Runt Domain Transcription Factor Research Articles

Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia

AbstractRUNX3/AML2 is a Runt domain transcription factor like RUNX1/AML1 and RUNX2/AML3. Regulated by 2 promoters P1 and P2, RUNX3 is frequently inactivated by P2 methylation in solid tumors. Growing evidence has suggested a role of this transcription factor in hematopoiesis. However, genetic alterations have not been reported in blood cancers. In this study on 73 acute myeloid leukemia (AML) patients (44 children and 29 adults), we first showed that high RUNX3 expression among childhood AML was associated with a shortened event-free survival, and RUNX3 was significantly underexpressed in the prognostically favorable subgroup of AML with the t(8;21) and inv(16) translocations. We further demonstrated that this RUNX3 repression was mediated not by P2 methylation, but RUNX1-ETO and CBFβ-MYH11, the fusion products of t(8;21) and inv(16), via a novel transcriptional mechanism that acts directly or indirectly in collaboration with RUNX1, on 2 conserved RUNX binding sites in the P1 promoter. In in vitro studies, ectopically expressed RUNX1-ETO and CBFβ-MYH11 also inhibited endogenous RUNX3 expression. Taken together, RUNX3 was the first transcriptional target found to be commonly repressed by the t(8;21) and inv(16) fusion proteins and might have an important role in core-binding factor AML.

Cancer inhibition by normal differentiated cells

The Runt domain transcription factors, RUNX1, RUNX2 and RUNX3, are integral components of signaling cascades mediated by both TGF-β and bone morphogenetic proteins (BMPs) in several important biological systems. RUNX2 functions synergistically with Smad1 and Smad5 to regulate bone-specific genes when BMP induces osteogenesis. RUNX3, which has been mapped to locus 1p36, is a major tumor suppressor of gastric cancer and appears to be an important component of the TGF-β-induced tumor suppressor pathway. A possible relationship between the TGF-β-induced tumor-suppressor pathway and a postulated tumor suppressor gene on 1p36 must be examined.

Phosphorylation of Runx1 at Ser249, Ser266, and Ser276 is dispensable for bone marrow hematopoiesis and thymocyte differentiation

Runx1, one of three mammalian runt-domain transcription factor family proteins, is essential for definitive hematopoiesis. Based on transfection assays, phosphorylation of Runx1 at the three serine residues, Ser249, Ser266, and Ser276, was thought to be important for trans-activation activity of Runx1. By using “knock-in” gene targeting, we generated mouse strains expressing mutant Runx1 protein that harbored a combined serine-to-alanine substitution at either of two residues, Ser249/Ser266 or Ser249/Ser276. Either mutation resulted in a lack of major phosphorylated form of Runx1. However, while loss of definitive hematopoiesis and impaired thymocyte differentiation was observed following the loss of Runx1, these phenotypes were rescued in those mice lacking the major phosphorylated form of Runx1. These results not only challenge the predicted regulation of Runx1 activity by phosphorylation at these serine residues, but also reaffirm the effectiveness of “knock-in” mutagenesis as a powerful tool for addressing the physiological relevance of post-translation modifications.

Two of four alternatively spliced isoforms of RUNX2 control osteocalcin gene expression in human osteoblast cells

Runx2 is a Runt domain transcription factor that transcriptionally regulates osteoblast differentiation and bone formation. In this study, we show that human chondro- and osteosarcoma cell lines, human mesenchymal stem cells (hMSC) and a human primary chondrocytes (HC), osteoblst cells (HOb) express an intact isoform (RUNX2wt) and 3 alternatively spliced isoforms (RUNX2Δ5, Δ7, and Δ5Δ7) that are generated by skipping exon 5 and/or exon 7. Two of the truncated forms of RUNX2 (RUNX2Δ5 and RUNX2Δ5Δ7) did not localize in the nucleus and had lost their DNA binding activity. In cotransfection experiments with an osteocalcin ( OC) promoter construct, we confirmed that only RUNX2wt and RUNX2Δ7 could upregulate the OC promoter activity in the osteosarcoma cell line. In addition, the coactivator CBP/p300 enhanced the transcriptional activity of the OC promoter when coexpressed with RUNX2wt or RUNX2Δ7, but not when coexpressed with RUNX2Δ5 or RUNX2Δ5Δ7. In contrast, the corepressor HDAC3 only repressed the activation from the OC promoter when coexpressed with RUNX2wt. These results support the hypothesis that RUNX2 both up- and downregulates its target gene promoters, as exemplified by the OC gene, using various isoforms and context-dependent formation of transcriptional complexes.

