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Análisis Funcional De MCPH1 En La Condensación Cromosómica Y El Control Del Ciclo Celular

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Análisis Funcional De MCPH1 En La Condensación Cromosómica Y El Control Del Ciclo Celular Área de Genética, Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén. Análisis funcional de MCPH1 en la condensación cromosómica y el control del ciclo celular TESIS DOCTORAL María de la Cabeza Arroyo López Jaén 2018 Área de Genética Departamento de Biología Experimental Facultad de Ciencias Experimentales Universidad de Jaén TESIS DOCTORAL Análisis funcional de MCPH1 en la condensación cromosómica y el control del ciclo celular MARIA DE LA CABEZA ARROYO LOPEZ 2018 Análisis funcional de MCPH1 en la condensación cromosómica y el control del ciclo celular Dirigida por los doctores: J. Alberto Marchal Ortega Antonio Sánchez Baca Memoria presentada por la licenciada María de la Cabeza Arroyo López para optar al Grado de Doctor Internacional en Biología. Junio, 2018 El presente trabajo ha sido realizado en el Área de Genética del Departamento de Biología Experimental de la Universidad de Jaén durante los años 2014-2018, período durante el cual el Doctorando disfrutó de una Ayuda para la Formación de Personal Investigador con cargo al Plan de Apoyo a la Investigación, Desarrollo Tecnológico e Innovación de la Universidad de Jaén. Además, parte de este trabajo se ha llevado a cabo en el “Department of Genetics, Cell Biology and Developoment, University of Minnesota”, gracias a unas ayudas para estancias breves proporcionadas por la organización EMBO y la Escuela de Doctorado de la Universidad de Jaén. La investigación realizada ha sido financiada en parte por la Universidad de Jaén a través del proyecto UJA/2011/12/36, y la Junta de Andalucía a través del programa “Ayudas a grupos de Investigación (BIO-220). Parte de los resultados de este estudio han sido recogidos en las siguientes publicaciones científicas: Chromosoma (2015) 124:491–501 DOI 10.1007/s00412- 015-0512-2; Scientific Reports (2017) 7: 13019 DOI:10.1038/s41598-017-12793-7. Además, también han sido presentados en el “19th International Chromosome Conference”; en el “EMBO Workshop on Cell Division: Molecular Machineries and Cancer Targeted Therapies”; en el “XL Congreso de la Sociedad Española de Genética”; y en las ediciones VIII y IX del Seminario de Citogenética. AGRADECIMIENTOS Quiero expresar mi más sincero agradecimiento: A los doctores Juan Alberto Marchal Ortega y Antonio Sánchez Baca, porque no habría podido caer en mejores manos. Esta tesis doctoral no habría sido posible sin su apoyo y su dedicada dirección. Con ellos di mis primeros pasos en la ciencia, de ellos he aprendo todo y espero poder seguir aprendiendo aún más. Agradezco y admiro su actitud siempre positiva, amable y humilde, porque no solo me han enseñado protocolos, también me han transmitido sus valores personales y su motivación incansable. Ellos han hecho que la ciencia sea mi principal vocación. A Duncan J. Clarke y Ryoko Kuriyama. Jamás habría imaginado que al otro lado del mundo iba a encontrar otro laboratorio con dos personas tan dedicadas y amables, que se implicarían tanto en mi trabajo. Su colaboración y su experiencia ha sido fundamental y lo seguirá siendo. A Jesús Calahorra, por su ayuda y sobre todo su paciencia con los muchos y muchos WB que hemos hecho (y los que nos queden…), siempre buscando el mejor resultado. A Ana Rosa Cañuelo Navarro, por todo lo que he aprendido de ella, los conocimientos y la buena energía transmitida a nivel personal y profesional. A Jorge Nicolás Dominguez Macías, por su gran sentido del humor y por crear un buen ambiente de trabajo con esas bromas que a veces tardo en pillar. Y por supuesto por su ayuda y su orientación como el gran profesional que es. A los doctores Heidemarie Neitzel, Karl Sperling y Marc Trimborn (Charite Hospital, Berlín) por haber cedido las líneas de linfoblastos utilizadas en este trabajo, así como diversos reactivos, anticuerpos y oligos siRNA. Su asesoramiento en la realización de este estudio ha sido imprescindible. Al doctor Tatsuya Hirano (RIKEN Advanced Science Institute, Japón) por proporcionar el anticuerpo frente a MCPH1, y por los comentarios y sugerencias aportadas. Al doctor J. Francisco Giménez-Abián (CIB, Madrid), por las ideas y discusiones científicas aportadas, que han sido muy importante en los aspectos relacionados con el ciclo celular. A todos mis compañeros de la sala de becarios y del labo, a los que están y a los que no. Gracias a Ismael y a Maribel, que fueron los que me dieron la mano durante mis primeros pasos en el laboratorio. Aunque haya llovido mucho, no me olvido de ellos. A Vero, por ser la más simpática del Área de Genética y ayudarme a socializar con todos, por echarme una mano siempre que he necesitado algo. A mi pareja, por aguantar tantas charlas de ciencia, agobios a veces, por cruzar el charco conmigo y ayudarme a desconectar siempre que lo necesito. En fin, por estar al pie del cañón las 24 horas. A Armando y Mariloli, que desde que entré en sus vidas me han animado y apoyado, convirtiéndose en una parte fundamental de mi vida. A mi familia, porque qué sería yo sin mi familia. Los que lo dan todo por mí, los que han estado siempre ahí, pacientemente, aguantando mis malos momentos y mis errores sin esperar nada a cambio. ÍNDICE ÍNDICE ÍNDICE RESUMEN/ABSTRACT………………………………………………….