diversifying selection. To gain power over averaging all sites in a gene together, examination of sites in primary sequence windows has frequently been employed. However, selection acts on folded proteins and sites that are close in tertiary space may not be close in primary sequence. A new method for the examination of K(a)/K(s) ratios based upon windows in tertiary structure is introduced and applied to the leptin gene family in mammals. Tertiary sequence windowing detects new sites under positive diversifying selection and detects positive diversifying selection with a more significant signal along various branches of the leptin gene family tree.

Dramatic variation of the vomeronasal pheromone receptor gene repertoire among five orders of placental and marsupial mammals.

Pheromones are chemicals emitted and sensed by conspecifics to elicit social and sexual responses and are perceived in terrestrial vertebrates primarily by the vomeronasal organ (VNO). Pheromone receptors in the mammalian VNO are encoded by the V1R and V2R gene superfamilies. The V1R superfamily contains 187 and 102 putatively functional genes in the mouse and rat, respectively. To investigate whether this large repertoire size is typical among mammals with functional VNOs, we here describe the V1R repertoires of dog, cow, and opossum based on their draft genome sequences. The dog and cow have only 8 and 32 intact V1R genes, respectively. Thus, the intact V1R repertoire size varies by at least 23-fold among placental mammals with functional VNOs. To our knowledge, this size ratio represents the greatest among-species variation in gene family size of all mammalian gene families. Phylogenetic analysis of placental V1R genes suggests multiple losses of ancestral genes in carnivores and artiodactyls and gains of many new genes by gene duplication in rodents, manifesting massive gene births and deaths. We also identify 49 intact opossum V1R genes and discover independent expansions of the repertoire in placentals and marsupials. We further show a concordance between the V1R repertoire size and the complexity of VNO morphology, suggesting that the latter could indicate the sophistication of pheromone communications within species. In sum, our results demonstrate tremendous diversity and rapid evolution of mammalian V1R gene inventories and caution the generalization of VNO biology from rodents to all mammals.

TRP channels as new pharmacological targets

Changes in intracellular ion concentrations are universal triggers of cell activation. Ca, for instance, is one of the most versatile intracellular second messengers in eukaryotic cells involved in a wide range of cellular processes including exocytosis, contraction, cell proliferation, development and apoptosis. Stimulation of target cells with neurotransmitters, hormones or growth factors leads to an elevation of the intracellular Ca concentration brought about by release from intracellular stores and influx via Ca-permeable cation channels in the cell membrane (Clapham et al. 2001). While excitable cells like neurons are well equipped with voltage-gated ion channels mediating the bulk of cation influx, the molecular identity of cation entry channels in non-excitable cells has been completely mysterious for quite some time. A major leap forward in our understanding of agonist-triggered cation influx was achieved by the discovery of Drosophila melanogaster visual transduction channels associated with the transient receptor potential (trp) and the trp-like (trpl) mutant phenotypes. Since then a new mammalian gene family of TRP homologs has been identified by homology screening (Montell et al. 2002; Clapham et al. 2003). Based on structural homology and on systematic glycosylation scanning, TRP proteins are allocated to the structural superfamily of six-transmembrane spanning ion channels like voltage-gated K channels, the cyclic nucleotide-gated channel family, and single cassettes of voltage-activated Ca and Na channels (Fig. 1). Both Nand C-termini of TRP proteins are assumed to be localised intracellularly, and a channel pore loop appears to be bordered by transmembrane domains 5 and 6. In specialised cells, some members of the TRP family participate in sensory processes like vision and hearing as well as temperature, pain, gustatory and pheromone perception. In addition, many TRP proteins are involved in ubiquitous, very basic cellular processes like sensing of osmotic or oxidative stress and mediating agonist-induced cation entry (Clapham 2003). Based on primary amino acid homology, the conventional TRP proteins can be classified into three subfamilies: TRPC, TRPV, and TRPM (see Fig. 1). Three additional, more distantly related subfamilies have recently been defined: ANKTM1, a Ca-permeant, non-selective cation channel, is the only mammalian member of the TRPA branch of TRP proteins. It is activated by noxious cold temperature, pungent mustard oil compounds and may additionally serve as a mechanosensitive transduction channel in auditory hair cells as well as an ionotropic cannabinoid receptor. The three mucolipins, TRPML1, 2, and 3, appear to be ion channels in intracellular vesicles. Mutations in TRPML1 cause mucolipidosis type IV, a neurodegenerative lysosomal storage disorder, while genetic defects in TRPP2, a member of the TRPP subfamily, are frequently encountered in patients suffering from autosomal dominant polycystic kidney disease. The current compilation of review articles covers salient features of TRPC, TRPV, TRPM, and TRPP proteins at the cutting edge of science. In particular, the authors continue to explore the pharmacotherapeutic potential of this new class of ion channels, a fruitful endeavor that was started at the 25th Symposium at Rauischholzhausen Castle on “TRP channels as Pharmacological Targets” which took place on June 10–12, 2004. TRPC proteins all share a common gating mechanism that involves the activation of phospholipase C isoforms and thus constitute prime molecular candidates responsible for phospholipase C-dependent cation influx. The molecThis revised version was published online in June 2005 with corrections to the Reference list

