Functional Differentiation of tbf1 Orthologues in Fission and Budding Yeasts

Abstract

In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.The phylogenetic pedigree of Pneumocystis species implies they are likely to employ conserved fungus-specific proteins in pathways that are essential for viability. Identification of such modules in the genome of the rat-specific pathogen P. carinii may help to identify novel targets for directed pharmaceutical intervention to treat opportunistic infection of immunocompromised human hosts by P. jirovecii. To identify conserved functional modules, we are assessing the ability of genes from P. carinii to function in the budding yeast Saccharomyces cerevisiae as well as in the fission yeast Schizosaccharomyces pombe, the closest evolutionary relationship for which molecular genetic analysis is well developed (25,36).In the course of this study, we identified a potential homologue of the S. pombe tbf1 (Sptbf1) and S. cerevisiae TBF1 (ScTBF1) genes. Sequence comparisons indicate that both SpTbf1p and ScTbf1p are members of a conserved fungusspecific family (50, 51). All the Tbf1 family members contain a C-terminal "telobox" DNA binding domain (8, 9) but bear additional significant homology throughout their coding sequences. The telobox, a particular variant of the Myb family motif, is also found in the mammalian telomere factors TRF1 and TRF2 as well as in the fission yeast telomeric protein Taz1p (13). The telobox recognizes sequences similar to the mammalian telomeric repeat TTAGGG. High-affinity binding sites for ScTbf1p have also been identified in the STARs (subtelomeric antisilencing regions) of the subtelomeric X and YЈ elements. Several studies have proposed a regulatory role for ScTbf1p at telomeres (2,6,19,33). The ScTBF1 gene is essential, but this is commonly assumed to be due to its function as a global transcriptional regulator that binds to many sites throughout chromatin rather than to direct effects on telomeres (10, 33).SpTbf1p was identified in S. pombe whole-cell extracts as one of several activities that exhibited differential affinity in vitro for tandem copies of the human and S. pombe telomere repeat sequences (55, 62). The Sptbf1 gene is essential, although its full range of cellular functions is unknown. Overexpression of Sptbf1 has been shown to slightly increase the mean length of telomeres in vivo (51).We have examined whether the P. carinii tbf1 homologue (Pctbf1) can rescue ...

Characterization of the Aspergillus nidulans Monodictyphenone Gene Cluster

“…AN0153.4 encodes a putative 852-amino-acid (aa) protein which contains a Myb-like DNA binding domain that has high amino acid identity to telomeric repeat binding factor 1 (Tbf1) (as revealed by a BLAST search of the NCBI database). A tbf1 homologue (Pneumocystis carinii tbf1 [Pctbf1]) having high amino acid identity with AN0153.4 (E value, 1eϪ59) has been identified as an essential gene in Pneumocystis carinii (10). We conclude that AN0153.4 is almost certainly an essential gene that is not involved in monodictyphenone synthesis.…”

Antifungal compound honokiol triggers oxidative stress responsive signalling pathway and modulates central carbon metabolism

“…We also observed several modules which are related to signalling transduction. For examples, there is a predicted transcriptional regulation complex including DNA binding factor (Tbf1 and Spt2) (Cockell et al 2009), transcription factor TFIIH complex subunit (Rad15) (Murray et al 1992), histone acetyltransferase (Mst2) (Gómez et al 2005) and Jmjc domain chromatin-associated protein (Epe1) (Trewick et al 2007). There is also a potential signalling module associated with protein kinase and secondary messenger cAMP, which consists of cAMP-dependent protein kinase catalytic subunit (Pka1) (Matsuo et al 2008), serine/threonine protein kinase Ppk18 (Beltrao et al 2009) and a G-protein coupled receptor, Git3, which can activate the adenylate cyclase activity (Wang et al 2005).…”

Evidence for Proinflammatory β-1,6 Glucans in the Pneumocystis carinii Cell Wall

“…Due to the inability to culture and genetically manipulate P. carinii in vitro, traditional methods to characterize gene function in the organism are not possible at this time. To circumvent this, we used the heterologous expression and complementation of genetic knockouts in tractable fungi, such as Candida glabrata, S. cerevisiae, or S. pombe, to assess gene function in P. carinii (33,50,55,56). Using this approach, the biological activity of Pckre6 was determined by assessing the gene's ability to complement budding S. cerevisiae kre6⌬ cells in the presence of calcofluor white M2R, a compound that binds strongly to cell wall cellulose and chitins (57).…”