Definição do perfil transcricional da sequência de DNA satélite major de Rattus norvegicus num modelo celular tumoral
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2014
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As sequências de DNA satélite são sequências repetidas em tandem, presentes em regiões heterocromáticas do genoma como os centrómeros ou os telómeros. Devido à sua localização em regiões de elevada condensação da cromatina, eram tidas como sequências não transcritas, e foram, como tal, consideradas dispensáveis no contexto genómico e celular, sendo adereçadas como “junk” genómico. Contudo, diversas evidências da sua expressão foram recentemente relatadas, tendo-lhes sido atribuídas funcionalidades no contexto celular, algumas das quais associadas com o desenvolvimento e/ou progressão de cancro.
A sequência de DNA satélite em foco neste trabalho é a sequência sat I de Rattus norvegicus, e é até ao momento a única sequência deste tipo identificada na referida espécie.
Os nossos grandes objetivos foram a avaliação do perfil transcricional, dos mecanismos de regulação e da função da sequência DNA satélite “major” centromérica sat I num modelo celular tumoral de Rattus norvegicus.
No presente trabalho, foi feita a análise comparativa da sequência de DNA satélite I de Rattus norvegicus na linha tumoral (CLS-ACI-1) desta espécie e numa linha normal (RNO).
Com este trabalho foi conseguida a determinação do perfil transcricional de sat I no modelo em estudo. Verificou-se a ocorrência de expressão da sequência satélite sat I de R. norvegicus em ambos os genomas analisados, embora apresentem níveis diferentes de expressão. A expressão desta sequência parece ser regulada através de alterações epigenéticas, nomeadamente a metilação do DNA. Contudo, este mecanismo epigenético aparenta ser um facilitador, mas não o mecanismo de maior importância na regulação da expressão desta sequência especificamente. Como tal, são de apontar as modificações histónicas também como um possível regulador das mesmas. Os transcritos observados in situ apresentam sinal em “cluster”, com natureza madura e co-localização com a região centromérica. A expressão de sat I não aparenta dependência de ciclo e aponta-se uma possível função na manutenção da heterocromatina. Na análise comparativa do nível de expressão na linha normal e tumoral, foi verificada a diminuição desta em cancro, explicada pelo aumento da percentagem de metilação da sequência de DNA satélite I. A expressão diferencial relatada leva-nos a supor uma possível função de supressor tumoral para sat I (como TERRA, sequência repetitiva telomérica, cuja expressão aparenta estar reduzida em cancro devido a suposta função reguladora negativa da telomerase) e/ou possível dependência do tipo de tecido para a sua transcrição. Pelo ensaio de stresse de “choque térmico”, verifica-se a não dependência do fator HSF 1 para a expressão de sat I, mas sim um mecanismo independente deste fator como a alteração epigenética. Desta forma, este trabalho levou-nos à conclusão que as sequências de DNA satélite apresentam uma regulação altamente controlada e complexa, em que sua compreensão inclui o conhecimento aprofundado e amplo de diversos mecanismos e vias de regulação transcricional.
