RESUMEN
BACKGROUND: Growing evidence has implicated core-binding factor beta (Cbfb) as a contributor to osteoblast differentiation, which plays a key role in fracture healing. Herein, we aimed to assess whether Cbfb affects osteoblast differentiation after fibula fracture. METHODS: Initially, we established a Cbfb conditional knockout mouse model for subsequent studies. Immunohistochemical staining was conducted to detect the expression of proliferating cell nuclear antigen (PCNA) and collagen II in the fracture end. Next, we isolated and cultured osteoblasts from specific Cbfb conditional knockout mice for BrdU analysis, alkaline phosphatase (ALP) staining, and von Kossa staining to detect osteoblast viability, differentiation, and mineralization, respectively. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression of osteoblast differentiation-related genes. RESULTS: The Cbfb conditional knockout mice exhibited downregulated expression of PCNA and collagen II, reduced ALP activity, and mineralization, as well as diminished expression of osteoblast differentiation-related genes. Further, Cbfb knockout exerted no obvious effects on osteoblast proliferation. CONCLUSIONS: Overall, these results substantiated that Cbfb could promote fibula fracture healing and osteoblast differentiation and thus provided a promising therapeutic target for clinical treatment of fibula fracture.
Asunto(s)
Diferenciación Celular/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Peroné/lesiones , Curación de Fractura/fisiología , Osteoblastos/metabolismo , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , RatonesRESUMEN
The core binding factor composed of CBFß and RUNX subunits plays a critical role in most hematopoietic lineages and is deregulated in acute myeloid leukemia (AML). The fusion oncogene CBFß-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells and induces AML. This review focuses on novel insights regarding the molecular mechanisms involved in CBFß-SMMHC-driven leukemogenesis and recent advances in therapeutic approaches to target CBFß-SMMHC in inv(16) AML.
Asunto(s)
Transformación Celular Neoplásica/genética , Inversión Cromosómica , Cromosomas Humanos Par 16/genética , Subunidad beta del Factor de Unión al Sitio Principal/genética , Inmunoterapia/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Terapia Molecular Dirigida , Cadenas Pesadas de Miosina/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cromosomas Humanos Par 16/ultraestructura , Terapia Combinada , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Predicción , Gemtuzumab/uso terapéutico , Regulación Leucémica de la Expresión Génica , Técnicas de Sustitución del Gen , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is often characterized by the presence of specific and recurrent chromosomal abnormalities. Current treatments have greatly increased remission rate, but relapse still occurs. Therefore, novel therapeutic approaches are required. Previously, using a conditional Cbfb-MYH11 knockin mouse model, we showed that Cbfb-MYH11 induces the expression of a cytokine receptor, IL1RL1. Treatment with IL-33, the only known ligand of IL1RL1, promotes leukemia cell survival in vitro. We further found that IL1RL1+ cells survive better with chemotherapy than IL1RL1- population. However, the mechanism is not clear. Here, we show that IL-33 treatment decreased drug sensitivity in the human inv(16) AML cell line ME-1. By RT-PCR, we found that IL-33 increased the expression of IL-4 and IL-6 and led to the activation of both p38 MAPK and NF-κB. We also showed that IL-33 decreased apoptosis with increased phosphorylation of p38 MAPK. Moreover, pre-treatment with MAPK inhibitor attenuated the phosphorylation of p38 enhanced by IL-33 and reversed the anti-apoptotic effect by IL-33. Taken together, our findings give news insights into the potential mechanism of the anti-apoptotic effect by IL-33/IL1RL1 axis in AML which will help in future drug development.
