Characterization and modeling of a specific transcriptional regulatory network required for multidrug resistance in yeast.

Principal Investigator: Isabel Sá-Correia

Contract: PTDC/BIO/72063/2006 - Biochemistry Engineering and Biotechnology

Start date: 01/01/2008

Duration: 36 months

 

The objective of this project is to unveil the hierarchy and synergy that lies behind the combined action of the transcription factors (TF) involved in yeast response to drugs and other chemical aggressions and to develop computational tools for modeling the dynamic of these transcription regulatory networks. The transcription level from a gene results from the balance of the actions of various transcriptional regulators, either promoting or repressing its transcription. However, how transcriptional regulatory networks overlap and cross-talk to influence transcription, under optimal growth conditions or under environmental stress, and how the promoter context affects the binding
and action of each transcription factor is scarcely understood. The regulatory network that mediates the activation of the drug: H+antiporter encoding gene FLR1 in the presence of the fungicide mancozeb, widely used in Agriculture, is chosen as the first and main experimental platform of this project. This option is due to the theoretical and practical importance of this regulatory response and to the previous experience of the IST team with this particular system. Specifically, we want to clarify the hypothesized combinatorial action of the TFs Yap1, Pdr3 and Yrr1, on FLR1 activation and the possible role, in yeast stressed cells, of other selected TFs that have potential binding sites in FLR1 promoter region or that regulate TFs of this network. Gene transcript levels will be compared in wild-type and derived mutant strains, in which the different transcription factors encoding genes were individually deleted, or in double or multiple mutants. The time course of the dynamic transcriptional response to mancozeb in the different mutant strains will be analysed. Chromatine immunoprecipitation (IP) and real time-PCR will be used to determine the in vivo occupancy of gene promoter sequence by the TFs in response to the drug. During this research project, we will develop computational methods and tools for the modeling of the behavior of the FLR1 regulatory network. Based on the models obtained, the regulation of other multidrug resistance (MDR) transporters (e.g. those encoded by SNQ2, PDR5 and YOR1, of the ABC Superfamily, or by TPO1, also belonging to the major facilitator superfamily-MFS, like Flr1p) or in the regulation of FLR1 in the presence of different chemical stress agents (e.g. fluconazole, a clinical fungicide) will be predicted and these predictions experimentally tested. This approach will help to improve, extend and validate the established models. In the long term, this study will provide useful indications that may lead to the delineation of strategies to control fungicide resistance with impact in Biotechnology, Agriculture and Health. It is also expected that results coming out of this project focused on the eukaryotic experimental model S. cerevisiae will be a step forward in our efforts to understand and model the complex biological networks that control life processes.