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Mecanismo de cooperación beta c-Myb-C/EBP desde sitios separados en un promotor
Tahir H Tahirov1, Ko Sato, Emi Ichikawa-Iwata
1Kanagawa Academy of Science and Technology, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. tahir@med.yokohama-cu.ac.jp
Cell
|January 17, 2002
Resumen
El factor de transcripción de la mieloblastosis celular (c-Myb) coopera con C/EBP beta para regular la expresión génica, a diferencia de su contraparte viral (v-Myb). El análisis estructural revela una interacción clave que permite esta diferencia funcional.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- Biología Estructural Biología estructural.
- Regulación genética Reglamento genético.
Sus antecedentes:
- La mieloblastosis celular (c-Myb) y el virus de la mieloblastosis aviar (AMV) v-Myb son factores de transcripción involucrados en la regulación del gen mieloide.
- c-Myb, pero no v-Myb, coopera con C/EBP beta para activar la transcripción de genes específicos del mieloide.
- La base estructural de esta diferencia funcional sigue sin estar clara.
Objetivo del estudio:
- Aclarar los mecanismos estructurales que subyacen a la cooperación diferencial entre c-Myb y C/EBP beta.
- Para comparar las interfaces de interacción de los complejos c-Myb/C/EBP beta y v-Myb/C/EBP beta.
Principales métodos:
- Estructuras cristalinas determinadas de complejos que involucran los dominios de unión al ADN (DBD) de c-Myb o v-Myb, C/EBP beta DBD, y el ADN promotor.
- Utilizó ensayos de pull-down GST para confirmar las interacciones proteína-proteína.
- Se realizan ensayos de transactivación de luciferasa para evaluar la actividad transcripcional.
- Empleó microscopía de fuerza atómica para visualizar el bucle del ADN y las interacciones de largo alcance.
Principales resultados:
- Las estructuras cristalinas revelaron que en el complejo c-Myb, C/EBP beta unido al ADN interactúa con la región R2 de c-Myb unido a un fragmento de ADN separado.
- Las mutaciones puntuales en la región v-Myb R2 abolieron esta interacción en el complejo v-Myb.
- Los ensayos de GST pull-down y luciferasa confirmaron el significado funcional de la interacción c-Myb/C/EBP beta.
- La microscopía de fuerza atómica demostró que las interacciones cristalinas observadas imitan las interacciones de largo alcance en el promotor, que involucran el bucle de ADN.
Conclusiones:
- La región R2 de c-Myb es crítica para su interacción cooperativa con C/EBP beta.
- Esta interacción facilita el bucle de ADN de largo alcance, lo que permite una mejor regulación transcripcional de los genes mieloides.
- La incapacidad de v-Myb para interactuar efectivamente con C/EBP beta, debido a mutaciones en R2, explica su falta de cooperación.
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