Mechanisms of Compartmentalized Expression of Mrg Class G-Protein-Coupled Sensory Receptors

Mrg class G-protein-coupled receptors (GPCRs) are expressed exclusively in sensory neurons in the trigeminal and dorsal root ganglia. Pharmacological activation of Mrg proteins is capable of modulating sensory neuron activities and elicits nociceptive effects. In this study, we illustrate a control mechanism that allows the Runx1 runt domain transcription factor to generate compartmentalized expression of these sensory GPCRs. Expression of MrgA, MrgB, and MrgC subclasses is confined to an "A/B/C" neuronal compartment that expresses Runx1 transiently (or does not express Runx1), whereas MrgD expression is restricted to a "D" compartment with persistent Runx1 expression. Runx1 is initially required for the expression of all Mrg genes. However, during late development Runx1 becomes a repressor for MrgA/B/C genes. As a result, MrgA/B/C expression persists only in the Runx1- "A/B/C" compartment. In delta446 mice, in which Runx1 lacks the C-terminal repression domain, expression of MrgA/B/C genes is dramatically expanded into the Runx1+ "D" compartment. MrgD expression, however, is resistant to Runx1-mediated repression in the "D" compartment. Therefore, the creation of Runx1+ and Runx1- compartments, in conjunction with different responses of Mrg genes to Runx1-mediated repression, results in the compartmentalized expression of MrgA/B/C versus MrgD genes. Within the MrgA/B/C compartment, MrgB4-expressing neurons innervate exclusively the hairy skin. Here we found that Smad4, a downstream component of bone morphological protein-mediated signaling, is required selectively for the expression of MrgB4. Our study suggests a new line of evidence that specification of sensory subtypes is established progressively during perinatal and postnatal development.

AML1/RUNX1, One of the Most Common Targets of Aberration in Acute Myeloid Leukemia as a Transcriptional Regulator of Vascular Endothelial Growth Factor (VEGFA).

Cellular VEGFA is an independent adverse prognostic factor related to worse outcome in AML and is significantly upregulated in leukemic blasts (de bont ES. et al., 2002). Transcriptional activation of the VEGFA promoter represents the core mechanism through which expression of VEGFA can be regulated, but is still not identified in AML (Carmeliet P. and Jaine RK., 2000; Kolch W. et al., 1995; Yancoupolus GD. et al., 2000). One of the most common targets of aberrations in acute leukemia is Runt domain transcription factor (AML1/RUNX1), due to point mutations (Roumier C. et al., 2003) or disruption of chromosome 21, which occurs in about 10%-15% of all de novo AML patients. In the current study, functional 5′ deletion analysis of the VEGFA promoter was performed to identify VEGFA promoter regions involved in VEGFA transcriptional regulation in AML. Loss of the region spanning −2274/−507 resulted in a significant increase of promoter activity in two AML cell lines, whereas the promoter activity dropped after deletion of the promoter region −286 to −52. Computer-assisted sequence analysis of the −2247/−507 fragment of the VEGFA promoter, revealed three perfect AML1/RUNX1-binding sites in the first three deletion regions. The loss of each AML1/RUNX1-binding site corresponded to an (further) increase in VEGFA promoter activity. siRNA mediated AML1/RUNX1 depletion caused a 24% to 36% increase in VEGFA mRNA expression in two different AML cell lines (HL-60 and TF-1), whereas VEGFA promoter activity was two-fold increased in HL-60 cells treated with AML1/RUNX1 siRNA compared to the non-silencing control siRNA. In addition, mutation of all three AML1/RUNX1 sites resulted in a 22 fold increase of VEGFA promoter activity in HL-60 cells. VEGFA mRNA expression was found to be higher in AML patients with a t(8;21) translocation compared to patients without this translocation. To investigate whether blocking of the fusion protein AML1-ETO, generated by the t(8;21) translocation, would have an effect on VEGFA mRNA we have used a siRNA against AML-ETO in Kasumi-1. siRNA mediated depletion of AML1-ETO caused a 40% decrease in VEGFA mRNA expression. AML-ETO has the potential to interact with AML1/RUNX1 co-factors such as histone deacetylase (HDAC) which results in a dominant negative effect on AML1/RUNX1 transcriptional regulation. To test whether AML1/RUNX1 transcription repression of VEGFA is histone deacetylase (HDAC) dependent HL-60 cells were treated with 200 nM Trichostatin A (TSA), a potent inhibitor of HDAC, this resulted in an 75% increase of luciferase activity after 24h. In contrast, TSA treatment of Kasumi-1 cells containing the AML-ETO fusion protein had no effect on VEGFA expression. These results indicate that VEGFA is transcriptionally regulated by AML1/RUNX1, one of the most common targets of aberrations in AML, through three AML1/RUNX1 repressor elements located in the promoter of VEGFA. Furthermore, prognostically unfavorable high VEGFA expression in AML is caused by a dominant negative effect of the fusion protein AML-ETO on AML1/RUNX1 mediated repression of VEGFA transcription. These results underscore the importance of AML1/RUNX1 aberrations in AML development and progression and better understanding the molecular basis for aberrant AML/RUNX1 signaling pathway may help design more effective treatment strategies.

