Abstract : Rim101p is a conserved fungal transcription factor that becomes activated through C-terminal cleavage under neutral to alkaline conditions. The identification and analysis of Rim101p targets in Candida albicans was the main subject of the PhD thesis.
A constitutively active truncated version of Rim101p (Rim101SLp) was introduced under the control of the MET3-promotor into a rim101 null mutant to monitor Rim101-dependent transcriptional changes independently of other pH-dependent regulatory events. Transcriptional changes were recorded using microarrays along a time course following induction of RIM101SL transcription.
After filtering the data, the transcriptional patterns of 133 selected genes was clustered into five distinct classes. Significantly more putative Rim101p binding sites were detected in the promoters of these genes than in a randomly chosen set of genes. Further analysis permitted to identify a putative extended Rim101p binding motif. Putative Rim101p targets were examined for predicted functions and amino acid landmarks like transmembrane domains and signal peptides that could indicate localization at the cell surface and thus a possible involvement in host interaction.
Microarray results were confirmed on 20 selected genes by quantitative real-time PCR. Furthermore, the relevance of the microarray data for the pH response of C. albicans was assessed by monitoring transcriptional changes of these genes in a wild type strain grown at pH 4 or pH 8. In spite of these experimental setup differences, a clear correlation of the results was observed for a large majority of the tested genes.
Microarray data suggested that Rim101p activity had a strong impact on the expression of genes of the ALS (Agglutinin-Like Sequence) gene family. The extremely high sequence conservation within this family hampered however a gene-specific analysis. Using a gene-specific primer set, transcription of each member of the ALS gene family was analyzed by real-time qPCR. Four ALS genes were shown to be transcribed in a pH-dependent manner, and Rim101p was found to be required for the alkaline induction of ALS1 and the repression of ALS4. The two other genes, ALS2 and ALS9, were also repressed at alkaline pH, but their regulation was at least partially independent of Rim101p.
Finally, the mechanism of ALS1 and ALS4 regulation was addressed by two different approaches. First, reporter strains that put a modified bacterial Β-galactosidase gene under the control of ALS promoters were constructed in order to monitor more easily pH and Rim101p effects on ALS1 and ALS4 expression. Second, a tagged version of Rim101p was used to demonstrate in vivo binding of Rim101p to ALS promoters by Chromatin Immunoprecipitation (ChIP): however, no clear specific binding could be observed.