טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKahana Smadar
SubjectA New Role for Kss1/Fus3 MAPK Cascade in Regulating
Meiosis
DepartmentDepartment of Biology
Supervisor Professor Emeritus Yona Kassir
Full Thesis textFull thesis text - English Version


Abstract

In this study, we applied both bioinformatics and functional genomic approaches to scrutinize the unusually large and complex promoter of the IME1 gene, the master regulator of meiosis in budding yeast. Using a fluorescent protein-based reporter screen (R-SGA), we assessed the effect of viable deletion mutants on the transcription of various IME1 promoter-reporter genes. Using ChIP assay, we directly demonstrate the binding of three identified transcription factors (TFs): Sum1, Swi4, and Ste12, to non-perfect consensuses in UASru, a 167bp region in the promoter region of IME1. Most of the cis-regulatory sequences with perfect homology to known TF consensus were found to be nonfunctional in the R-SGA analysis. In addition, our results suggest that lack of conservation may not discriminate against a TF regulatory role at a specific promoter. We demonstrate that IREu, a specific functional element in the promoter of IME1, and the TFs which bind to and regulate its activity (Sok2, Msn2, and Ime1) are not conserved in the sensu stricto Saccharomyces strains.

The UASru element exhibits Upstream Activation Sequence (UAS) driving transcription under all growth conditions, although in the presence of acetate its activity is significantly induced. UASru composes of at list three distinct regions: (i) UASru(A) functions as a URS element in the presence of glucose and as s UAS element in its absence depending on the TF Com2; (ii) UASru(C) possesses URS activity under conditions leading to vegetative growth and UAS activity upon nitrogen depletion depending on Sum1; (iii) A third region with UAS activity resides between UASru(C) and an additional non-functional region, UASru(B).

Eukaryotic cells use signal transduction pathways such as MAPK cascades to specifically respond to a large number of external signals. The CWI pathway positively regulates UASru activity via the direct binding of Swi4-Mlp1 and Swi4-Mpk1. The filamentation pathway transmits in diploids an acetate signal to UASru through Kss1, Tec1 and Ste12. We showed that Ste12 directly binds to UASru only under these conditions. The pheromone response signal pathway regulates the activity UASru in haploids through Fus3 and Ste12, independent of nutrients. The osmoregulatory signal pathway positively regulates UASru, most probably indirectly through its effect on Kss1.

Finally, we showed that UASru's activity is specific to the acetate signal and that there is no cross-talk with the filamentation and pheromone pathways. Our research identified a new developmental pathway, meiosis, which is being regulated by the same MAPK cascades via specific signals.