Promoter 2 -1025 T/C Polymorphism in the RUNX2 Gene Is Associated with Femoral Neck BMD in Spanish Postmenopausal Women

Stimulation of bone formation is a key therapeutic target in osteoporosis. Runx2 is a runt domain transcription factor essential to osteoblast differentiation, bone remodeling, and fracture healing. Runx2 knockout mice exhibit a complete lack of ossification, while overexpression of this gene in transgenic mice results in an osteoporotic phenotype. Thus, RUNX2 is a good candidate for the genetic determination of osteoporosis. In this association study, the effects of the -330 G/T polymorphism in promoter 1 and the -1025 T/C polymorphism (rs7771980) in promoter 2 of RUNX2 were tested in relation to lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) in a cohort of 821 Spanish postmenopausal women. The minor allele frequencies for the two polymorphisms were 0.15 and 0.07, respectively. The two polymorphisms, located more than 90 kb apart, were not in linkage disequilibrium (D' = 0.27, r (2) = 0.028). In an ANCOVA test adjusting by weight, height, age, and years since menopause, the -330 G/T polymorphism was not associated with any of the phenotypes analyzed, while we found the -1025 T/C polymorphism to be associated with FN BMD (p = 0.001). In particular, individuals carrying the TC genotype had higher mean adjusted FN BMD values than those bearing the TT genotype. Our results highlight the importance of this RUNX2 promoter 2 polymorphism in FN BMD determination.

The Transcription Factor Runx3 Represses the Neurotrophin Receptor TrkB during Lineage Commitment of Dorsal Root Ganglion Neurons

Runx3, a Runt domain transcription factor, determines neurotrophin receptor phenotype in dorsal root ganglion (DRG) neurons. Molecular mechanisms by which Runx3 controls distinct neurotrophin receptors are largely unknown. Here, we show that RUNX3 abolished mRNA induction of TRKB expression, and concomitantly altered the neurotrophin response in a differentiating neuroblastoma cell line. In contrast, RUNX3 did not play a significant role in TRKC regulation even under the relevant BMP signaling pathway. We identified putative regulatory elements of Ntrk2/NTRK2 (a gene that codes for TrkB) using an unbiased computational approach. One of these elements was a highly conserved intronic sequence that contains a cluster of Runx binding sites. In a primary culture of DRG neurons, endogenous Runx3 bound to the consensus cluster, which had repressor activity against the Ntrk2 promoter under the control of NT-3 signaling. Consistent with these findings, Runx3-deficient embryos showed an increased number of trkB+ DRG neurons and failed to maintain trkC expression. Taken together, Runx3 determines TrkC positive sensory neuron identities through the transcriptional repression of TrkB when Trk-BTrkC double positive neurons differentiate into TrkC single positive neurons.

RUN-CBFβ Interaction in C. elegans: Computational Prediction and Experimental Verification

