Abstract
Background Epigenetics of human cancer is an area of emerging research due to a growing understanding of specific epigenetic pathways and rapid development of detection technologies. Aberrant promoter hypermethylation is a prevalent phenomenon in human cancers. The best known epigenetic change in cancer is tumor suppressor genes inactivation by DNA hypermethylation due to the increased activity or deregulation of DNA methyl transferases. Increasing evidence also reveals that viral genes are one of the key players in regulating DNA methylation.
Worldwide, it has been estimated that viral infection is linked to about 15% of cancer cases. Since only a minority of human tissues affected with these viruses inevitable progress to cancer, this association is very intriguing. Thus, the molecular reasons why the infection is controlled in some cases or instead progresses to subsequent stages of tumorigenesis are largely unknown. Methylation of the viral genome appears to play a crucial role in many aspects including the regulation of viral gene expression, escape of infected cells from immune surveillance and the resistance of infected tumor cells to antiviral compounds. For the first time, the complete DNA methylomes in the mayor virus associated with human cancer -Papillomavirus and Epstein Barr- are described in this thesis. These findings might provide important clues for a better understanding of the puzzling association between cancer and viral infections.
Methods and Findings Using bisulphite genomic sequencing of multiple clones, which allow performing a systematic DNA hypermethylation analysis, we have obtained the DNA methylation status of every CpG dinucleotide present in the genome of the Human Papilloma Viruses 16 and 18, and in all the transcription start sites of the Epstein Barr virus. The samples analyzed here were associated with infectious diseases (such as infectious mononucleosis) and human tumours (cervical, nasopharyngeal, and lymphoma).
We have also analyzed the active changes in the viral DNA methylome and their functional relevance in the natural history of the disease.
The DNA methylomes presented here provide evidence of the dynamic nature of the epigenome in the evolution of tumorigenesis.
Conclusions The results obtained in this thesis indicate that the DNA methylome of the above mentioned viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and premalignant lesions, to the full-blown invasive tumour. The observed DNA methylation changes have a major functional impact in the biological behaviour of the viruses.
These data also provide a proof of principle for the importance of sequencing complete DNA methylomes of other organisms involved in human diseases, and of human healthy cells as well.
The study of epigenetic changes should provide a global view of gene profile in cancer, and epigenetic markers could be used for early detection, prognosis, and therapy of cancer.
Esta introducción se divide en tres partes: en la primera se revisan los aspectos generales de la epigenética, en la segunda se profundiza en la relación de la epigenética con el cáncer, y en la tercera se introduce a los virus asociados a cáncer objeto de estudio en esta tesis.
1.1. Primera Parte: Epigenética
1.1.1. El nucleosoma El ADN genómico de las células eucariotas se condensa y organiza en una estructura denominada cromatina en la cual interacciona con ciertas proteínas, entre las que se destacan las histonas. El nucleosoma, descrito originalmente por Kornberg en 1974, constituye la unidad fundamental y esencial de esta cromatina y está constituido por un núcleo proteico formado por un octámero de histonas. Este octámero incluye dos copias de la histona tipo H2A, dos de la H2B, dos de la H3 y dos de la H4, dando lugar a lo que se conoce como "core" alrededor del cual se dispone el ADN (FIG. 1).
FIG. 1 A: Representación esquemática del ensamblaje de las histonas nucleares en el nucleosoma. B: Estructura del nucleosoma a 2,7 Å de resolución.
Entre dos nucleosomas consecutivos existe un fragmento de ADN asociado a la histona H1 (linker) que conecta cada partícula del core con la adyacente. Cada octámero de histonas está rodeado por 1,7 vueltas de ADN bicatenario. La doble hélice de ADN enrollada sobre los sucesivos octámeros de histonas junto con el ADN linker, da un aspecto de "collar de perlas" cuando se observan bajo microscopio electrónico (FIG. 2)
FIG. 2. Compactación del DNA. Cada molécula de DNA se empaqueta para formar la fibra de cromatina y posteriormente un cromosoma mitótico condensado que es 10.000 veces más corto que su longitud original.
1.1.2. Epigenética La epigenética puede ser definida como el estudio de los cambios heredables en la expresión de los genes que no van a acompañados por alteraciones en la secuencia de ADN y todos los mecanismos implicados en la regulación de las funciones de la cromatina. (Wolffe y Matke, 1999).
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