RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation.

Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation, and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice.

Although the function of Runt-related (RUNX) transcription factors has been well characterized in leukemogenesis and regarded as an ideal target in antileukemia strategies, the effect of RUNX-inhibition therapy on bone marrow niche cells andr its impact on the engraftment of acute myeloid leukemia (AML) cells have largely been unknown. Here, we provide evidence suggesting the possible involvement of RUNX transcription factors in the transactivation of E-selectin, a member of selectin family of cell adhesion molecules, on the vascular endothelial cells of the mice bone marrow niche. In our experiments, gene switch-mediated silencing of RUNX downregulated E-selectin expression in the vascular niche and negatively controlled the engraftment of AML cells in the bone marrow, extending the overall survival of leukemic mice. Our work identified the novel role of RUNX family genes in the vascular niche and showed that the vascular niche, a home for AML cells, could be strategically targeted with RUNX-silencing antileukemia therapies. Considering the excellent efficacy of RUNX-inhibition therapy on AML cells themselves as we have previously reported, this strategy potentially targets AML cells both directly and indirectly, thus providing a better chance of cure for poor-prognostic AML patients.

La denervación 5-HTérgica córtico-frontal induce cambios en la expresión de las subunidades alfa 4 y alfa 7 de los receptores de acetilcolina tipo nicotínico en la corteza prefrontal de la rata adulta / 5-HT denervation of the adult rat prefrontal cortex induces changes in the expression of ¦Á4 and ¦Á7 nicotinic acetylcholine receptor subtypes

Introducción: Los receptores de la acetilcolina de tipo nicotínico (R-Ach-n) son expresados ampliamente en diferentes regiones del cerebro. Particularmente, la conformación de los subtipos α4β2 y la α7 ha sido involucrada con la organización de diferentes tipos de memoria. Además, debido a su localización, estos pueden controlar la liberación de diferentes tipos de neurotransmisores, así como su participación en la plasticidad sináptica. Métodos: Se conformaron 3 grupos de trabajo, un grupo experimental (E), un grupo control (C) y un grupo testigo (T). Al grupo E se le realizó la lesión farmacológica por vía estereotáxica en la región anteroventral del núcleo del rafe dorsal (NRD) con 1μ/μl de 5,7-dihidroxitriptamina. Al grupo C, se le sometió a cirugía y se le aplicó la solución vehículo y finalmente el grupo T no recibió ningún tratamiento; 20 días después de la cirugía, los animales de los 3 grupos fueron sacrificados por decapitación para el análisis de la expresión de las subunidades, α4 y α7 de los R-Ach-n mediante la técnica de biología molecular. Resultados: La denervación 5-HTérgica a la CPF de la rata modifica la expresión de los receptores α4 y α7 de manera diferencial. La expresión de las subunidades α4 se incrementa, mientras que las subunidades α7 disminuyen. Conclusión: Las diferencias de expresión que tuvieron las 2 subunidades podrían deberse a la localización que presentan. La subunidad α4 se localiza en sitios post sinápticos y podría estar relacionada con cambios post sinápticos adaptativos, en tanto que la de la α7 se localiza en sitios presinápticos, por lo que la lesión y eliminación de fibras 5-HTérgicas en la CPF provoca su disminución (AU)