The Runt domain proteins are eukaryotic transcription factors that regulate major developmental pathways. All members of this family contain a highly-conserved sequence-specific DNA binding domain: the Runt domain (RD). Structural and biochemical studies have shown that the Runt domain undergoes a conformational transition upon binding to DNA and that this process is regulated by an unrelated partner protein CBFβ that enhances the DNA binding affinity of RD. Most of the reported studies on the Runt domain transcription factors were performed on proteins from mammals and Drosophila whereas very little has been known about the C. elegans RD protein, RUN, which provides the simplest model system for understanding the function of this class of transcription factors. We performed computational studies on RD domains from various species including C. elegans, Drosophila, and human, using the atom-atom contact surface area scoring method. The scoring analysis indicates that the DNA binding regulation of the C. elegans RD protein (CeRD) occurs via its interaction with a CBFβ-like partner, as found for the human proteins, whereas a different mode of regulation may occur in the Drosophila system. Sequence, secondary structure and fold analyses of a putative CBFβ protein identified in the C. elegans genome, CeCBFβ, sharing a 22% identity with the human protein, predict a similar structure of this protein to that of the human CBFβ protein. We produced the C. elegans proteins CeRD and CeCBFβ in bacteria and confirmed their physical interaction as well as cross interactions with the corresponding human proteins. We also confirmed the structural similarity of CBFβ and CeCBFβ by circular dichroism analysis. The combined results suggest that a similar mechanism of regulation operates for the human and the C. elegans RD proteins despite the low sequence identity between their CBFβ proteins and the evolutionary distance between the two systems.

Identification of a Promoter Element within the Zebrafish colXα1 Gene Responsive to Runx2 Isoforms Osf2/Cbfa1 and til-1 but not to pebp2αA2

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Cyclin D1-Cdk4 Induce Runx2 Ubiquitination and Degradation

Runx2 is a Runt domain transcription factor involved in the activation of genes encoding osteoblast and chondrocyte-specific proteins. Runx2 activity is regulated by transcriptional and post-transcriptional mechanisms. The functional significance of the post-translational modification of Runx2 has not been fully defined. We show that cyclin D1-Cdk4 induce Runx2 degradation in an ubiquitination-proteasome-dependent manner. Mutagenesis of Runx2 serine-472, a consensus Cdk site, to alanine increases the half-life of Runx2 and causes loss of sensitivity to cyclin D1-induced Runx2 degradation. The targeted Runx2 degradation by cyclin D1 identifies a novel mechanism through which Runx2 activity is regulated coordinately with the cell cycle machinery in bone cells.

Groucho/transducin-like Enhancer-of-split (TLE)-dependent and -independent transcriptional regulation by Runx3

Regulation of gene expression by tissue-specific transcription factors involves both turning on and turning off transcription of target genes. Runx3, a runt-domain transcription factor, regulates cell-intrinsic functions by activating and repressing gene expression in sensory neurons, dendritic cells (DC), and T cells. To investigate the mechanism of Runx3-mediated repression in an in vivo context, we generated mice expressing a mutant Runx3 lacking the C-terminal VWRPY, a motif required for Runx3 interaction with the corepressor Groucho/transducin-like Enhancer-of-split (TLE). In contrast with Runx3(-/-) mice, which displayed ataxia due to the death of dorsal root ganglia TrkC neurons, Runx3(VWRPY-/-) mice were not ataxic and had intact dorsal root ganglia neurons, indicating that ability of Runx3 to tether Groucho/TLE is not essential for neurogenesis. In the DC compartment, the mutant protein Runx3(VWRPY-) promoted normally developed skin Langerhans cells but failed to restrain DC spontaneous maturation, indicating that this latter process involves Runx3-mediated repression through recruitment of Groucho/TLE. Moreover, in CD8(+) thymocytes, Runx3(VWRPY-) up-regulated alphaE/CD103-like WT Runx3, whereas unlike wild type, it failed to repress alphaE/CD103 in CD8(+) splenocytes. Thus, in CD8-lineage T cells, Runx3 regulates alphaE/CD103 in opposing regulatory modes and recruits Groucho/TLE to facilitate the transition from activation to repression. Runx3(VWRPY-) also failed to mediate the epigenetic silencing of CD4 gene in CD8(+) T cells, but normally regulated other pan-CD8(+) T cell genes. These data provide evidence for the requirement of Groucho/TLE for Runx3-mediated epigenetic silencing of CD4 and pertain to the mechanism through which other Runx3-regulated genes are epigenetically silenced.

Runx1 Determines Nociceptive Sensory Neuron Phenotype and Is Required for Thermal and Neuropathic Pain

In mammals, the perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptive sensory neurons. The molecular mechanisms responsible for the specification of distinct sensory modality are, however, largely unknown. We show here that Runx1, a Runt domain transcription factor, is expressed in most nociceptors during embryonic development but in adult mice, becomes restricted to nociceptors marked by expression of the neurotrophin receptor Ret. In these neurons, Runx1 regulates the expression of many ion channels and receptors, including TRP class thermal receptors, Na+-gated, ATP-gated, and H+-gated channels, the opioid receptor MOR, and Mrgpr class G protein coupled receptors. Runx1 also controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. Thus, Runx1 coordinates the phenotype of a large cohort of nociceptors, a finding with implications for pain therapy.

