Zinc cluster proteins form a family of fungal-specific transcriptional regulators characterized by having a well-conserved zinc-cluster motif involved in DNA recognition and binding. These factors are involved in transcriptional control of genes in a wide variety of cellular processes such as gluconeogenesis, amino acid biosynthesis, multi-drug resistance, and gluconeogenesis. Saccharomyces cerevisiae adapts to depletion of glucose through various mechanisms including massive reprogramming of gene expression. Ethanol regulator of translation (Ert1) is a newly characterized zinc cluster protein found in S. cerevisiae that shows strong similarity to AcuK, a zinc cluster protein involved in regulating the transcription of gluconeogenic genes in the filamentous fungi Aspergillus nidulans. In an effort to understand the functionality of Ert1 and its potential involvement in transcriptional regulation of gluconeogenic genes under non-fermentable carbon condition, ChIP-chip analysis was performed for cells grown in ethanol. Results suggest that Ert1 binds to the promoter of the gluconeogenic gene PCK1 and promote its expression in ethanol medium. Primer extension analysis suggests that Ert1 is involved in promoting the active expression of PCK1 under glucose depressed conditions. Besides binding to gluconeogenic genes under ethanol condition, Ert1 also binds to genes involved in mitochondrial function, translation and transcription by RNA polymerase III. From the ChIP-chip results obtained, Ert1 appears to link glucose exhaustion to modulation of translation and mitochondrial function.S. cerevisiae has the ability to use a variety of nitrogen-containing compounds as nitrogen source. Non-preferred nitrogen sources such as GABA can induce the de-repression of genes involved in the utilization of these compounds. The zinc cluster protein Uga3 was implicated in regulating expression of genes involved in γ-aminobutyrate (GABA) catabolism. Studies from our lab have mapped a regulatory and activation region of this factor and demonstrated that activation of Uga3 is independent of GABA catabolism. In this study, to investigate the potential interaction of Uga3 with cofactors Dal81 and Gal11 in vivo, chromatin immunoprecipitation was performed (ChIP). Analysis revealed that Uga3 is constitutively bound to target promoter and allows recruitment of cofactor Dal81 and Gal11 in the presence of GABA. Results suggested that Dal81 may enhance the transcriptional activity of Uga3, recruit Gal11 to promoter and these factors may act in concert by targeting common components of the transcriptional machinery. Investigation of these fungal-specific zinc cluster proteins will pave the way to understanding the role of homologous transcriptional regulators present in the human pathogen Candida albicans and their roles in mediating drug resistance. / Les protéines à grappe de zinc forment une famille de protéines régulatrices fongiques qui sont caractérisées par un domaine à doigt de zinc binucléaire impliqué dans la reconnaissance et la fixation à l'ADN. Ces régulateurs transcriptionnels sont responsables de la régulation de l'expression de gènes impliqués dans une grande variété de processus cellulaires, comme le métabolisme primaire et secondaire, la biosynthèse d'acides aminés, la gluconéogenèse, et la multi-résistance aux médicaments. Une protéine de cette famille chez Saccharomyces cerevisiae, Ert1, dont l'homologue chez Aspergillus nidulans participe à la régulation de la gluconéogenèse, a été analysé par la technique ChIP-chip. Les résultats démontrent que Ert1 se fixe au promoteur du gène gluconéogenique PCK1 et augmente son expression en présence d'éthanol. Donc, un autre régulateur de la gluconéogénèse a été identifié chez Saccharomyces cerevisiae. Une autre protéine à grappe de zinc, Uga3, est impliquée dans la régulation de l'expression des gènes qui participent dans le catabolisme de l'acide γ-aminobutyrique (GABA). Nous avons identifié une région régulatrice et une région activatrice pour ce facteur et nous avons démontré que l'activation de Uga3 est indépendante du métabolisme du GABA. Une analyse par immunoprécipitation de la chromatine (ChIP) a démontré que Uga3 est fixé de façon constitutive au promoteur d'un gène cible et permet le recrutement du co-facteur Dal81 et de Gal11, un composant du complexe médiateur.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.110428 |
| Date | January 2012 |
| Creators | Liang, Xiao Bei |
| Contributors | Bernard Turcotte (Internal/Supervisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | French |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Microbiology & Immunology) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | Electronically-submitted theses. |
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