Runx1 mediates the development of the granular convoluted tubules in the submandibular glands.
ABSTRACT: The mouse granular convoluted tubules (GCTs), which are only located in the submandibular gland (SMG) are known to develop and maintain their structure in an androgen-dependent manner. We previously demonstrated that the GCTs are involuted by the epithelial deletion of core binding factor ? (CBF?), a transcription factor that physically interacts with any of the Runt-related transcription factor (RUNX) proteins (RUNX1, 2 and 3). This result clearly demonstrates that the Runx /Cbfb signaling pathway is indispensable in the development of the GCTs. However, it is not clear which of the RUNX proteins plays useful role in the development of the GCTs by activating the Runx /Cbfb signaling pathway. Past studies have revealed that the Runx /Cbfb signaling pathway plays important roles in various aspects of development and homeostatic events. Moreover, the Runx genes have different temporospatial requirements depending on the biological situation. In the present study, the GCTs of the SMG showed a remarkable phenotype of, which phenocopied the epithelial deletion of Cbfb, in epithelial-specific Runx1 conditional knock-out (cKO) mice. The results indicate that Runx1 works as a partner of Cbfb during the development of the GCTs. We also discovered that the depletion of Runx1 resulted in the reduced secretion of saliva in male mice. Consistent with this finding, one of the water channels, Aquaporin-5 (AQP5) was mislocalized in the cytoplasm of the Runx1 mutants, suggesting a novel role of Runx1 in the membrane trafficking of AQP5. In summary, the present findings demonstrated that RUNX1 is essential for the development of the GCTs. Furthermore, RUNX1 could also be involved in the membrane trafficking of the AQP5 protein of the acinar cells in the SMG in order to allow for the proper secretion of saliva.
Project description:Core binding factor ? (Cbfb) is a cofactor of the Runx family of transcription factors. Among these transcription factors, Runx1 is a prerequisite for anterior-specific palatal fusion. It was previously unclear, however, whether Cbfb served as a modulator or as an obligatory factor in the Runx signaling process that regulates palatogenesis. Here, we report that Cbfb is essential and indispensable in mouse anterior palatogenesis. Palatal fusion in <i>Cbfb</i> mutants is disrupted owing to failed disintegration of the fusing epithelium specifically at the anterior portion, as observed in <i>Runx1</i> mutants. In these mutants, expression of TGFB3 is disrupted in the area of failed palatal fusion, in which phosphorylation of Stat3 is also affected. TGFB3 protein has been shown to rescue palatal fusion <i>in vitro</i> TGFB3 also activated Stat3 phosphorylation. Strikingly, the anterior cleft palate in <i>Cbfb</i> mutants is further rescued by pharmaceutical application of folic acid, which activates suppressed Stat3 phosphorylation and <i>Tgfb3</i> expression <i>in vitro</i> With these findings, we provide the first evidence that Cbfb is a prerequisite for anterior palatogenesis and acts as an obligatory cofactor in the Runx1/Cbfb-Stat3-Tgfb3 signaling axis. Furthermore, the rescue of the mutant cleft palate using folic acid might highlight potential therapeutic targets aimed at Stat3 modification for the prevention and pharmaceutical intervention of cleft palate.
Project description:Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor ? (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.
Project description:Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.
Project description:The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFb was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFb is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFb holoenzyme forms a wellde?ned heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFb. Heterodimers of CBFb and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFb is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways. Analysis of Vif-dependent effects on gene expression in Jurkats when cells are activated for 4 or 6 hours with PMA and PHA
Project description:Runx1 and Cbf? are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbf? is essential for the development of Flt3(+) macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105(+) erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbf? restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia.
Project description:Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration.Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration.We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation.Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.
Project description:Natural killer (NK) cells are innate lymphocytes that have features of adaptive immunity such as clonal expansion and generation of long-lived memory. Interleukin-12 (IL-12) signaling through its downstream transcription factor signal transducer and activator of transcription 4 (STAT4) is required for the generation of memory NK cells after expansion. We identify gene loci that are highly enriched for STAT4 binding using chromatin immunoprecipitation sequencing for STAT4 and the permissive histone mark H3K4me3 in activated NK cells. We found that promoter regions of Runx1 and Runx3 are targets of STAT4 and that STAT4 binding during NK cell activation induces epigenetic modifications of Runx gene loci resulting in increased expression. Furthermore, specific ablation of Runx1, Runx3, or their binding partner Cbfb in NK cells resulted in defective clonal expansion and memory formation during viral infection, with evidence for Runx1-mediated control of a cell cycle program. Thus, our study reveals a mechanism whereby STAT4-mediated epigenetic control of individual Runx transcription factors promotes the adaptive behavior of antiviral NK cells.
Project description:CBF?-SMMHC (core-binding factor ?-smooth muscle myosin heavy chain), the fusion protein generated by the chromosome 16 inversion fusion gene, CBFB-MYH11, is known to initiate leukemogenesis. However, the mechanism through which CBF?-SMMHC contributes to leukemia development is not well understood. Previously, it was proposed that CBF?-SMMHC acts by dominantly repressing the transcription factor RUNX1 (Runt-related protein 1), but we recently showed that CBF?-SMMHC has activities that are independent of RUNX1 repression. In addition, we showed that a modified CBF?-SMMHC with decreased RUNX1-binding activity accelerates leukemogenesis. These results raise questions about the importance of RUNX1 in leukemogenesis by CBF?-SMMHC. To test this, we generated mice expressing Cbfb-MYH11 in a Runx1-deficient background, resulting from either homozygous Runx1-null alleles (Runx1(-/-)) or a single dominant-negative Runx1 allele (Runx1(+/lz)). We found that loss of Runx1 activity rescued the differentiation defects induced by Cbfb-MYH11 during primitive hematopoiesis. During definitive hematopoiesis, RUNX1 loss also significantly reduced the proliferation and differentiation defects induced by Cbfb-MYH11. Importantly, Cbfb-MYH11-induced leukemia had much longer latency in Runx1(+/lz) mice than in Runx1-sufficient mice. These data indicate that Runx1 activity is critical for Cbfb-MYH11-induced hematopoietic defects and leukemogenesis.
Project description:Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-? (CBFB) play pivotal roles in leukemogenesis, and inhibition of RUNX1 has now been widely recognized as a novel strategy for anti-leukemic therapies, it has been elusive how leukemic cells could acquire the serious resistance against RUNX1-inhibition therapies and also whether CBFB could participate in this process. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion, and their mutual interaction causes the physiological resistance against chemotherapy for acute myeloid leukemia (AML) cells. Mechanistically, p53 induced by RUNX1 gene silencing directly binds to CBFB promoter and stimulates its transcription as well as its translation, which in turn acts as a platform for the stabilization of RUNX1, thereby creating a compensative RUNX1-p53-CBFB feedback loop. Indeed, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis, and these CBFB expressions were positively correlated to those of p53. Our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted at any sides of this triangle in strategizing anti-leukemia therapies.
Project description:Gene expression profiles of Cbfb-deficient and control Treg cells were compared. Abstract: Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here we showed that Treg cell-specific deficiency of Cbfβ, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyper-production of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfβ heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3. Experiment Overall Design: CD4+CD25hi cells, most of which were Foxp3+ Treg cells, were isolated from Cbfb-flox/flox: Foxp3-ires-Cre (n = 3) and control Cbfb-flox/wt: Foxp3-ires-Cre (n = 3) mice. Total RNA was extracted from those purified Cbfb-deficient or control Treg cells.