Molecular biological analysis of cell adhesion in Ashbya gossypii

Ashbya gossypii is a filamentous fungus of the family Saccharomycetaceae, and about 95% of its genes have a homologue in the yeast Saccharomyces cerevisiae. S. cerevisiae can turn into a filamentous growth form for which the expression of adhesion molecules is necessary. Some of these so called FLO genes have homologues in A. gossypii, which are AgFLO5-1 and AgFLO5-2, AgFLO11 and AgFIG2. Genes encoding the transcriptional regulators AgFlo8, AgSfl1 as well as AgTec1 and AgSte12 are present as well. Unlike in S. cerevisiae, flocculation gene products in A. gossypii do not bind to mannose. Deletion of A. gossypii adhesins and most of their transcriptional regulators did not alter the colony phenotype. However, Agflo8, Agsfl1 and Agtec1 showed a growth delay. Formation of aerial hyphal aggregations and the attachment to the growth substrate was not impaired. The mentioned mutants sporulated in an abundant manner compared to the precursor strain but spore phenotypes were normal. Reporter gene assays and promoter analysis revealed that AgFlo8 induces the expression of AgSFL1 which in turn represses AgFLO5-2, AgFLO8 and AgFIG2. The AgFLO8 and AgSFL1 deletion mutants seemed to possess certain insensitivity to elevated sugar levels, indicating non sufficient stress response in the absence of AgSfl1. Overexpression of AgSFL1 resulted in a wild-type-like phenotype. A C-terminal GFP fusion to AgSfl1 shows that the protein is localized in nuclei. In S. cerevisiae, pheromone stimulation leads to expression of ScFIG2 via Ste12. AgFIG2 seems to be permanently expressed at high levels via Tec1 without involvement of Ste12. Reporter gene assays and promoter analysis revealed conserved Tec1 and Ste12 binding sites between A. gossypii and S. cerevisiae. Out of 4726 protein coding genes in the genome of A. gossypii at least 16.6% show Tec1 binding sites in their promoter.Ashbya gossypii ist ein filamentöser Pilz der Familie Saccharomycetaceae, und für etwa 95% seiner Gene ist ein homologes Gen in Saccharomyces cerevisiae zu finden. S. cerevisiae kann eine filamentöse Wachstumsform ausbilden, wofür die Expression von Adhäsionsmolekülen notwendig ist. Einige dieser sogenannten FLO-Gene besitzen Homologe in A. gossypii, welche von AgFLO5-1 und AgFLO5-2, AgFLO11 und AgFIG2 kodiert werden. Sowohl die Transkriptionsregulatoren AgFlo8, AgSfl1 als auch AgTec1 und AgSte12 sind vorhanden. Anders als in S. cerevisiae binden die Flockulierungsmoleküle in A. gossypii nicht an Mannose. Deletion sowohl der Adhäsine als auch deren Transkriptionsregulatoren in A. gossypii führte nicht zu einer Änderung des Koloniephänotyps. Hingegen wiesen Agflo8, Agsfl1 und Agtec1 Wachstumsverzögerungen auf. Lufthyphenaggregationen und das Anhaftungsverhalten blieb jedoch unverändert. Die genannten Deletionsmutanten zeigten vermehrte Sporenbildung, jedoch waren Sporenphänotypen unverändert. Reportergen-Experimente und Promotoranalysen deuten auf AgFlo8-induzierte AgSFL1 Expression hin. AgSfl1 wiederum reprimiert die Expression von AgFLO5-2, AgFLO8 und AgFIG2. Die Deletionsmutanten Agflo8 und Agsfl1 schienen eine gewisse Insensitivität gegenüber erhöhten Zuckermengen aufzuweisen, was auf unzureichende Stressabwehr in Abwesenheit von AgSfl1 deutet. Überexpression von AgSFL1 resultierte in wildtypartigem Phänotyp. C-terminale GFP Markierung von AgSfl1 zeigt die Lokalisierung des Proteins im Zellkern. In S. cerevisiae wird ScFIG2 nach Pheromon-Induktion mit Hilfe von Ste12 exprimiert. AgFIG2 scheint permanent in hohem Maße mit Hilfe von Tec1 jedoch ohne Ste12 exprimiert zu werden. Mit Hilfe von Reportergen-Experimenten und Promotoranalysen wurde die Konservierung von Tec1 und Ste12 Bindestellen zwischen A. gossypii und S. cerevisiae nachgewiesen. Das Genom von A. gossypii enthält 4726 protein-kodierende Gene. Von diesen weisen mindestens 16,6% Tec1-Regulierung auf

A steady state model for the transcriptional regulation of filamentous growth in Saccharomyces cerevisiae

Occurrence of multiple Upstream Activation Sites (UASs) is a structural motif that is observed within the promoter of eukaryotic genes for coordinating gene expression. Transcriptional activation depends on the ability of transcriptional activators to bind to its specific UASs, which are kept inaccessible due to the nucleosomal organization of the chromatin. Targeting of chromatin remodeling complexes by a sequence specific transcriptional activator is shown to be detrimental for transcriptional initiation. Here, we analyze such a regulatory structure involving ordered recruitment of transcriptional activators and chromatin remodeling complexes with respect to activation of a flocculin gene, FLO11 involved in the filamentous growth to gain insights into its regulation. We develop a steady state model for the transcriptional regulation of FLO11 by primary transcriptional activators Flo8p, Ste12p, Tec1p and Mss11p, which are under a complex network comprising of cAMP and MAPK pathways. Our analysis predicts that the FLO11 promoter should undergo varying chromatin remodeling activity from partial to complete disassembly depending upon the concentration of Ste12p. This variation should be sensitive and sharply shift to saturate with Ste12p concentration. Overexpression of Ste12p can increase the overall chromatin remodeling activity by increasing the local concentration of remodeling complex through active recruitment. Further, we demonstrate that the chromatin remodeling activity brings about amplification of cAMP and MAPK signal and in absence of either of the signals, the input signal required for the other increases. We also discuss the results obtained from our steady state analysis in respect to other eukaryotic genes