Host factor Senataxin negatively regulates incoming DNA expression

• Autores: Andoni Gómez Moreno
• Directores de la Tesis: Amelia Nieto Martín (dir. tes.), Urtzi Garaigorta de Dios (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2023
• Idioma: español
• Número de páginas: 172
• Títulos paralelos:
  • El factor celular senataxin regula negativamente la expresión de DNA entrante
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • In an eukaryotic cell, DNA is the molecular template encoding the necessary information to produce all the RNAs and proteins needed for life. The vast majority of cellular DNA is spatially organized in the form of chromosomes in the nucleus of quiescent cells. Viruses are intracellular parasites that hijack many cellular processes to complete their replicative cycle. Some viruses lack the necessary enzymatic activities to produce viral mRNAs and they completely depend on cellular transcriptional machinery to complete their cycles. Therefore, they developed different strategies to ensure that the viral genetic material reaches the cellular transcription machinery. Regardless the nature of the viral genetic material delivered in the nucleus of infected cell, a common feature for some virus infections is the production of covalently closed circular DNAs (cccDNA) that persist as episomes during these viral infections. In fact, hepatitis B virus (HBV) cccDNA, is a hallmark of its persistence in infected patients and approved therapies for the treatment of chronic HBV infection are not curative because they do not completely eliminate the cccDNA from infected hepatocytes. In this thesis, we identified host factor SETX as a restriction factor of episomal DNA expression. The absence of SETX increased: HBV infection, integration deficient lentiviral and retroviral transduction, and adeno-associated viral vector transduction. Moreover, the absence of SETX also increased transfected plasmid DNA expression independently of its promoter and reporter. Since SETX interacts with cellular RNA polymerase II and it is involved in transcription termination, in situ RNA-labeling experiments, using ethyl uridine click technology, were performed to analyze the effect of SETX silencing on cellular transcription. Unlike the effect observed on previous models, SETX silencing did not increase cellular transcription, suggesting a selective effect on episomal DNAs. To demonstrate this hypothesis a GFP-expressing plasmid was transfected in a stably mCherry-expressing cell line and the expression of both reporters was quantitated upon SETX silencing. GFP expression increased 4-fold in contrast to the 1.23-fold increase observed in integrated mCherry expression in SETX-silenced cells. Collectively these results suggest a restrictive role of SETX on incoming DNA expression. In order to elucidate the mechanism by which SETX restricts episomal DNA expression, we studied the effect of the SETX silencing on DNA integration of a retroviral vector. The absence of SETX did not increase integration of retroviral DNA, suggesting that integration of episomal DNA into the host cell genome is not responsible of the observed phenotype. On the other hand, only overexpression of the wild-type SETX, but not a mutant deficient in RNA binding activity, was able to restrict transfected plasmid expression. These results suggest that RNA binding activity of SETX is required for restriction of episomal DNA. The knowledge generated during this thesis might be useful in the future to generate new therapeutic strategies for the development of curative treatments against chronic infections such as HBV, as well as for the improvement of viral gene therapy strategies