Influencia de secuencias de ADN en procesos activos que determinan el posicionamiento nucleosomal.
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Date
2023
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Universidad de Concepción
Abstract
La posición de los nucleosomas tiene un gran impacto en procesos celulares que involucran interacción proteína-ADN, como es el caso de la replicación, reparación y transcripción. En Saccharomyces cerevisiae los promotores de genes se encuentran enriquecidos en secuencias
homopoliméricas de ADN denominados tractos poli (dA:dT), las cuales se relacionan con la presencia de regiones libres de nucleosomas (NDRs). En otros organismos, como ratón y humano, la presencia de tractos poli (dA:dT) es menor y su distribución es más amplia; sin embargo, mantienen su capacidad de excluir nucleosomas, lo que podría significar que existen mecanismos comunes que se han conservado durante la evolución. ¿Cómo se forman y mantienen los NDRs? Estudios a nivel de genoma completo han determinado que los complejos remodeladores de cromatina dependientes de ATP juegan un papel clave en este fenómeno, donde existen complejos denominados “despejadores”, que abren y forman los NDRs, y
complejos denominados “cubridores”, que cierran los NDRs. Además, otros estudios han encontrado una relación entre la acción de los complejos remodeladores de cromatina dependientes de ATP y la presencia de los tractos poli (dA:dT), donde la acción de factores de transcripción también estaría implicada. Sin embargo, aún se desconocen los mecanismos moleculares de la comunicación que existe entre los diferentes elementos que se encuentran en
los NDRs. Por este motivo, en el presente trabajo buscamos determinar la influencia de los tractos poli (dA:dT) sobre la actividad remodeladora de nucleosomas de los complejos ISW1a, RSC y ySWI/SNF, los cuales cumplen diferentes roles en la mantención de los NDRs. Además, estudiamos cómo factores de transcripción pueden influir en la dinámica entre estas secuencias y los complejos estudiados. Para llevar a cabo nuestro estudio, usamos sondasmononucleoso-males reconstituidas in vitro que poseían diferentes configuraciones de los tractos poli (dA:dT), con las que se ejecutaron ensayos de remodelación analizando la acción de los complejos purificados. En nuestros resultados hallamos que estas secuencias pueden afectar de manera directa varios aspectos de la acción de estos complejos, como: la unión al nucleosoma, la intensidad de la actividad de remodelación y la dirección en que ejecutan ésta. Además, observamos que la orientación de los tractos poli (dA:dT) afecta diferencialmente a los complejos remodeladores de cromatina estudiados. Por otro lado, encontramos que los factores de
transcripción podrían ejercer efectos diferenciales sobre ISW1a y RSC. Estos resultados realzan la importancia y el impacto que tienen los tractos poli (dA:dT) en el posicionamiento nucleosomal y ayudan a dilucidar los mecanismos mediante los cuales estas secuencias ejercen sus efectos.
Nucleosome positioning is crucial for cellular processes involving proteinDNA interactions, as in the case of replication, repair, and transcription. Gene promoters in Saccharomyces cerevisiae are highly enriched in homopolymeric DNA sequences called poly (dA:dT) tracts. These sequences are correlated with the presence of nucleosome-depleted regions (NDRs). In organisms like human and mouse, poly (dA:dT) tracts are less abundant and their distribution is wider. However, their ability to exclude nucleosomes is still present, which could mean that common molecular mechanisms have been conserved during evolution. How are NDRs around poly (dA:dT) tracts formed and maintained? Genome wide analyses have found that ATP-dependent chromatin-remodeling complexes are key in these processes. There are complexes called “pushers” which can open and maintain NDRs, and “pullers” which can close NDRs. In addition, these studies have shown a correlation between the action of remodeling complexes and the presence of poly (dA:dT) tracts, where the action of transcription factors can be also involved. Nevertheless, the molecular mechanisms of by which these different elements act are still unknown. In this context, our goal was to determine the influence of poly (dA:dT) tracts on the remodeling activity of ISW1a, RSC and ySWI/SNF complexes. We chose these complexes because they play different roles at NDRs. In addition, transcription factors were analyzed to understand their contribution in NDR dynamics. To answer our questions, we used in vitro reconstituted mononucleosomal probes, which harbor poly (dA:dT) tracts in different configurations, analyzing the action of the purified complexes. We found that poly (dA:dT) tracts can influence the action of these complexes in several aspects, such as: binding to nucleosome, strength of remodeling and the direction in which the histone octamer is moved. In addition, we observed differential effects due to orientation of poly (dA:dT) tracts. Furthermore, we found that transcription factors can exert differential effects on RSC and ISW1a activities. In summary, our results highlight the role of poly (dA:dT) tracts on nucleosome positioning and help in the elucidation of the molecular mechanisms by which these sequences exert their effects.
Nucleosome positioning is crucial for cellular processes involving proteinDNA interactions, as in the case of replication, repair, and transcription. Gene promoters in Saccharomyces cerevisiae are highly enriched in homopolymeric DNA sequences called poly (dA:dT) tracts. These sequences are correlated with the presence of nucleosome-depleted regions (NDRs). In organisms like human and mouse, poly (dA:dT) tracts are less abundant and their distribution is wider. However, their ability to exclude nucleosomes is still present, which could mean that common molecular mechanisms have been conserved during evolution. How are NDRs around poly (dA:dT) tracts formed and maintained? Genome wide analyses have found that ATP-dependent chromatin-remodeling complexes are key in these processes. There are complexes called “pushers” which can open and maintain NDRs, and “pullers” which can close NDRs. In addition, these studies have shown a correlation between the action of remodeling complexes and the presence of poly (dA:dT) tracts, where the action of transcription factors can be also involved. Nevertheless, the molecular mechanisms of by which these different elements act are still unknown. In this context, our goal was to determine the influence of poly (dA:dT) tracts on the remodeling activity of ISW1a, RSC and ySWI/SNF complexes. We chose these complexes because they play different roles at NDRs. In addition, transcription factors were analyzed to understand their contribution in NDR dynamics. To answer our questions, we used in vitro reconstituted mononucleosomal probes, which harbor poly (dA:dT) tracts in different configurations, analyzing the action of the purified complexes. We found that poly (dA:dT) tracts can influence the action of these complexes in several aspects, such as: binding to nucleosome, strength of remodeling and the direction in which the histone octamer is moved. In addition, we observed differential effects due to orientation of poly (dA:dT) tracts. Furthermore, we found that transcription factors can exert differential effects on RSC and ISW1a activities. In summary, our results highlight the role of poly (dA:dT) tracts on nucleosome positioning and help in the elucidation of the molecular mechanisms by which these sequences exert their effects.
Description
Tesis para optar al grado de Doctor en Ciencias Biológicas
Keywords
ADN Biosíntesis, Moléculas biológicas, Nucleótidos