Coordinación de los mecanismos reguladores que median en Aspergillus nidulans la respuesta a la alcalinización extracelular
[EN] Changes in the transcriptional and translational patterns of an organism are the result of a response to changes in the intra- or extracellular environment, allowing the organism to adapt and survive. The transcriptional pattern is dynamic and subject to the activity of regulatory elements such...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/353506 |
| Acceso en línea: | http://hdl.handle.net/10261/353506 |
| Access Level: | acceso abierto |
| Palabra clave: | Regulación transcripcional Factor de transcripción Estrés ambiental Transcriptómica Genética comparativa Modificacion postraduccional http://metadata.un.org/sdg/3 Ensure healthy lives and promote well-being for all at all ages |
| Sumario: | [EN] Changes in the transcriptional and translational patterns of an organism are the result of a response to changes in the intra- or extracellular environment, allowing the organism to adapt and survive. The transcriptional pattern is dynamic and subject to the activity of regulatory elements such as transcription factors (TFs). The main characteristic of TFs is their DNA-binding domains, which recognize target sequences in the genes under their domain, driving their expression in a positively or negatively manner (Lacthman 2003, Etxebeste and Espeso 2019). In addition, the function of some TFs is only activated under certain environmental conditions that trigger signaling pathways resulting in post-translational modifications of the primary forms of the TFs. Among the many transcriptional regulatory processes that can be studied, environmental stress is a powerful inducer of these pathways, leading to coordination between different transcriptional regulatory systems mediated by TFs forming gene regulatory networks (GRNs) (Etxebeste 2021, Brown et al., 2017). One of the most numerous and diverse groups of the Fungi kingdom is the Ascomycete phylum due to their ability to adapt to all types of environments, some of which are extreme. The filamentous fungus Aspergillus nidulans is a haploid model organism used to study these transcriptional regulatory networks because of its ease of handling in the laboratory, which allows the use of classical and reverse genetics techniques (Mellado 2014). Similarly to the rest of the fungi of the phylum, they are very versatile in a wide variety of environments and tolerating environmental conditions such as water and osmotic stresses, alkaline environments and UV radiation. The main objective of this work was to study the transcriptional changes in A. nidulans under environmental stress conditions caused by high sodium concentrations or alkaline environmental pH. And how the TF-mediated systems involved in this response coordinate with each other. In A. nidulans the response to alkaline pH is mediated by at least three systems whose TFs contain a DNA-binding domain of the C2H2 zinc finger type: PacC, SltA and CrzA (Tilburn et al., 1995, Spielvogel et al., 2008). These TFs carry out their function as transcriptional regulators under conditions of alkalinity and/or high cation stress, but so far it has not been determined whether these systems are interconnected or whether there is a hierarchy among them. The Slt system is the least known of the three TFs and of which only two elements have been described: the transcription factor SltA and its processing protease SltB (O’neil et al., 2002, Mellado et al 2015). This system, unlike PacC and CrzA, is found only in fungi of the Pezizomycotina subphylum, where it is essential for fungal development in cationic or alkaline stress environments. Both the transcriptional response to environmental stress and the regulatory role of SltA, were studied by massive RNA sequencing. The different transcriptional patterns in a wild-type strain and in a mutant strain lacking the sltA gene (sltAΔ) were determined by analyzing the differences between standard and stress conditions induced by 1M sodium cations and alkaline pH (Picazo et al., 2020). This technique allowed us to identify clusters of genes with different degrees of dependence on SltA function, either through direct or indirect regulation. Among these genes we found clusters with an expression profile similar to that of sltB gene, whose expression is fully dependent on SltA function. These selected genes were studied using reverse genetics techniques, as well as phenotypic and microscopy assays to determine whether any of them were part of the Slt system. Among the genes whose expression was affected by the sltAΔ genetic background, a large number of genes encoding TFs of different families were found, including PacC. This transcription factor has a 72kDa primary form that is signaled by the Pal pathway when the environmental pH is alkalinized, triggering proteolysis to a 53kDa intermediate form that is processed independently of pH in the proteasome to its active 27kDa form. Processing of the primary form of PacC72kDa allows pacC gene expression levels to rise at alkaline pH (Peñalva et al., 2014, Bussink et al., 2015). The absence of SltA affects this system both at the transcriptional level by preventing the upregulation of pacC expression levels at alkaline pH and at the translational level showing an anomalous PacC processing pattern. A strategy using different mutants in elements of the Pal/PacC and Slt systems allowed us to delimit those steps in the PacC-signaling pathway where the role of the SltA protein appears to be determinant. The absence of PacX function, the negative regulator of pacC, (Bussink et al., 2015), did not rescue the anomalous PacC processing pattern or the alkaline pH tolerance phenotype in the null sltA background. Also, the study of mutants in the Pal pathway evidenced that despite deficient PalF signaling and post-translational modification, Pal pathway elements were correctly recruited upstream of the PacC72kDa processing step in the sltAΔ background. Mutations in pacC generating intermediate-like and fully processed forms displayed a correct PacC processing but did not suppressed the alkaline pH tolerance phenotype observed in the sltAΔ background. These results evidenced the coordination between the Slt and Pal/PacC systems in the alkaline pH response, where the function of SltA is epistatic over the Pal/PacC alkaline pH response system. |
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