Introduction: Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout several brain regions. Formation of the α4β2 and α7 subtypes in particular is involved in the organisation of different types of memory. Furthermore, due to their location, these receptors can control the release of various types of neurotransmitters and contribute to synaptic plasticity. Methods: Rats were divided into three groups, an experimental group (E), a sham-operated group, (S) and an intact group (T). In group E, stereotactic guidance was used to induce a chemical lesion with 1 μ/μL of 5,7-dihydroxytryptamine (5,7-DHT) in the anteroventral part of the dorsal raphe nucleus (DRN). In the sham-operated group (S), animals underwent surgery including delivery of the same excipient solution to the same site. The intact group (T) received no treatment whatsoever. Twenty days after surgery, animals in all groups were euthanised by decapitation to evaluate the expression of α4 and α7 nAChRs by means of molecular biology techniques. Results: 5-HT denervation of the rat PFC differentially modified the expression of α4 and α7 receptors: while α4 receptor expression increased, α7 expression decreased. Conclusion: Expression differences observed between the two subtypes may be due to their separate locations. The α4 subtype is found in postsynaptic locations and may be related to adaptive changes in postsynaptic cells, while the location of α7 is presynaptic. This explains why the lesion and the elimination of 5-HT fibres in the CPF would cause a decrease in α7 expression (AU)

RUNX family: Regulation and diversification of roles through interacting proteins.

The Runt-related transcription factors (RUNX) belong to an ancient family of metazoan genes involved in developmental processes. Through multiple protein-interacting partners, RUNX proteins have been implicated in diverse signaling pathways and cellular processes. The frequent inactivation of RUNX genes in cancer indicates crucial roles for RUNX in tumor suppression. This review discusses the abilities of RUNX proteins, in particular RUNX3, to integrate oncogenic signals or environmental cues and to initiate appropriate tumor suppressive responses.

Signal transduction pathways and transcriptional regulation in Th17 cell differentiation.

Over the last decade, our understanding of helper/effector T cell differentiation has changed dramatically. The discovery of interleukin (IL-)17-producing T cells (Th17) and other subsets has changed our view of T cell-mediated immunity. Characterization of the signaling pathways involved in the Th17 commitment has provided exciting new insights into the differentiation of CD4+ T cells. Importantly, the emerging data on conversion among polarized T helper cells have raised the question how we should view such concepts as T cell lineage commitment, terminal differentiation and plasticity. In this review, we will discuss the current understanding of the signaling pathways, molecular interactions, and transcriptional and epigenetic events that contribute to Th17 differentiation and acquisition of effector functions.

Runx family genes, niche, and stem cell quiescence.

In multicellular organisms, terminally differentiated cells of most tissues are short-lived and therefore require constant replenishment from rapidly dividing stem cells for homeostasis and tissue repair. For the stem cells to last throughout the lifetime of the organism, however, a small subset of stem cells, which are maintained in a hibernation-like state known as stem cell quiescence, is required. Such dormant stem cells reside in the niche and are activated into proliferation only when necessary. A multitude of factors are required for the maintenance of stem cell quiescence and niche. In particular, the Runx family genes have been implicated in stem cell quiescence in various organisms and tissues. In this review, we discuss the maintenance of stem cell quiescence in various tissues, mainly in the context of the Runx family genes, and with special focus on the hematopoietic system.

DNA specificity determinants associate with distinct transcription factor functions.

To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited, because a single factor can use degenerate sequence motifs and because related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation; thus, unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, a genome-wide approach, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and also improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T cell-specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of DNA binding motifs may enable variable transcription factor function at different genomic sites.

Runx transcription factors: lineage-specific regulators of neuronal precursor cell proliferation and post-mitotic neuron subtype development.

Runt-related (RUNX) genes encode evolutionarily conserved transcription factors that play essential roles during development and adult tissue homeostasis. RUNX proteins regulate the transition from proliferation to differentiation in a variety of cell lineages. Moreover, they control the diversification of distinct cellular phenotypes in numerous tissues. Alterations of RUNX functions are associated with several cancers and other human pathologies, underscoring the vital roles of these transcription factors in adult organs. Insights into the functions and regulations of mammalian RUNX proteins have been provided mostly by studies of RUNX involvement in mechanisms of hematopoietic and skeletal development and disease. A growing number of recent investigations are revealing new functions for RUNX family members during the development of the mammalian nervous system. This review will discuss recent progress in the study of RUNX protein involvement in mammalian neural development, with emphasis on the differentiation of olfactory, sensory, and motor neuron lineages.