Asunto(s)
Apoptosis , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Interleucina-33/farmacología , Leucemia Mieloide Aguda/patología , Cadenas Pesadas de Miosina/fisiología , FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Regulación Leucémica de la Expresión Génica , Proteína 1 Similar al Receptor de Interleucina-1/genética , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/genética , Fosforilación , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Fertilidad , Células de la Granulosa/metabolismo , Infertilidad Femenina/fisiopatología , Luteinización , Ovulación , Animales , Femenino , Células de la Granulosa/citología , Ratones , Ratones Noqueados , ReproducciónRESUMEN
Epithelial to mesenchymal transition (EMT) is a dynamic process that drives cancer cell plasticity and is thought to play a major role in metastasis. Here we show, using MDA-MB-231 cells as a model, that the plasticity of at least some metastatic breast cancer cells is dependent on the transcriptional co-regulator CBFß. We demonstrate that CBFß is essential to maintain the mesenchymal phenotype of triple-negative breast cancer cells and that CBFß-depleted cells undergo a mesenchymal to epithelial transition (MET) and re-organise into acini-like structures, reminiscent of those formed by epithelial breast cells. We subsequently show, using an inducible CBFß system, that the MET can be reversed, thus demonstrating the plasticity of CBFß-mediated EMT. Moreover, the MET can be reversed by expression of the EMT transcription factor Slug whose expression is dependent on CBFß. Finally, we demonstrate that loss of CBFß inhibits the ability of metastatic breast cancer cells to invade bone cell cultures and suppresses their ability to form bone metastases in vivo. Together our findings demonstrate that CBFß can determine the plasticity of the metastatic cancer cell phenotype, suggesting that its regulation in different micro-environments may play a key role in the establishment of metastatic tumours.
Asunto(s)
Neoplasias de la Mama/patología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Metástasis de la Neoplasia , Animales , Factor de Unión a CCAAT , Línea Celular Tumoral , Femenino , Humanos , Ratones , FenotipoRESUMEN
The transcription factor RUNX1 is required in the embryo for formation of the adult hematopoietic system. Here, we describe the seminal findings that led to the discovery of RUNX1 and of its critical role in blood cell formation in the embryo from hemogenic endothelium (HE). We also present RNA-sequencing data demonstrating that HE cells in different anatomic sites, which produce hematopoietic progenitors with dissimilar differentiation potentials, are molecularly distinct. Hemogenic and non-HE cells in the yolk sac are more closely related to each other than either is to hemogenic or non-HE cells in the major arteries. Therefore, a major driver of the different lineage potentials of the committed erythro-myeloid progenitors that emerge in the yolk sac versus hematopoietic stem cells that originate in the major arteries is likely to be the distinct molecular properties of the HE cells from which they are derived. We used bioinformatics analyses to predict signaling pathways active in arterial HE, which include the functionally validated pathways Notch, Wnt, and Hedgehog. We also used a novel bioinformatics approach to assemble transcriptional regulatory networks and predict transcription factors that may be specifically involved in hematopoietic cell formation from arterial HE, which is the origin of the adult hematopoietic system.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Hemangioblastos/fisiología , Hematopoyesis/fisiología , Animales , Arterias/citología , Arterias/embriología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Proteínas de Drosophila/genética , Sangre Fetal/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Leucemia Experimental/genética , Leucemia Experimental/virología , Leucemia Mieloide Aguda/genética , Ratones , Ratones Noqueados , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Transcripción Genética , Saco Vitelino/citologíaRESUMEN
