Estudios de la expresión y la función de la variante de histona macroH2a, en la adaptación estacional de cyprinus carpio
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2008
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es
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Universidad Andrés Bello
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Resumen
Los seres vivos se encuentran constantemente sometidos a las presiones selectivas que
imponen las fluctuaciones dinámicas del medio ambiente, frente a las cuales deben generar repuestas
compensatorias que les permitan sobrevivir. Organismos ectotérmicos que viven en hábitats con
ciclos estacionales alternados, como el pez Cyprinus carpio son un buen modelo para el estudio de
las respuestas adaptativas no genéticas conocidas como plasticidad fenotípica. Fundamentalmente,
estas respuestas se expresan como ajustes coordinados de tipo fisiológico y molecular. En la carpa, a
nivel de la ultraestructura celular, la característica fenotípica más destacable es la transformación de
la citoarquitectura del nucléolo entre un estado segregado en invierno y otro reticular en verano,
alternancia de estados que trae asociada modificaciones en la estructura de la cromatina nucleolar.
En este contexto, la presente tesis abordó la caracterización de algunos de los componentes
estructurales de la cromatina que participan en la plasticidad fenotípica de este dominio nuclear,
asociada al fenómeno de climatización estacional. Entre los componentes de la cromatina, las
variantes de histonas parecen ser uno de los mecanismos epigenéticos relevantes en la configuración
de sus diferentes estados. En el marco de la segregación de los componentes nucleolares en células
hepáticas, planteamos como hipótesis que el reemplazo de histonas normales por la variante
macroH2A forma parte de los mecanismos que permiten la adaptación invernal de la carpa.
Nuestros resultados muestran que la histona variante macroH2A está sobreexpresada durante
el invierno y que se localiza a lo largo del gen ribosomal. Este efecto está en consonancia con la
actividad represora atribuida a esta variante y a la disminución de la tasa de transcripción del gen
ribosomal observada en invierno. En conjunto con la sobreexpresión de mH2A, hemos evidenciado
un aumento del nivel de metilaciones a nivel del promotor del gen y un aumento de la modificación
post-traduccional trimetil lisina 4 de histona H3. Mediante ChIP-chop-qPCR demostramos que
parte de las secuencias del promotor del rDNA asociadas a mH2A además están metiladas, por lo
cual postulamos que existe más de un mecanismo represor de la transcripción del rDNA.
En verano hay un aumento generalizado de la monometilación de lisina 36 en histona H3 y
de acetilación en lisina 12 en histona H4, esta última también está aumentada en el cistrón
ribosomal. Estas características ocurren en concomitancia con un mayor nivel de expresión del gen
ribosomal durante esta estación.
Todos estos antecedentes permiten apoyar el modelo propuesto recientemente por Huang y
colaboradores (2006), que postula la existencia de tres estados en la configuración de la cromatina
del rDNA: un estado heterocromático transcripcionalmente inactivo, un estado eucromático activo y
un tercer estado inte1medio con características del estado eucromático pero transcripcionalmente
inactivo, situación que concuerda con la estructura de la cromatina nucleolar de la carpa y sus
cambios fenotípicos.