Effects of osteogenic inducers on cultures of canine mesenchymal stem cells

To examine age-related efficacy of bone morphogenetic protein (BMP)-2, ascorbate, and dexamethasone as osteogenic inducers in canine marrow-derived stromal cells (MSCs). Samples of femoral bone marrow obtained from 15 skeletally immature (< 1 year old) and 4 skeletally mature (> 1.5 years old) dogs. First-passage canine MSC cultures were treated with 100 microg of ascorbate phosphate/mL, 10(-7)M dexamethasone, 100 ng of BMP-2/mL, or a combination of these osteoinducers. On day 6, cultures were harvested for quantitation of alkaline phosphatase (ALP) activity and isolation of RNA to prepare cDNA for real-time polymerase chain reaction analyses of osteoblast markers. Early markers of osteogenesis were induced in canine MSCs by BMP-2 but not dexamethasone. In young dogs, the combination of BMP-2 and ascorbate yielded the highest ALP mRNA concentrations and activity. This combination also induced significant increases in mRNA for osteopontin and runt-domain transcription factor 2. In comparison to MSCs from immature dogs, those from mature dogs had diminished ALP activity in response to BMP and ascorbate. Results for cultures treated with 3,4-dehydroproline suggested that ascorbate-induced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs. BMP-2 was capable of inducing markers of osteogenesis in short-term cultures of canine MSCs. In MSCs obtained from skeletally immature dogs, ascorbate was required for maximal effects of BMP These results define optimal conditions for stem cell osteogenesis in dogs and will facilitate development of stem cell-based treatments for dogs with fractures.

Involvement of Runx1 in the down-regulation of fetal liver kinase-1 expression during transition of endothelial cells to hematopoietic cells

AbstractDuring early mouse embryogenesis, fetal liver kinase-1 (Flk-1), a receptor for vascular endothelial growth factor, and Runx1, a runt domain transcription factor, have prerequisite roles in the generation of hematopoietic lineages. Flk-1 expression is maintained in successive stages from mesodermal to endothelial cells and is down-regulated in nascent hematopoietic cells, whereas Runx1 (Runt-related transcription factor 1) is expressed in embryonic sites of hematopoietic cell de novo generation and in practically all hematopoietic organs. Here we show that Runx1 represses Flk-1 during the development of hemogenic endothelial cells into hematopoietic cells. We established embryonic stem cell clones carrying the Venus gene, a modified version of yellow fluorescence protein, in the Runx1 locus and cultured them on OP9 cells. Flk-1+ cells appeared on day 3.5, and Runx1+ cells first appeared from the Flk-1+ fraction on day 4.5. The Flk-1+Runx1+ cells rapidly stopped expressing Flk-1 with further incubation and eventually gave rise to CD45+ or TER119+ cells. Runx1 repressed Flk-1 promoter transcriptional activity in an endothelial cell line, and this repression required intact DNA-binding and transactivating domains of Runx1 protein. The repressor activity of Runx1 endogenous Flk-1 was also confirmed overexpressing Runx1 in embryonic stem cell differentiation cultures. These results provide novel insight into the role Runx1 during the development of hematopoietic cell lineages.

Shared and distinct roles mediated through C-terminal subdomains of acute myeloid leukemia/Runt-related transcription factor molecules in murine development

AbstractAML1/Runx1 is a frequent target of human leukemia–associated gene aberration and encodes a transcription factor with nonredundant biologic functions in initial development of definitive hematopoiesis, T-cell development, and steady-state platelet production. AML1/Runx1 and 2 closely related family genes, AML2/Runx3 and AML3/Runx2/Cbfa1, present in mammals, comprise the Runt-domain transcription factor family. Although they have similar structural and biochemical properties, gene-targeting experiments have identified distinct biologic roles. To directly determine the presence of functional overlap among runt-related transcription factor (Runx) family molecules, we replaced the C-terminal portion of acute myeloid leukemia 1 (AML1) with that derived from its family members, which are variable in contrast to conserved Runt domain, using the gene knock-in method. We found that C-terminal portions of either AML2 or AML3 could functionally replace that of AML1 for myeloid development in culture and within the entire mouse. However, while AML2 substituted for AML1 could effectively rescue lymphoid lineages, AML3 could not, resulting in a smaller thymus and lymphoid deficiency in peripheral blood. Substitution by the C-terminal portion of AML3 also led to high infantile mortality and growth retardation, suggesting that AML1 has as yet unidentified effects on these phenotypes. Thus, the C-terminal portions of Runx family members have both similar and distinct biologic functions.

