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Epigenetic changes in a mouse model of Rett syndrome : optimizing a technique to evaluate DNA methylation
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A Síndrome de Rett é uma doença genética causada, em 90% dos casos, por uma mutação no gene MECP2, localizado no cromossoma X, que codifica a proteína com o mesmo nome. A Mecp2 tem inúmeras funções, em particular ser um importante “reader” da metilação de ADN. Sabe-se que a metilação de ADN está alterada na epilepsia; a hipótese da metilação na epileptogénese sugere que convulsões em si podem induzir modificações epigenéticas na cromatina agravando a condição epileptogénica. A adenosina, anticonvulsionante endógeno, foi implicada na epileptogénese pela sua capacidade de modular a metilação do ADN no cérebro. Em condições epiléticas os níveis de adenosina estão reduzidos, impedindo a sua ação anticonvulsionante a partir do controlo do tónus inibitório sináptico e pela promoção da hipometilação do ADN no processo epileptogénico. Recentemente, foi demonstrado que a adenosina está diminuída no hipocampo e córtex de modelos de ratinho Mecp2 knockout, hipotetizando-se, assim, que a condição epilética na RTT pode ter contribuição da subregulação de adenosina. Com a o objetivo de explorar uma possível relação entre a severidade da condição epilética associada a cada fenótipo RTT e mudanças na metilação do ADN no Sistema Nervoso Central e periferia (sangue), decidimos otimizar a metodologia necessária para observar mudanças na metilação do ADN em dois fenótipos diferentes (mild – symptomatic heterozygous (HET) females and severe – knockout (KO) symptomatic males) de ratinho modelo Mecp2 Knockout (B6.129P2 (C)-Mecp2tm1.1Bird/J). Assim, diferentes métodos de purificação de ADN foram testados, usando dois “buffers” de lise, procedidos de dot blot usando o anticorpo N6-metiladenosina. No fim, podemos confirmar que deteção foi possível com o uso de dot blot para as diferentes amostras e concentrações. Comparando os buffers, chegámos à conclusão inicial que o TDB, face ao RIPA, oferece resultados melhores no que toca à concentração e ratios da espectrofotometria obtidos, indicativos de maior pureza.
RTT is a genetic disorder caused, in 90% of cases, by a genetic mutation in the MECP2 gene, located on the X chromosome, which encodes the protein of the same name. MECP2 has innumerous roles, namely being an important reader of DNA methylation. It is known that DNA methylation is altered in epilepsy; the methylation hypothesis of epileptogenesis suggests that seizures by themselves can induce epigenetic chromatin modifications and thereby aggravate the epileptogenic condition. Adenosine, an endogenous anticonvulsant, has been implicated in epileptogenesis by its ability to modulate DNA methylation in the brain. In epileptic conditions adenosine levels are decreased, which impairs its proper anticonvulsant action by controlling both synaptic inhibitory tonus and epileptogenesis process by promoting DNA hypomethylation. Recently, it was shown that adenosine is decreased in the hippocampus and cortex of Mecp2 knockout mouse model hypothesizing that epileptic condition in RTT could have a contribution of adenosine deregulation. With the intention of exploring a possible relationship between epileptic condition severity associated with each RTT phenotype and DNA methylation changes in central nervous system and at periphery (blood), we set ourselves to optimize the methodology needed to observe changes on DNA methylation in two different phenotypes (mild – symptomatic heterozygous (HET) females and severe – knockout (KO) symptomatic males) of the Mecp2 KO mouse model (B6.129P2 (C)-Mecp2tm1.1Bird/J). With this in mind, different DNA purifications methods were tested, using two different lyses buffers, proceeded by a dot blot technique using the the N6-methyladenosine antibody (6ma). In the end, we can confirm that detection was possible by the use of a dot blot technique for the different samples and concentrations. When comparing the buffers we’ve reached an initial conclusion that TDB, opposed to RIPA, offers better results when it comes to the achieved DNA concentration and the microvolume spectrophotometry ratios, indicators of purity.
RTT is a genetic disorder caused, in 90% of cases, by a genetic mutation in the MECP2 gene, located on the X chromosome, which encodes the protein of the same name. MECP2 has innumerous roles, namely being an important reader of DNA methylation. It is known that DNA methylation is altered in epilepsy; the methylation hypothesis of epileptogenesis suggests that seizures by themselves can induce epigenetic chromatin modifications and thereby aggravate the epileptogenic condition. Adenosine, an endogenous anticonvulsant, has been implicated in epileptogenesis by its ability to modulate DNA methylation in the brain. In epileptic conditions adenosine levels are decreased, which impairs its proper anticonvulsant action by controlling both synaptic inhibitory tonus and epileptogenesis process by promoting DNA hypomethylation. Recently, it was shown that adenosine is decreased in the hippocampus and cortex of Mecp2 knockout mouse model hypothesizing that epileptic condition in RTT could have a contribution of adenosine deregulation. With the intention of exploring a possible relationship between epileptic condition severity associated with each RTT phenotype and DNA methylation changes in central nervous system and at periphery (blood), we set ourselves to optimize the methodology needed to observe changes on DNA methylation in two different phenotypes (mild – symptomatic heterozygous (HET) females and severe – knockout (KO) symptomatic males) of the Mecp2 KO mouse model (B6.129P2 (C)-Mecp2tm1.1Bird/J). With this in mind, different DNA purifications methods were tested, using two different lyses buffers, proceeded by a dot blot technique using the the N6-methyladenosine antibody (6ma). In the end, we can confirm that detection was possible by the use of a dot blot technique for the different samples and concentrations. When comparing the buffers we’ve reached an initial conclusion that TDB, opposed to RIPA, offers better results when it comes to the achieved DNA concentration and the microvolume spectrophotometry ratios, indicators of purity.
Descrição
Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2021
Palavras-chave
Síndrome de Rett (RTT) Gene MECP2 Metilação de ADN Adenosina