The mouse Langerhans cell (LC) network is established through the differentiation of embryonic LC precursors. BMP7 and TGFß1 initiate cellular signaling that is essential for inducing LC differentiation and preserving LCs in a quiescent state, respectively. Here we show that loss of Cbfß2, one of two RNA splice variants of the Cbfb gene, results in long-term persistence of embryonic LC precursors after their developmental arrest at the transition into the EpCAM+ stage. This phenotype is caused by selective loss of BMP7-mediated signaling essential for LC differentiation, whereas TGFßR signaling is intact, maintaining cells in a quiescent state. Transgenic Cbfß2 expression at the neonatal stage, but not at the adult stage, restored differentiation from Cbfß2-deficient LC precursors. Loss of developmental potential in skin-residential precursor cells was accompanied by diminished BMP7-BMPR1A signaling. Collectively, our results reveal an essential requirement for the Cbfß2 variant in LC differentiation and provide novel insight into how the establishment and homeostasis of the LC network is regulated.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Células de Langerhans/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Animales , Proteína Morfogenética Ósea 7/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/fisiología , Diferenciación Celular/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiologíaRESUMEN
CBFß-SMMHC (core-binding factor ß-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 CBFß-SMMHC contributes to leukemia development is not well understood. Previously, it was proposed that CBFß-SMMHC acts by dominantly repressing the transcription factor RUNX1 (Runt-related protein 1), but we recently showed that CBFß-SMMHC has activities that are independent of RUNX1 repression. In addition, we showed that a modified CBFß-SMMHC with decreased RUNX1-binding activity accelerates leukemogenesis. These results raise questions about the importance of RUNX1 in leukemogenesis by CBFß-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.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Hematopoyesis/fisiología , Leucemia/metabolismo , Leucemia/patología , Cadenas Pesadas de Miosina/fisiología , Animales , Apoptosis , Western Blotting , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Técnicas de Sustitución del Gen , Humanos , Inmunoprecipitación , Leucemia/mortalidad , RatonesRESUMEN
Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA-binding capacity. Cbfb-deficient (Cbfb(-/-) ) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb(-/-) mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb(fl/fl) mice with Dermo1 Cre knock-in mice. Cbfb(fl/fl/Cre) mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb(fl/fl/Cre) chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb(fl/fl/Cre) embryos compared with the respective protein in the respective tissue of Cbfb(fl/fl) embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb(fl/fl/Cre) primary osteoblasts, and Runx2 protein was less stable in Cbfb(fl/fl/Cre) osteoblasts than Cbfb(fl/fl) osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.
Asunto(s)
Desarrollo Óseo/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Animales , Diferenciación Celular , Condrocitos/citología , Subunidad beta del Factor de Unión al Sitio Principal/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/citologíaRESUMEN
Core binding factor beta (Cbfß), the partner protein of Runx family transcription factors, enhances Runx function by increasing the binding of Runx to DNA. Null mutations of Cbfb result in embryonic death, which can be rescued by restoring fetal hematopoiesis but only until birth, where bone formation is still nearly absent. Here, we address a direct role of Cbfß in skeletal homeostasis by generating osteoblast-specific Cbfß-deficient mice (Cbfb(Δob/Δob) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col1a1 promoter. Cbfb(Δob/Δob) mice showed normal growth and development but exhibited reduced bone mass, particularly of cortical bone. The reduction of bone mass in Cbfb(Δob/Δob) mice is similar to the phenotype of mice with haploinsufficiency of Runx2. Although the number of osteoblasts remained unchanged, the number of active osteoblasts decreased in Cbfb(Δob/Δob) mice and resulted in lower mineral apposition rate. Immunohistochemical and quantitative real-time PCR analyses showed that the expression of osteogenic markers, including Runx2, osterix, osteocalcin, and osteopontin, was significantly repressed in Cbfb(Δob/Δob) mice compared with wild-type mice. Cbfß deficiency also reduced Runx2 protein levels in osteoblasts. The mechanism was revealed by forced expression of Cbfß, which increased Runx2 protein levels in vitro by inhibiting polyubiquitination-mediated proteosomal degradation. Collectively, these findings indicate that Cbfß stabilizes Runx2 in osteoblasts by forming a complex and thus facilitates the proper maintenance of bone mass, particularly cortical bone.