………....………….……...pág. 3. INTRODUCCIÓN………………………………………………………………...………….…..……..pág. 9. 1. Microcefalia primaria autosómica recesiva (MCPH)………………………….….……pág. 11. 1.1 Bases clínicas………………………………………………………………………….………. pág. 11. 1.2 Bases genéticas…………………………………………………………………..……………..pág.12. 1.2.1 Genes MCPH y fenotipo clínico……………………………….…………….pág. 13. 1.2.2 Genes MCPH: función molecular………………………………….……….pág. 14. 1.3 Bases ambientales……………………………………………………………………..……....pág. 18. 2. Microcefalia primaria MCPH1……………………………………………………..………pág. 19. 2.1 MCPH1 y microcefalia primaria: mutaciones descritas…………………………………pág. 21. 2.2 Expresión y localización subcelular de MCPH1………………………………..…………pág. 23. 3. Funciones moleculares de MCPH1………………………………………………….…… pág. 24. 3.1 Condensación cromosómica………………………………………………………………….pág. 24. 3.1.1 Bases moleculares del proceso de condensación cromosómica………. pág. 25. 3.1.2 Papel de MCPH1 en la condensación cromosómica………………..….. pág. 28. 4. MCPH1 y control del ciclo celular………………………………………………………… pág. 30. 4.1 Bases moleculares de la entrada en mitosis………………………………………………. pág. 30. 4.2 MCPH1 y control de la entrada en mitosis……………………………………………...…. pág. 32. 4.3 Papel de MCPH1 en el control del daño en ADN…………………………………………pág. 33. 4.3.1 Punto de control por roturas en ADN dependiente de ATR/ATM……… pág. 34. 4.3.2 Papel de MCPH1 en los puntos de control dependientes de ATR y ATM………………………………………………………….………………………..pág. 35. 4.4 Otros puntos de control en G2: “Decatenation Checkpoint”…………………………… pág. 37. 4.5 Adaptación celular al punto de control……………………………………………………. pág. 41. 4.6 MCPH1 y control del ciclo celular: importancia en cáncer………………………………pág. 42. i ÍNDICE_________________________________________________________________________ OBJETIVOS/AIMS…………………………………………………………………………….………pág. 45. CAPÍTULO I/CHAPTER I……………………………………………………………………………pág. 49. - Chromosome structure deficiencies in MCPH1 syndrome……………………..………...pág. 51. CAPÍTULO II/CHAPTER II…………………………………………………………………………pág. 63. - MCPH1, mutated in primary microcephaly, is required for efficient chromosome alignment during mitosis………….………………………………………………………………………pág. 65. - Supplementary information……..…………………………………………………………….pág.77. CAPÍTULO III/CHAPTER III………………….……………………………………………………pág. 87. - MCPH1 is essential for cellular adaptation to the G2-phase decatenation checkpoint……………………………………………………………………………………...pág. 89. CAPÍTULO IV/CHAPTER IV…………….………………………………………………………..pág. 125. - PLK1 acts downstream of MCPH1 in a single pathway that confers adaptation to the G2 decatenation checkpoint….………………………………………………………………....pág. 127. DISCUSIÓN…………………………………………………………………………………...………pág. 159. - Capítulo I………………………………………………………………...……….…………..pág. 161. - Capítulo II……………………...…………………………….……………………….………pág. 164. - Capítulo III y IV………………………………………………………………………………pág. 167. CONCLUSIONES/CONCLUSIONS………………………………………………………………pág. 173. BIBLIOGRAFÍA DE LA INTRODUCCIÓN Y DE LA DISCUSIÓN…..……………...……pág. 179. CURRICULUM VITAE……………………………………………………………………………...pág. 209. ii RESUMEN/ABSTRACT RESUMEN/ABSTRACT___________________________________________________________ RESUMEN El gen MCPH1 es uno de los implicados en el síndrome de microcefalia primaria (MCPH), una enfermedad rara consecuencia de una alteración en la neurogénesis que determina una reducción drástica en el tamaño cerebral, con el consiguiente retraso mental asociado. A diferencia de los otros genes MCPH identificados, los pacientes por mutaciones en MCPH1 muestran una alteración en el ciclo celular que afecta a todos los tipos celulares analizados. Por tanto, la importancia de este gen trasciende su papel como regulador de la neurogenésis humana, siendo un elemento clave para la coordinación del proceso de condensación cromosómica con el resto de mecanismos que participan de la mitosis. Además, su función también es importante en la respuesta celular frente al daño en el ADN. Por todo ello el síndrome asociado al gen MCPH1 es un modelo experimental interesante para el estudio de las bases moleculares de la división mitótica, y su importancia en los mecanismos reguladores que controlan el desarrollo cerebral humano. En esta tesis doctoral se presentan nuevos resultados sobre la función específica del gen MCPH1 durante la mitosis y su contribución en las vías moleculares que controlan su puesta
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