Browsing by Author "Munoz-Guzman, Felipe"

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    A global search for novel transcription factors impacting the Neurospora crassa circadian clock
    (2021) Munoz-Guzman, Felipe; Caballero, Valeria; Larrondo, Luis F.
    Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors (TFs) knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and -2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 TFs that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated TFs, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.
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    A global search for novel transcription factors impacting the Neurospora crassa circadian clock
    (2021) Munoz-Guzman, Felipe; Caballero, Valeria; Larrondo, Luis F.
    In order to identify new players modulating the circadian clock in the model Neurospora crassa, we adopted a reverse genetics strategy. Thus, we focused on transcription factors knockouts and crossed them to strains containing circadian luciferase reporters. Our screen covered close to 60% de the 302 genes encoding for such proteins in Neurospora, identifying that 23 of them appear to modulate period, while none of the tested ones (besides the classic core-clock components) are essential for daily rhythms.
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    Developing a Temperature-Inducible Transcriptional Rheostat in Neurospora crassa
    (2023) Tabilo-Agurto, Cyndi; Del Rio-Pinilla, Veronica; Eltit-Villarroel, Valeria; Goity, Alejandra; Munoz-Guzman, Felipe; Larrondo, Luis F.
    Heat shock protein (HSP)-encoding genes (hsp), part of the highly conserved heat shock response (HSR), are known to be induced by thermal stress in several organisms. In Neurospora crassa, three hsp genes, hsp30, hsp70, and hsp80, have been characterized; however, the role of defined cis elements in their responses to discrete changes in temperature remains largely unexplored. To fill this gap, while also aiming to obtain a reliable fungal heat shock-inducible system, we analyzed different sections of each hsp promoter by assessing the expression of real-time transcriptional reporters. Whereas all three promoters and their resected versions were acutely induced by high temperatures, only hsp30 displayed a broad range of expression and high tunability, amply exceeding other inducible promoter systems existing in Neurospora, such as quinic acid- or light-inducible ones. As proof of concept, we employed one of these promoters to control the expression of clr-2, which encodes the master regulator of Neurospora cellulolytic capabilities. The resulting strain fails to grow on cellulose at 25 degrees C, whereas it grows robustly if heat shock pulses are delivered daily. Additionally, we designed two hsp30 synthetic promoters and characterized them, as well as the native promoters, using a gradient of high temperatures, yielding a wide range of responses to thermal stimuli. Thus, Neurospora hsp30-based promoters represent a new set of modular elements that can be used as transcriptional rheostats to adjust the expression of a gene of interest or for the implementation of regulated circuitries for synthetic biology and biotechnological strategies.
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    Domestication signatures in the non-conventional yeast Lachancea cidri
    (2024) Villarreal, Pablo; O'Donnell, Samuel; Agier, Nicolas; Munoz-Guzman, Felipe; Benavides-Parra, Jose; Urbina, Kami; Pena, Tomas A.; Solomon, Mark; Nespolo, Roberto F.; Fischer, Gilles; Varela, Cristian; Cubillos, Francisco A.
    Evaluating domestication signatures beyond model organisms is essential for a thorough understanding of the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation (similar to 16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role in the strain's phenotypic variation. The set of SVs identified directly impacts dozens of genes and likely underpins the greater fermentation performance in the L. cidri CBS2950. In addition, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to a human-related environment and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species.