Asunto(s)
Huesos/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Osteoblastos/metabolismo , Animales , Desarrollo Óseo/fisiología , Núcleo Celular/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Ratones , Ratones Endogámicos C57BL , Tamaño de los ÓrganosRESUMEN
Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-ß) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-ß is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-ß. In addition, CBF-ß did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-ß silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. Importance: The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-ß was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-ß knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-ß to degrade APOBEC3. CBF-ß did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vif-CBF-ß interaction.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Citosina Desaminasa/metabolismo , Productos del Gen vif/fisiología , Lentivirus/fisiología , Desaminasas APOBEC , Secuencia de Bases , Citidina Desaminasa , Cartilla de ADN , Células HEK293 , Humanos , Lentivirus/clasificación , Proteolisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mitotic bookmarking is an epigenetic control mechanism that sustains gene expression in progeny cells; it is often found in genes related to the maintenance of cellular phenotype and growth control. RUNX transcription factors regulate a broad spectrum of RNA Polymerase (Pol II) transcribed genes important for lineage commitment but also regulate RNA Polymerase I (Pol I) driven ribosomal gene expression, thus coordinating control of cellular identity and proliferation. In this study, using fluorescence microscopy and biochemical approaches we show that the principal RUNX co-factor, CBFß, associates with nucleolar organizing regions (NORs) during mitosis to negatively regulate RUNX-dependent ribosomal gene expression. Of clinical relevance, we establish for the first time that the leukemogenic fusion protein CBFß-SMMHC (smooth muscle myosin heavy chain) also associates with ribosomal genes in interphase chromatin and mitotic chromosomes to promote and epigenetically sustain regulation of ribosomal genes through RUNX factor interactions. Our results demonstrate that CBFß contributes to the transcriptional regulation of ribosomal gene expression and provide further understanding of the epigenetic role of CBFß-SMMHC in proliferation and maintenance of the leukemic phenotype.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Epigénesis Genética , Cadenas Pesadas de Miosina/fisiología , Proteínas de Fusión Oncogénica/fisiología , Ribosomas/genética , Línea Celular Tumoral , Cromosomas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , ADN Polimerasa I/metabolismo , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia , Mitosis , Ribosomas/metabolismoRESUMEN
The pathogenesis of cleidocranial dysplasia (CCD) as well as the specific role of core binding factor ß (Cbfß) and the Runt-related transcription factor (RUNX)/Cbfß complex in postnatal skeletogenesis remain unclear. We demonstrate that Cbfß ablation in osteoblast precursors, differentiating chondrocytes, osteoblasts, and odontoblasts via Osterix-Cre, results in severe craniofacial dysplasia, skeletal dysplasia, abnormal teeth, and a phenotype recapitulating the clinical features of CCD. Cbfß(f/f)Osterix-Cre mice have fewer proliferative and hypertrophic chondrocytes, fewer osteoblasts, and almost absent trabecular bone, indicating that Cbfß may maintain trabecular bone formation through its function in hypertrophic chondrocytes and osteoblasts. Cbfß(f/f)Collagen, type 1, alpha 1 (Col1α1)-Cre mice show decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage, indicating that osteoblast lineage-specific ablation of Cbfß results in milder bone defects and less resemblance to CCD. Activating transcription factor 4 (Atf4) and Osterix protein levels in both mutant mice are dramatically reduced. ChIP assays show that Cbfß directly associates with the promoter regions of Atf4 and Osterix. Our data further demonstrate that Cbfß highly up-regulates the expression of Atf4 at the transcriptional regulation level. Overall, our genetic dissection approach revealed that Cbfß plays an indispensable role in postnatal skeletal development and homeostasis in various skeletal cell types, at least partially by up-regulating the expression of Atf4 and Osterix. It also revealed that CCD may result from functional defects of the Runx2/Cbfß heterodimeric complex in various skeletal cells. These insights into the role of Cbfß in postnatal skeletogenesis and CCD pathogenesis may assist in the development of new therapies for CCD and osteoporosis.
Asunto(s)
Condrocitos/fisiología , Displasia Cleidocraneal/fisiopatología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Osteoblastos/fisiología , Osteogénesis/fisiología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Condrocitos/metabolismo , Displasia Cleidocraneal/genética , Displasia Cleidocraneal/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Osteoblastos/metabolismo , Osteogénesis/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Multimerización de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/citología , Cráneo/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Different mechanisms for CBFß-MYH11 function in acute myeloid leukemia with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFß-MYH11-binding site analysis and quantitative interaction proteomics, we found that CBFß-MYH11 localizes to RUNX1 occupied promoters, where it interacts with TAL1, FLI1 and TBP-associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the coregulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knockdown, a small subset of the CBFß-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFß-MYH11 target genes, including genes implicated in hematopoietic stem cell self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knockdown. Together these results suggest an essential role for CBFß-MYH11 in regulating the expression of genes involved in maintaining a stem cell phenotype.
