RESUMEN
MTG8 (RUNX1T1) is a fusion partner of AML1 (RUNX1) in the leukemic chromosome translocation t(8;21). The AML1-MTG8 fusion gene encodes a chimeric transcription factor. One of the highly conserved domains of MTG8 is TAFH which possesses homology with human TAF4 [TATA-box binding protein-associated factor]. To obtain specific inhibitors of the AML1-MTG8 fusion protein, we isolated RNA aptamers against the MTG8 TAFH domain using systematic evolution of ligands by exponential enrichment. All TAF aptamers contained guanine-rich sequences. Analyses of a TAF aptamer by NMR, CD, and mutagenesis revealed that it forms a parallel G-quadruplex structure in the presence of K+ . Furthermore, the aptamer could bind to the AML1-MTG8 fusion protein and dissociate the AML1-MTG8/DNA complex, suggesting that it can inhibit the dominant negative effects of AML1-MTG8 against normal AML1 function and serve as a potential therapeutic agent for leukemia.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , ADN/metabolismo , G-Cuádruplex , Proteínas de Fusión Oncogénica/metabolismo , ARN/química , ARN/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/química , Proteína 1 Compañera de Translocación de RUNX1/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/farmacología , Secuencia de Bases , G-Cuádruplex/efectos de los fármacos , Humanos , Leucemia/metabolismo , Mutación , Unión Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacosAsunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Regulación Neoplásica de la Expresión Génica , Leucemia/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/química , Proteína 1 Compañera de Translocación de RUNX1/química , Translocación Genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Proteína 1 Inhibidora de la Diferenciación/genética , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Leucemia/genética , Leucemia/metabolismo , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/genética , Proteína 1 Compañera de Translocación de RUNX1/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chromosomal translocations represent frequent events in leukemia. In t(8;21)+ acute myeloid leukemia, RUNX1 is fused to nearly the entire ETO protein, which contains four conserved nervy homology regions, NHR1-4. Furthermore RUNX1/ETO interacts with ETO-homologous proteins via NHR2, thereby multiplying NHR domain contacts. As shown recently, RUNX1/ETO retains oncogenic activity upon either deletion of the NHR3 + 4 N-CoR/SMRT interaction domain or substitution of the NHR2 tetramer domain. Thus, we aimed to clarify the specificities of the NHR domains. A C-terminally NHR3 + 4 truncated RUNX1/ETO containing a heterologous, structurally highly related non-NHR2 tetramer interface translocated into the nucleus and bound to RUNX1 consensus motifs. However, it failed to interact with ETO-homologues, repress RUNX1 targets, and transform progenitors. Surprisingly, transforming capacity was fully restored by C-terminal fusion with ETO's NHR4 zinc-finger or the repressor domain 3 of N-CoR, while other repression domains failed. With an inducible protein assembly system, we further demonstrated that NHR4 domain activity is critically required early in the establishment of progenitor cultures expressing the NHR2 exchanged truncated RUNX1/ETO. Together, we can show that NHR2 and NHR4 domains can be replaced by heterologous protein domains conferring tetramerization and repressor functions, thus showing that the NHR2 and NHR4 domain structures do not have irreplaceable functions concerning RUNX1/ETO activity for the establishment of human CD34+ cell expansion. We could resemble the function of RUNX1/ETO through modular recomposition with protein domains from RUNX1, ETO, BCR and N-CoR without any NHR2 and NHR4 sequences. As most transcriptional repressor proteins do not comprise tetramerization domains, our results provide a possible explanation as to the reason that RUNX1 is recurrently found translocated to ETO family members, which all contain tetramer together with transcriptional repressor moieties.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Leucemia Mieloide Aguda/patología , Proteínas de Fusión Oncogénica/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/metabolismo , Antígenos CD34 , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Dominios Proteicos , Proteína 1 Compañera de Translocación de RUNX1/química , Proteína 1 Compañera de Translocación de RUNX1/genéticaRESUMEN
Runt-related transcription factor 1 translocation partner 1 (RUNX1T1), a potential novel regulator of adipogenesis, exists in two splice variants: a long (RUNX1T1-L) and a short (RUNX1T1-S) isoform. However, there is no data showing the existence of RUNX1T1 in ovine subcutaneous fat at different stages of developmental and its role on ovine adipogenesis. Therefore, the objectives of this study were to evaluate the presence of RUNX1T1 in subcutaneous fat of five-day-old to 24-month-old sheep and to investigate the role of RUNX1T1 in ovine adipogenesis. In this study, we detected a 1829 bp cDNA fragment of RUNX1T1 which contains a 1815 bp coding sequence that encodes 602-amino acid and 14 bp of 5' untranslated region, respectively. The amino acid sequence of RUNX1T1 has 31.18â»94.21% homology with other species' protein sequences. During fat development, the RUNX1T1 protein expression was higher in subcutaneous fat of 24-month-old Hu sheep. In addition, the expression of RUNX1T1-L mRNA decreased first, then subsequently increased during ovine preadipocyte differentiation. Knockdown of RUNX1T1-L in ovine preadipocytes promoted preadipocyte differentiation and lipid accumulation. Taken together, our data suggests that RUNX1T1 is an important functional molecule in adipogenesis. Moreover, it showed for the first time that RUNX1T1-L was negatively correlated with the ovine preadipocyte differentiation.
