The molecular characterisation of Mss11p, a transcriptional activator of the Saccharomyces cerevisiae MUC1 and STA1-3 genes

[摘要] ENGLISH ABSTRACT:Upon nutrient limitation, normal cells of the budding yeast, Saccharomyces cerevisiae,undergo a transition from ovoid cells that bud in an axial (haploid) or bipolar (diploid) fashionto elongated cells that bud in a unipolar fashion. The daughter cells stay attached to themother cells, resulting in chains of cells referred to as pseudohyphae. These filaments cangrow invasively into the growth substrate (haploid), or away from the colony (diploid), andare hypothesised to be an adaptation of yeast cells that enables them to search for nutrientrichsubstrates. This filamentous growth response to nutrient limitation was shown to bedependent on the expression of, amongst others, the MUC1 gene.MUC1 (also known as FL011) encodes a large, cell wall-associated, GPI-anchoredthreonine/serine-rich protein that bears structural resemblance to mammalian mucins and tothe yeast flocculins. Deletion and overexpression studies demonstrated that it is critical forpseudohyphal differentiation and invasive growth, and that overexpression of the gene alsoresults in strongly flocculating yeast strains. The upstream regulatory region of MUC1comprises the largest yeast promoter identified to date and areas as far as 2.4 kb upstream ofthe translational start site have been shown to confer regulation on MUC1 expression. Thelarge promoter region is not unique to MUC1, however, since it is almost identical to that ofthe functionally unrelated STA2 gene. The STA2 gene, as well as the identical STA1 and STA3genes, encodes extracellular glucoamylase isozymes that enable the yeast cell to utilisestarch as a carbon source. Glucoamylases liberate glucose residues from the non-reducing endof the starch molecule, thereby making it accessible to yeast cells.The high identity between the promoters of MUC1 and STA1-3 suggests that the two genesare co-regulated. In addition, several transcription factors that regulate the transcriptionallevels of both MUC1 and STA2 have been identified and include Msn1p and the previouslyuncharacterised Mss11p. Overexpression of either Msn1p or Mss11p results in elevated levelsof MUC1 and STA2 transcription and a dramatic increase in flocculation, invasive growth,pseudohyphal differentiation and the ability to utilise starch, suggesting that the two genesare indeed co-regulated. The main objective of this study was to characterise Mss11p and itsrole in the co-regulation of MUC1 and STA2 (as a representative member of the STA genefamily).A detailed expression analysis, using Northern blots and Lacl reporter gene expressionstudies in different media, confirmed that these genes are indeed co-regulated to a largeextent. MUC1 and STA2 are also regulated by the same transcriptional regulators, whichinclude not only Msn1pand Mss11p, but also Ste12p, the transcription factor of the matingpheromone/filamentous growth signalling cascade, and Flo8p, a transcriptional activator ofthe flocculation genes. Overexpression of the genes encoding these factors results in elevatedexpression levels of both MUC1 and STA2 in most nutritional conditions and enhances thefilamentous growth phenotypes of the strain, as well as the ability to degrade starch. On theother hand, the deletion thereof results in severe reductions in the transcription levels ofMUC1 and STA2, with equally severe reductions in filamentous growth and the ability tohydrolyse starch. These expression studies also showed that the repressive effect of STA10, apreviously uncharacterised negative regulator of STA2, is actually a phenotype conferred by aFLOB mutation in some laboratory strains of S. cerevisiae.The upstream regulatory regions of MUC1 and STA2 are the largest promoters in the yeastgenome. By sequencing the upstream areas of STA2 and STA3 and comparing them to thesequence of MUC 1, it was shown that these upstream areas are 99.7%identical over morethan 3 900 base pairs (bp) upstream of the translational start. With the exception of a fewminor substitutions, the only significant difference between the MUC1 and STA2 promoters isthe presence of a 20-bp and a 64-bp sequence found in the MUC1 promoter, but not in thepromoters of any of the STA1-3 genes.Through a promoter-deletion analysis, it was shown