Living organisms are constantly subjected to selective pressures imposed by environmental dynamic fluctuations, which must generate compensatory responses that allow their adaptation in order to survive. Ectothe1mal organisms that live in habitats with cyclical altemate seasons, such as the fish Cyprinus carpio, are a good model to study non-genetic adaptative responses known as phenotypic plasticity. Essentially, these responses are expressed as coordinated physiological and molecular adjushnents. In the carp, at a leve! of cellular ultrastructure, the most distinguishable phenotypic characteristic is the transformation of the nucleolus cytoarchitechture between a segregated state during winter and reticular state during summer, alternation associated with modifications of the nucleolar chromatin structure. Within this context, the present thesis characterized sorne of the chromatin structural components that play a role in the phenotypic plasticity of this nuclear domain, associated to the phenomena of seasonal acclimatization. Among the chromatin components, histone variants have appeared as one of the relevant epigenetical mechanisms in the configuration of the different states. It is within the segregation of the nucleolar components in hepatic cells that we propose as an hypothesis that the replacement of normal histones by the variant macroH2A forms part of the mechanisms that allow winter adaptation in carp. Our results show that the histone variant macroH2A is over expressed during winter and it localizes along the ribosomal gene. This effect agrees with the repressor activity attributed to this variant and to the reduction in the transcription rate of the ribosomal gene. Together, with the over expression of mH2A, we have observed an increase in the leve! of methylations at promoter leve! of this gene and an increase in post-translational modification of trimethyl lysine 4 of histone H3. Using ChIP-chop-qPCR, we demonstrated that most of the promoter sequences of rDNA associated to mH2A are methylated, thus this would constitute an additional repressor mechanism of rDNA transcription .. During summer it is a general increase at the leve! of histone H3 monomethylation of lysine 36 and acetylation of lysine 12 at histone H4, which is also increased in ribosomal gene. These events take place in parallel with a higher leve! of expression of the ribosomal gene during this season. Ali these evidence supports the model recently proposed by Huang et al. (2006), that propases the existence of three states of rDNA chromatin configuration; a transcriptionally inactive heterochromatic state, an active euchromatic state and a third intermediate state with characteristics of a euchromatic state but transcriptionally inactive, situation that agrees with the nucleolar chromatin structure in carp and its phenotypic changes.
Living organisms are constantly subjected to selective pressures imposed by environmental dynamic fluctuations, which must generate compensatory responses that allow their adaptation in order to survive. Ectothe1mal organisms that live in habitats with cyclical altemate seasons, such as the fish Cyprinus carpio, are a good model to study non-genetic adaptative responses known as phenotypic plasticity. Essentially, these responses are expressed as coordinated physiological and molecular adjushnents. In the carp, at a leve! of cellular ultrastructure, the most distinguishable phenotypic characteristic is the transformation of the nucleolus cytoarchitechture between a segregated state during winter and reticular state during summer, alternation associated with modifications of the nucleolar chromatin structure. Within this context, the present thesis characterized sorne of the chromatin structural components that play a role in the phenotypic plasticity of this nuclear domain, associated to the phenomena of seasonal acclimatization. Among the chromatin components, histone variants have appeared as one of the relevant epigenetical mechanisms in the configuration of the different states. It is within the segregation of the nucleolar components in hepatic cells that we propose as an hypothesis that the replacement of normal histones by the variant macroH2A forms part of the mechanisms that allow winter adaptation in carp. Our results show that the histone variant macroH2A is over expressed during winter and it localizes along the ribosomal gene. This effect agrees with the repressor activity attributed to this variant and to the reduction in the transcription rate of the ribosomal gene. Together, with the over expression of mH2A, we have observed an increase in the leve! of methylations at promoter leve! of this gene and an increase in post-translational modification of trimethyl lysine 4 of histone H3. Using ChIP-chop-qPCR, we demonstrated that most of the promoter sequences of rDNA associated to mH2A are methylated, thus this would constitute an additional repressor mechanism of rDNA transcription .. During summer it is a general increase at the leve! of histone H3 monomethylation of lysine 36 and acetylation of lysine 12 at histone H4, which is also increased in ribosomal gene. These events take place in parallel with a higher leve! of expression of the ribosomal gene during this season. Ali these evidence supports the model recently proposed by Huang et al. (2006), that propases the existence of three states of rDNA chromatin configuration; a transcriptionally inactive heterochromatic state, an active euchromatic state and a third intermediate state with characteristics of a euchromatic state but transcriptionally inactive, situation that agrees with the nucleolar chromatin structure in carp and its phenotypic changes.
Notas
Tesis (Doctor en Biociencias Moleculares)
Palabras clave
Histopatologías, Cromatina, Plasticidad fenotípica, Factores de la climatización estacional, Chile