Connecting transcriptional dynamics with stress response: Insights into the yeast factors MOG1 and AIM4

• Autores: Joan Serrano Quílez
• Directores de la Tesis: Susana Rodríguez Navarro (dir. tes.)
• Lectura: En la Universitat de València ( España ) en 2024
• Idioma: español
• Tribunal Calificador de la Tesis: Francisco Navarro Gomez (presid.), Mercè Gomar Alba (secret.), Hannah Elisabeth Mischo (voc.)
• Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
• Materias:
  • Ciencias económicas
    • Economía sectorial
      • Sanidad
• Enlaces
  • Tesis en acceso abierto en: TESEO
• Resumen

  • Post-translational histone modifications are essential for chromatin dynamics. Ubiquitination of H2B at lysine 123 and H3 trimethylation at K4 are particularly important for cellular processes such as transcription regulation and DNA repair. In Saccharomyces cerevisiae, H2B is ubiquitinated by Rad6/Bre1/Lge1 complex with the participation of other protein assemblies as PAF1c. In contrast, H2Bub is primarily removed by the enzyme Ubp8 from the SAGA complex. H3K4me3 is deposited by the COMPASS complex and eliminated by the demethylase Jhd2. Although Mog1 is a yeast factor that participates in protein import by binding to the small GTPase Ran, our previous research found that yeast cells lacking MOG1 had lower levels of these two modifications and a reduced rate of transcription.

    In chapter 1, this thesis presents evidence supporting Mog1's role in histone modification and the regulation of transcription elongation. We confirmed that Mog1 genetically and physically interacts with components of the H2Bub and H3K4me3 machinery and influences their recruitment to chromatin. Our results also show that Mog1's impact on methylation is limited to H3K4me3, while H3K4me2, H3K36me3, and H3K79me3 remained unaffected. We observed that mog1 cells are sensitive to hydroxyurea, which triggers replicative stress, and deletions of COMPASS suppress the growth defect phenotype of mog1 under various stress conditions. Moreover, elimination of demethylase JHD2 recovered the decreased levels of H3K4me3 in the mog1 mutants.

    We also report that Mog1 affects the recruitment of RNAPolII to chromatin and that mog1 cells display decreased levels of serine 2 phosphorylation in the RNAPolII CTD and sensitivity to 6-azauracil, indicative of transcriptional elongation impairment. Thus, we confirmed that the loss of MOG1 leads to decreased phosphorylation and recruitment to chromatin of Spt5, a crucial transcription elongation factor. The interaction between Mog1 and transcription factors was confirmed using TAP purification assays. Using in situ hybridisation analysis, we observed that Mog1 is necessary in coordination with the H2Bub machinery to facilitate the export of mRNA upon heat stress.

    We also assessed the independence of these Mog1 functions from its Ran-binding activity, confirming that the deletion of MOG1 does not affect the nuclear import of factors related to H2Bub and H3K4me3. Furthermore, we created a MOG1-E65K mutant, previously reported to impair binding to Ran, but found that it still interacts with factors involved in protein import.

    In chapter 2, we aimed to study the transcriptional response of aim4 mutants to osmotic stress since we found marked altered stress responses in this mutant in previous works. Bioinformatic analysis of the gene expression pattern of aim4 revealed a transcriptional signature very similar to that of WT under osmotic stress.