that Mss11p, Msn1pand Flo8p exerttheir control over the transcription of MUC1 and STA2 from an 90-bp sequence located at-1 160 to -1 070 in the STA2 and -1 210 to -1 130 in the MUC1 promoters. This sequence alsomediates the effect of carbon catabolite repression on the transcription of STA2 and MUC1.Despite the similarities in the expression patterns of MUC1 and STA2, some discrepanciesalso exist. The most significant difference is that, in wild-type cells and under all nutritionalconditions tested, MUC1 transcription is reduced significantly if compared to the transcriptionlevels of STA2. This was attributed to the presence of the 20- and 64-bp sequences, that arepresent in the promoter region of MUC1, but absent from that of STA2.To place the transcriptional regulators of MUC1 and STA2 in the context of known signaltransduction pathways, an epistasis analysis was conducted between MSN1, MSS11 andcomponents of the mating pheromone/filamentous response MAPkinase cascade and cAMPPKApathway that were shown to be required for the filamentous growth response. Thisanalysis revealed that Msn1p functions in a third, as yet uncharacterised, signal transductionpathway, also downstream of Ras2p,but independent of the two identified pathways, i.e. thecAMP-PKA and pheromone response/filamentous growth response MAP kinase pathways.However, Mss11p seems to function downstream of all three the identified pathways. Thissuggestsa critical and central role for Mss11p in determining the transcription levels of MUC1and STA2.To further characterise Mss11p and its role in the transcriptional regulation of MUC1 andSTA2, it was also subjected to a detailed deletion and mutation analysis. Mss11p was shownto harbour two distinct activation domains required for the activation of MUC1 and STA2, butalso able to activate a reporter gene expressed from under the GALl promoter. The moreprominent of the activation domains of Mss11p was shown to be one of the domains withhomology to Flo8p, designated H2. The H2 domain has significant homology to a number ofproteins of unknown function from a range of different organisms. A multi-sequencealignment allowed the identification of conserved amino acids in this domain. Mutations intwo of the four conserved amino acid pairs in the H2 domain completely eliminated theactivation function of Mss11p.The poly-glutamine and poly-asparagine domains of Mss11p are not required for itsactivation function. The deletion of these domains has no impact on the ability of Mss11p toactivate MUC1 or STA2 or of the Gal4p-Mss11p fusion to activate the lacl reporter geneexpressed from under the GAL7 promoter. Gal4p fusions of either of these domains were alsounable to trans-activate the PGAL7-lacl reporter gene. As such, it was concluded that neitherof these domains performs a function in the role of Mss11p as a transcriptional activator. Wealso demonstrated that the putative ATP/GTP-binding domain (P-loop) is not required for thetranscriptional activation function of Mss11p.In an attempt to identify other target genes of Mss11p, the use of micro-arrays wasemployed to assessthe impact of the overexpression and deletion of MSS11 on the total yeasttranscriptome. These results showed that MUC1 and STA2 are the only two genes in the ISP15genetic background that are significantly (more than 15-fold) enhanced by the overexpressionof MSS11. Mss11p therefore seemsto playa very specific or dedicated role in MUC1 and STA2transcription. This analysis also identified several genes (DBP2, ROM2, YPLOBOC, YGR053C,YNL179C, YGR066C) that are repressed by overexpression of MSS11 and activated when MSS11is deleted.To integrate all the results, three possible models for the activation of MUC1 and STA2transcription by Mss11p are proposed: (i) Mss11p performs the role of a transcriptionalmediator, possibly in a protein complex, to convey information from upstream regulatoryelements to the transcription machinery assembledat the core promoters of MUC1 and STA2;(ii) Mss11p plays a more direct role in transcriptional activation, possibly as a transcriptionfactor itself; and (iii) Mss11p facilitates transcription of the MUC1 and STA2 promoters as partof a larger complex that removes or releases the chromatin barrier over the MUC1 and STA2promoters in responseto specific nutritional signals.

[发布机构] Stellenbosch University