Runx and ThPOK: a balancing act to regulate thymocyte lineage commitment.

CD4-positive helper T cells and CD8-positive cytotoxic T cells comprise the majority of T lymphocytes present in secondary lymphoid organs and are essential for acquired immunity. These two populations are derived from common precursors in the thymus and selected through interaction between their clonal T-cell receptors and major histocompatibility complex molecules. Although intensely studied as a model system for binary cell fate decisions, the mechanisms underlying the helper versus cytotoxic lineage choice in the thymus has been elusive. In the past few years, it has been demonstrated that the Runx family of transcription factors, particularly Runx3, is essential for the generation of cytotoxic lineage T cells, whereas the ThPOK zinc finger transcription factor that plays a crucial role in the differentiation of the helper lineage. Recent works have implied that a cross-regulation between Runx and ThPOK contributes to appropriate thymocyte lineage commitment. In this article, recent findings on the transcription factor networks governing thymocyte lineage decisions are discussed, focusing on the two factors, and provide insights into mechanisms of lineage-specific gene regulation in the process of T-cell commitments.

Is Runx a linchpin for developmental signaling in metazoans?

The Runt domain (Runx) is a 128 amino acid sequence motif that defines a metazoan family of sequence-specific DNA binding proteins, which appears to have originated in concert with the intercellular signaling systems that coordinate multicellular development in animals. In the model organisms where they have been studied (fruit fly, mouse, sea urchin, and nematode) Runx genes are essential for normal development, and in humans they are causally associated with a variety of cancers, manifesting both oncogenic and tumor suppressive attributes. During development Runx proteins support both cell proliferation and differentiation, and function in both transcriptional activation and repression. Runx function is thus context-dependent, with the context provided genetically by cis-regulatory sequence architecture and epigenetically by development. This context dependency makes it difficult to formulate reductionistic generalizations concerning Runx function in normal and carcinogenic development. However, a growing body of literature links Runx function to each of the major intercellular signaling systems in animals, suggesting that the general function of Runx transcription factors may be to potentiate and govern genomic responsiveness to developmental signaling.

Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

BACKGROUND: The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Runx transcription factors in neuronal development.

Runt-related (Runx) transcription factors control diverse aspects of embryonic development and are responsible for the pathogenesis of many human diseases. In recent years, the functions of this transcription factor family in the nervous system have just begun to be understood. In dorsal root ganglion neurons, Runx1 and Runx3 play pivotal roles in the development of nociceptive and proprioceptive sensory neurons, respectively. Runx appears to control the transcriptional regulation of neurotrophin receptors, numerous ion channels and neuropeptides. As a consequence, Runx contributes to diverse aspects of the sensory system in higher vertebrates. In this review, we summarize recent progress in determining the role of Runx in neuronal development.

Worming out the biology of Runx.

Runx family transcription factors have risen to prominence over the last few years because of the increasing evidence implicating them as key regulators of the choice between cell proliferation and differentiation during development and carcinogenesis. Runx factors have been found to be involved in diverse developmental processes, ranging from hematopoiesis to neurogenesis, and are increasingly being linked with various human cancers. In this review, we examine the case for Runx factors as key regulators of cell proliferation in various developmental situations, a role that predisposes Runx mutations as causative agents in oncogenesis. We discuss the evidence that Runx factors regulate, and are regulated by, core components of the cell cycle machinery, and focus our attention on the solo Runx gene, rnt-1, in Caenorhabditis elegans, an organism that we feel has much to offer the Runx field.

[Study of cbfalpha1 on the expression of extracellular matrix proteins in dental papilla cells induced by BMP-2 in vitro].