Satellite DNAs are tandemly repeated sequences that are present in heterochromatic genome regions like the centromeres and telomeres. Resulting from their association with high condensed chromatin regions they were thought as non-transcribed and dispensable sequences in the genomic and cellular context. They were initially called genomic junk, however since that time much evidence has demonstrated their active transcription. To these transcripts were attributed some cellular functionalities, such us the development of cancer. The satellite DNA sequence in focus on this work is the sequence sat I of Rattus norvegicus and it is so far the only sequence of this type identified in this species. Our main goals were to determine the transcriptional profile, regulation mechanism and function of the centromeric major satellite DNA sequence sat I in a cancer cell line model of Rattus norvegicus, having as counterpart the normal genome. In the present work it was made the comparative analysis of the satellite DNA sequence sat I in the normal cell line RNO and tumor cell line CLS-ACI-1 of rat. With this work it was defined the transcriptional profile of sat I in the tumor cell model CLS-ACI-1. We verified the occurrence of expression of sat I in both genomes analyzed, although displaying differences in the expression levels exhibited. The expression of this sequence seems to be regulated by DNA methylation. This epigenetic mechanism appears to be the facilitator, but not the main epigenetic alteration behind the regulation of sat I. Therefore we can hypothesize the histonic modification as one possible regulator of this sequence. The transcripts observed in situ were identified as clusters, mature transcripts, with co-localization with the centromeric region. Sat I expression does not appear to be cycle dependent and we indicate a possible function for this sequence in the maintenance of heterochromatin. On the comparative expression analysis between normal and tumor cell line, it was verified a decrease of transcripts in cancer. This variation is explained by the increase of the methylation status in sat I. The differential expression leads to a possible tumor suppression function for sat I (as TERRA, a telomeric repeat-containing RNA whose expression appears decreased in cancer due to its supposed negative regulation of telomerase) and possible tissue dependent transcription for this satellite sequence. For the heat shock assay, it was verified the non-transcription dependence of HSF1, but a mechanism independent of this factor such as epigenomic mechanisms regulating sat I expression.As such, this work leads us to the conclusion that satellite expression presents a tight and complex regulation. The understanding of satellite DNA expression passes through a profound and ample knowledge of several mechanisms and pathways of transcriptional regulation.
Satellite DNAs are tandemly repeated sequences that are present in heterochromatic genome regions like the centromeres and telomeres. Resulting from their association with high condensed chromatin regions they were thought as non-transcribed and dispensable sequences in the genomic and cellular context. They were initially called genomic junk, however since that time much evidence has demonstrated their active transcription. To these transcripts were attributed some cellular functionalities, such us the development of cancer. The satellite DNA sequence in focus on this work is the sequence sat I of Rattus norvegicus and it is so far the only sequence of this type identified in this species. Our main goals were to determine the transcriptional profile, regulation mechanism and function of the centromeric major satellite DNA sequence sat I in a cancer cell line model of Rattus norvegicus, having as counterpart the normal genome. In the present work it was made the comparative analysis of the satellite DNA sequence sat I in the normal cell line RNO and tumor cell line CLS-ACI-1 of rat. With this work it was defined the transcriptional profile of sat I in the tumor cell model CLS-ACI-1. We verified the occurrence of expression of sat I in both genomes analyzed, although displaying differences in the expression levels exhibited. The expression of this sequence seems to be regulated by DNA methylation. This epigenetic mechanism appears to be the facilitator, but not the main epigenetic alteration behind the regulation of sat I. Therefore we can hypothesize the histonic modification as one possible regulator of this sequence. The transcripts observed in situ were identified as clusters, mature transcripts, with co-localization with the centromeric region. Sat I expression does not appear to be cycle dependent and we indicate a possible function for this sequence in the maintenance of heterochromatin. On the comparative expression analysis between normal and tumor cell line, it was verified a decrease of transcripts in cancer. This variation is explained by the increase of the methylation status in sat I. The differential expression leads to a possible tumor suppression function for sat I (as TERRA, a telomeric repeat-containing RNA whose expression appears decreased in cancer due to its supposed negative regulation of telomerase) and possible tissue dependent transcription for this satellite sequence. For the heat shock assay, it was verified the non-transcription dependence of HSF1, but a mechanism independent of this factor such as epigenomic mechanisms regulating sat I expression.As such, this work leads us to the conclusion that satellite expression presents a tight and complex regulation. The understanding of satellite DNA expression passes through a profound and ample knowledge of several mechanisms and pathways of transcriptional regulation.
Descrição
Dissertação de Mestrado em
Genética Molecular Comparativa e Tecnológica
Palavras-chave
DNA satelite , Transcrição , Metilação de DNA , Neoplasias mamárias animais , Adenocarcinomas , Ratazana , Modelo animal