Project description:The regulation of flocculation, surface adhesion and invasive growth in the fission yeast Schizosaccharomyces pombe has focused primarily at the transcriptional level, but little is known with regards to posttranscriptional control. Here, we identified the Pumilio protein Pfr1 as a novel posttranscriptional regulator of these processes. Deletion of pfr1+ prevented flocculation, surface adhesion and invasive growth under inducing conditions, while overexpression of pfr1+ was sufficient to trigger flocculation. The flocculent phenotype of pfr1+ overexpression was dependent on the presence of the Gsf2 flocculin, but not on the Mbx2, Cbf12 and Adn3 transcription factors. In addition, we used RNA immunoprecipitation and expression microarrays to identify pvg1+ and SPBPB7E8.01, which encode a galactose pyruvyltransferase and glycophosphatidylinositol membrane protein, respectively, as putative mRNA targets potentially degraded by Pfr1. The mRNAs of these genes were upregulated and downregulated in the pfr1 deletion and overexpression strains, respectively, and contained putative binding sites in the 3â-untranslated region. We also discovered that ccr4+ and ste13+, which encode components of the mRNA decay machinery, were required for these processes, but did not suppress the pfr1+ overexpression flocculent phenotype when deleted. This data suggest that these processes in S. pombe involve multiple posttranscriptional-regulatory pathways of which one requires Pfr1. We generated 2 overexpression microarrays with dye swap that were biological replicates, 2 deletion microarrys with dye swap. Mutants samples were compared to empty vector control or wild type. 1 RIP-chip array was generated with IP rna compared to total RNA from the sample.
Project description:The regulation of flocculation, surface adhesion and invasive growth in the fission yeast Schizosaccharomyces pombe has focused primarily at the transcriptional level, but little is known with regards to posttranscriptional control. Here, we identified the Pumilio protein Pfr1 as a novel posttranscriptional regulator of these processes. Deletion of pfr1+ prevented flocculation, surface adhesion and invasive growth under inducing conditions, while overexpression of pfr1+ was sufficient to trigger flocculation. The flocculent phenotype of pfr1+ overexpression was dependent on the presence of the Gsf2 flocculin, but not on the Mbx2, Cbf12 and Adn3 transcription factors. In addition, we used RNA immunoprecipitation and expression microarrays to identify pvg1+ and SPBPB7E8.01, which encode a galactose pyruvyltransferase and glycophosphatidylinositol membrane protein, respectively, as putative mRNA targets potentially degraded by Pfr1. The mRNAs of these genes were upregulated and downregulated in the pfr1 deletion and overexpression strains, respectively, and contained putative binding sites in the 3’-untranslated region. We also discovered that ccr4+ and ste13+, which encode components of the mRNA decay machinery, were required for these processes, but did not suppress the pfr1+ overexpression flocculent phenotype when deleted. This data suggest that these processes in S. pombe involve multiple posttranscriptional-regulatory pathways of which one requires Pfr1.
Project description:In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP-chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11 and Cbf12 regulators. Overexpression of mbx2+ and deletion of rfl1+ resulted in strong flocculation and transcriptional upregulation of gsf2+/pfl1+ and several other putative flocculin genes (pfl2+-pfl9+). Overexpression of the pfl+ genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl+ overexpression. Among the pfl1+ genes, only loss of gsf2+ abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2+ or adn3+ overexpression was likely mediated by the transcriptional activation of cell wall-remodeling genes including gas2+, psu1+, and SPAC4H3.03c. We also discovered that Rfl1, Mbx2, and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2+ expression in an inhibitory feed-forward loop involving mbx2+. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall-remodeling enzymes. Moreover, comparisons between the flocculation transcriptional regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences. We generated 2 overexpression microarrays and 1 deletion microarray with dye swaps, 2 overexpression single replicate microarray experiments, 3 deletion single replicate microarray experiments, and 3 single replicate ChIP-chip experiments. The effect of the mutant strains were all compared to wild type or empty vector strains.
Project description:In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP-chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11 and Cbf12 regulators. Overexpression of mbx2+ and deletion of rfl1+ resulted in strong flocculation and transcriptional upregulation of gsf2+/pfl1+ and several other putative flocculin genes (pfl2+-pfl9+). Overexpression of the pfl+ genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl+ overexpression. Among the pfl1+ genes, only loss of gsf2+ abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2+ or adn3+ overexpression was likely mediated by the transcriptional activation of cell wall-remodeling genes including gas2+, psu1+, and SPAC4H3.03c. We also discovered that Rfl1, Mbx2, and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2+ expression in an inhibitory feed-forward loop involving mbx2+. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall-remodeling enzymes. Moreover, comparisons between the flocculation transcriptional regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.