The connexin gene family in mammals

Unannotated mammalian genome databases (dog, cow, opossum) were searched for candidate connexin genes, using sequences from annotated genomes (man, mouse). All 18 'multi-species' connexin genes, i.e., orthologs of connexin26 , 29/31.3 (duplicated in opossum), 30, 30.2/31.9, 30.3, 31, 31.1, 32, 36, 37, 39/40.1, 40, 43, 45, 44/46, 47, 50, and 57/62 , were found in dog, cow and opossum. Connexin25 and 58 have been considered specific for man, but evident orthologs of connexin25 were found in dog, cow and opossum, and orthologs of connexin58 were found in dog and cow. Moreover, a connexin43 -like sequence (approx. 80% identical to connexin43 ) was found in man, chimpanzee, dog and cow. In the three former species, the sequences were located on the X chromosome. In man, chimpanzee and cow, there were stop codons in all reading frames; these sequences are therefore judged as pseudogenes, called here Cx43pX . In the dog, the sequence contained an open reading frame for a protein of 35.7 kDa (connexin35.7). We suggest that these sequences are orthologs of connexin33 , previously considered as a rodent-specific connexin gene. Thus, connexin25 , 33 and 58 are not species-specific genes. However, the opossum may possess a candidate, connexin39.2 , without obvious orthologs in other mammals. Furthermore, pseudogenes of primate connexin31.3 and opossum connexin35 (one of the two orthologs of primate connexin31.3) were detected. These results suggest that the structure of the mammalian connexin gene family should be revised, especially with regard to the so-called 'species-specific' connexins .

Supplementary material to the paper “The connexin gene family in mammals”

Supplementary material to the paper “The connexin gene family in mammals”

Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family

By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

Characterization of the baculovirus Bombyx mori nucleopolyhedrovirus gene homologous to the mammalian FGF gene family.

We characterized a gene of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) homologous to the mammalian fibroblast growth factor (FGF) family. We termed it vfgf, and examined its transcription and the properties of the gene product (vFGF). RT-PCR analysis showed that vfgf is one of the baculovirus early genes, although there are no consensus sequences of the baculovirus early gene promoters. 5'-RACE analysis revealed that its transcription started at 10 nucleotides upstream of the translation start codon. vFGF has a hydrophobic amino terminus (approximately 16 amino acids), which is a typical signal sequence. As expected, vFGF was efficiently secreted from BmNPV-infected BmN cells. Because possible glycosylation sites are found at positions 44 (Asn) and 171 (Asn), we examined whether BmNPV vFGF is glycosylated or not. Cleavage of recombinant vFGF with PNGase F revealed that BmNPV vFGF was glycosylated. We also found that secretion of vFGF is completely blocked by the treatment of Tunicamycin, which blocks N-linked glycosylation. This is the first report to characterize a virus-encoded FGF.

Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion.

The renal proximal tubule of vertebrates performs an essential role in controlling plasma SO(4)(2-) concentration ([SO(4)(2-)]). Although net tubular SO(4)(2-) reabsorption is the predominate control process in terrestrial vertebrates, a facilitated secretory flux is also present. In contrast, marine teleosts obtain excess SO(4)(2-) from drinking, and increased plasma [SO(4)(2-)] is prevented predominately through net tubular secretion. Tubular SO(4)(2-) secretion is accomplished by at least two electroneutral anion exchange processes in series. Movement of SO(4)(2-) into the cell across the basolateral membrane is pH dependent, suggesting SO(4)(2-)/OH(-) exchange. Luminal HCO(3)(-) and Cl(-) can facilitate SO(4)(2-) movement out of the cell across the brush-border membrane. The molecular identities of the anion exchangers are unknown but are probably homologues of SO(4)(2-) transporters in the mammalian SLC26 gene family. In all species tested, glucocorticoids increase renal SO(4)(2-) excretion. Whereas glucocorticoids downregulate SO(4)(2-) reabsorptive mechanisms in terrestrial vertebrates, they may also stimulate a mediated secretory flux. In the marine teleost, cortisol increases the level of SO(4)(2-)/HCO(3)(-) exchange at the brush-border membrane, tubular carbonic anhydrase (CA) activity, CAII protein, and a proportion of tubular SO(4)(2-) secretion that is CA dependent. CA activity is required for about one-half of this net SO(4)(2-) secretion but is also required for about one-half of the net reabsorption in bird proximal epithelium. A CA-SO(4)(2-)/anion exchanger metabolon arrangement is proposed that may speed both the secretory and reabsorptive processes.

Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.

Hyperpolarization-activated cation currents, termed Ih, are non-uniformly distributed along dendritic arbors with current density increasing with increasing distance from the soma. The non-uniform distribution of Ih currents contributes to normalization of location-dependent variability in temporal integration of synaptic input, but the molecular basis for the graded HCN distribution remains to be investigated. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs) underlie Ih currents and consist of four members (HCN1-HCN4) of the gene family in mammals. In this investigation, we report that HCN2 forms a protein assembly with tamalin, S-SCAM and Mint2 scaffold proteins, using several different approaches including immunoprecipitation of rat brain and heterologously expressing cell extracts and glutathione S-transferase pull-down assays. The PDZ domain of tamalin interacts with HCN2 at both the PDZ-binding motif and the internal carboxy-terminal tail of HCN2, whereas binding of the PDZ domain of S-SCAM occurs at the cyclic nucleotide-binding domain (CNBD) and the CNBD-downstream sequence of the carboxy-terminal tail of HCN2. A protein assembly between HCN2 and Mint2 is formed by the interaction of the munc18-interacting domain of Mint2 with the CNBD-downstream sequence of HCN2. The results demonstrate that HCN2 forms a protein complex with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.

RUNX1 transformation of primary embryonic fibroblasts is revealed in the absence of p53.

The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19(ARF)-p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently pro-oncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFbeta indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.