Asunto(s)
Inversión Cromosómica , Cromosomas Humanos Par 16 , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Leucemia Mieloide Aguda/genética , Cadenas Pesadas de Miosina/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Sitios de Unión , Factor de Transcripción GATA2/fisiología , Histona Desacetilasas/fisiología , Humanos , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-fli-1/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteína 1 de la Leucemia Linfocítica T Aguda , Activación TranscripcionalRESUMEN
HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor ß (CBFß) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFß (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFß. A comparison of heat capacity changes supports a model in which CBFß prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vif's C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Proteínas Cullin/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Termodinámica , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer's patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor γt (Rorγt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfß transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfß2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative α4ß7 integrin (α4ß7)(+) IL-7R α-chain (IL-7Rα)(+) population, can be subdivided into Rorγt-expressing IL-7Rα(high) cells and nonexpressing IL-7Rα(mid) cells. Whereas Id2 and Rorγt are required to direct α4ß7(+)IL-7Rα(mid) cells to become α4ß7(+)IL-7Rα(high) cells, Runx1/Cbfß2 complexes are necessary for the emergence of α4ß7(+)IL-7Rα(mid) cells. In addition, the loss of Cbfß2, but not P1-Runx1, resulted in an inefficient upregulation of Rorγt in residual α4ß7(+)IL-7Rα(+) LTi cells at anlagen. Our results thus revealed that Runx1/Cbfß2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorγt expression at anlagen.
Asunto(s)
Diferenciación Celular/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Tejido Linfoide/inmunología , Animales , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/genética , Variación Genética/inmunología , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/fisiología , Hígado/embriología , Hígado/inmunología , Hígado/patología , Tejido Linfoide/citología , Tejido Linfoide/embriología , Ratones , Ratones Mutantes , Células Madre Multipotentes/citología , Células Madre Multipotentes/inmunología , Células Madre Multipotentes/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/fisiologíaRESUMEN
BACKGROUND: The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFbeta is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFbeta is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFbeta-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFbeta. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFbeta expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFbeta. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. RESULTS: We show that CBFbeta is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFbeta contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFbeta is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFbeta is recruited to both the Osteopontin and the Galectin-3 promoters. CONCLUSIONS: CBFbeta is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFbeta is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFbeta is essential for invasion, not all Runx2-target genes require CBFbeta. We conclude that CBFbeta is required for a subset of Runx2-target genes that are sufficient to maintain the invasive phenotype of the cells. These findings suggest that the interaction between Runx2 and CBFbeta might represent a viable target for therapeutic intervention to inhibit bone metastasis.
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Neoplasias de la Mama/patología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Invasividad Neoplásica , Animales , Línea Celular Tumoral , Subunidad beta del Factor de Unión al Sitio Principal/genética , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , Interferencia de ARNRESUMEN
Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBF beta-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBF beta-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBF beta-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBF beta-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBF beta-SMMHC.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Leucemia Experimental/fisiopatología , Proteínas de Fusión Oncogénica/fisiología , Animales , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Humanos , Ratones , Ratones Transgénicos , Proteínas de Fusión Oncogénica/metabolismo , Fosforilación , Unión ProteicaRESUMEN
The transcription factor RUNX1 is essential for definitive hematopoiesis and is required for the expression of a number of important hematopoietic regulator genes. It was recently shown that RUNX1 acts within a narrow developmental window during which it cannot be replaced by other members of the RUNX transcription factor family. Studies of the molecular basis of this phenomenon revealed that RUNX1 is required for the opening of chromatin of important hematopoietic regulator genes and for the formation, but not the maintenance of stable transcription factor complexes on these genes. However, the chromatin opening activity of RUNX1 is context dependent, indicating that it cooperates with alternate transcription factors at different stages of hematopoietic development. This review summarizes recent results on the regulation of chromatin structure by RUNX1 in developing hematopoietic cells.