|Ph.D Thesis||Department of Biology|
|Supervisor:||Prof. Kassir Yona|
|Full Thesis text|
Transcriptional cascades which include positive and negative feedback loops are commonly involved in the robust execution of developmental pathways. Such cascades lead to an ordered transcription of networks of genes whose products are required simultaneously, or in successive stages. In this thesis, meiosis in budding yeast was used as a model system for studying a transcriptional cascade, and specifically for elucidating the correlation between the expression pattern of a "master" regulator and the efficiency of entry into and completion of a developmental pathway. In budding yeast, IME1 is a "master" gene which is transiently expressed under meiotic conditions and induces the transcriptional cascade of meiosis.
The first part of the thesis used systems biology tools. We are interested in studying how the transcription of meiosis-specific genes is regulated. We constructed a discrete computational model faithfully describing the expression pattern of two main positive regulators of meiosis, Ime1 and Ime2. This model, which was validated experimentally, has strong predictive aspects concerning the mode by which negative regulation through Ime1 and Ime2 are accomplished. Furthermore the model predicts that meiosis will be insensitive (robust) to modulations in the levels of Ime1 and Ime2. This prediction was confirmed by changing the copy number of the IME1 gene (1x, 2x, 3x, and 5x copies).
The aim of the second part of the thesis is to elucidate how this robust meiosis is accomplished. We found that although meiosis was robust to drastic changes in the levels of all analyzed positive regulators, their relative time of expression was essential. Premature transcription of NDT80, the transcriptional activator of the middle meiotic genes, led to loss of coordination between early and middle meiotic events, namely, DNA replication and nuclear division, and consequently to a defective meiosis. A switch from a gradient mode in the transcription of the early meiotic genes to a threshold mode in the transcription of NDT80 provides a key mechanism for robust meiosis. Our results indicate that this switch is mediated by an early meiotic gene - IME2. We showed that at early meiotic times the activity of Ime2 was inhibited, most probably following phosphorylation by Cdk1. Consequently, the graded level of Ime2 protein was switched to a threshold level of active Ime2 which regulated both initiation of DNA replication and the transcription of NDT80. Finally, we showed that Ndt80-dependent decline in the transcription of IME1 led to a shut-down of the transcriptional cascade and completion of meiosis.