PURPOSE: To evaluate the function of cbfalpha1 on BMP-2 signaling to extracellular matrix proteins in dental papilla cells in vitro. METHODS: RT-PCR and Western blot were performed to detect the expression of ALP, OC, ON, OPN, BSP, DMP-1 and DSPP in cultured dental papilla cells induced by 200ng/mL BMP-2 and/or down-regulated by cbfalpha1 antisense technology, the results were analysed with SPSS 11.0 software package. RESULTS: We found that the amount of ALP and OC and the expression of OPN, BSP and ON were upregulated significantly after the cells were treated with BMP-2. After transfected with antisense cbfalpha1, the cells downregulated the expression of ALP, OC, OPN and BSP significantly(P<0.01). CONCLUSIONS: As a kind of transcription factor, cbfalpha1 could be an important tache in the BMP-2 signal networks controlling cells differentiation and mineralization.

Nuclear microenvironments in biological control and cancer.

Nucleic acids and regulatory proteins are compartmentalized in microenvironments within the nucleus. This subnuclear organization may support convergence and the integration of physiological signals for the combinatorial control of gene expression, DNA replication and repair. Nuclear organization is modified in many cancers. There are cancer-related changes in the composition, organization and assembly of regulatory complexes at intranuclear sites. Mechanistic insights into the temporal and spatial organization of machinery for gene expression within the nucleus, which is compromised in tumours, provide a novel platform for diagnosis and therapy.

A GATA/RUNX cis-regulatory module couples Drosophila blood cell commitment and differentiation into crystal cells.

Members of the RUNX and GATA transcription factor families play critical roles during hematopoiesis from Drosophila to mammals. In Drosophila, the formation of the crystal cell hematopoietic lineage depends on the continuous expression of the lineage-specific RUNX factor Lozenge (Lz) and on its interaction with the GATA factor Serpent (Srp). Crystal cells are the main source of prophenoloxidases (proPOs), the enzymes required for melanization. By analyzing the promoter regions of several insect proPOs, we identify a conserved GATA/RUNX cis-regulatory module that ensures the crystal cell-specific expression of the three Drosophila melanogaster proPO. We demonstrate that activation of this module requires the direct binding of both Srp and Lz. Interestingly, a similar GATA/RUNX signature is over-represented in crystal cell differentiation markers, allowing us to identify new Srp/Lz target genes by genome-wide screening of Drosophila promoter regions. Finally, we show that the expression of lz in the crystal cells also relies on Srp/Lz-mediated activation via a similar module, indicating that crystal cell fate choice maintenance and activation of the differentiation program are coupled. Based on our observations, we propose that this GATA/RUNX cis-regulatory module may be reiteratively used during hematopoietic development through evolution.

T-lymphoid, megakaryocyte, and granulocyte development are sensitive to decreases in CBFbeta dosage.

The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non-DNA-binding partner CBFbeta. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBFbeta levels by 3- or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBFbeta levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBFbeta levels, and were virtually absent when CBFbeta levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4- CD8- double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBFbeta threshold for normal T-cell development, and situate an essential role for core-binding factors during the earliest stages of T-cell development.

Craniosynostosis-associated gene nell-1 is regulated by runx2.

UNLABELLED: We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. INTRODUCTION: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5' flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. MATERIALS AND METHODS: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2(-/-) calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. RESULTS: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. CONCLUSIONS: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis.

[Interactions of chondrocytes and osteoblasts during endochondral bone formation].

During endochondral bone formation, mesenchymal condensations, chondrocyte differentiation and proliferation, termination of proliferation, hypertrophic differentiation, and replacement by bone occur sequentially. This sequence is spatially represented by the structure of the growth plate of the embryo, and reflects the evolution of bone. Endochondral bone formation is mainly regulated by the interactions of chondrocytes and osteoblasts; a variety of signals are implicated in this regulation. The roles of factors regulating chondrocyte proliferation and hypertrophy including the Sox trio, PTH related peptide (PTHrP), Indian hedgehog (Ihh), the runt-related transcription factor (Runx) family, fibroblast growth factor receptor 3 (FGFR3) and bone morphogenetic protein (BMP) and other factors including hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) will be discussed.