Structures of the DNA-binding site of Runt-domain transcription regulators

Runt-domain (RD) proteins are transcription factors that play fundamental roles in various developmental pathways. They bind specifically to DNA sequences of the general form PyGPyGGTPy (Py = pyrimidine), through which they regulate transcription of target genes. The DNA duplex TCTGCGGTC/TGACCGCAG, incorporating the binding site for the RD transcription factors (bold), was crystallized in space group P4(3). X-ray analysis of two crystals diffracting to 1.7 and 2.0 angstroms resolution, which had slight variations in their unit-cell parameters, revealed two distinct conformations of the A-DNA helix. The two crystal structures possessed several structure and hydration features that had previously been observed in A-DNA duplexes. A comparative analysis of the present A-DNA structures and those of previously reported B-DNA crystal structures of RD-binding sites in free and protein-bound states showed the various duplexes to display several common features. Within this series, the present A-DNA duplexes adopt two conformations along the pathway from the canonical A-DNA to the B-DNA forms and the protein-bound helices display conformational features that are intermediate between those of the current A-DNA structures and that of the B-DNA-type helix of the free RD target. Based on these data and energy considerations, it is likely that the propensity of the RD-binding site to adopt the A-DNA or B-DNA conformation in solution depends on the sequence context and environmental conditions, and that the transition from either DNA form to the protein-bound conformation involves a small energy barrier.

Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells

We review here the development of the hematopoietic system and its relationship to the endothelium, with a special focus on the characterisation of the hemangioblast, the putative ancestor for endothelial cells and hematopoietic cells. Using the avian model, we have traced in vivo the progeny of embryonic endothelial cells and shown that aortic-born hematopoietic cells (known to generate the definitive hematopoietic lineage) derive from endothelial cells in the floor of the aorta. During this process, endothelial cells undergo a switch from endothelial cells to hematopoietic cells characterised by a downgrading of endothelial cell-specific genes and the parallel upgrading of hematopoietic cell-specific genes. Using a similar approach, we have shown that generation of hematopoietic cells from endothelial cells also takes place during mouse embryonic development. We have thoroughly characterised the dynamics of key molecules (several of which we have cloned) specifically expressed by the yolk sac or aortic hemangioblast. The yolk sac hemangioblast is characterized by the specific expression of SCL/Tal-1 and Lmo2, whereas the aortic hemangioblast expresses Runx-1 (a runt domain transcription factor). Finally, we have demonstrated the existence of a new site for hematopoiesis, namely the allantois. Using quail/chick grafts, we show that this embryonic appendage autonomously produces endothelial cells and hematopoietic cells, these latter being endowed with the attributes of the definitive hematopoietic lineage.

Control of RUNX2 isoform expression: The role of promoters and enhancers

The three mammalian RUNX genes constitute the family of runt domain transcription factors that are involved in the regulation of a number of developmental processes such as haematopoiesis, osteogenesis and neuronal differentiation. All three genes show a complex temporo-spatial pattern of expression. Since the three proteins are probably mutually interchangeable with regard to function, most of the specificity of each family member seems to be based on a tightly controlled regulation of expression. While RUNX gene expression is driven by two promoters for each gene, the promoter sequence alone does not seem to suffice for a proper expressional control. This review focuses on the available evidence for the existence of such control mechanisms and studies aiming at discovering cis-acting regulatory sequences of the RUNX2 gene.

O-3 Risk factors for myelodysplastic syndrome and leukemia after breast cancer: Results of a French multicenter case-control study

Alterations of the ets family transcription factor ETV6 (TEL) and the RUNT domain transcription factor RUNX1 (AML1) play pivotal roles in the leukemogenesis of various types of leukemia. While only three fusion partners of RUNX1 namely ETO, ETV6 and MTG16 have been described so far, there is a plethora of ETV6 fusion partners with about 20 partners described so far. Apart from forming fusion genes there are other genetic alterations of ETV6 including deletions, point mutations and possible alterations at the promoter level that might contribute to the malignant phenotype. This review will focus on ETV6 and on the different mechanisms that are used by this gene to cause leukemia.