1. Abstract The pheromone response pathway in S.cerevisiae is regulated on multiple levels and timescales, and by many biochemical mechanisms. In particular, several proteins that are directly involved in signal transduction through the pathway, as well as several positive and negative regulators of the pathway, are transcriptionally upregulated in response to pheromone. Previous work has focused on the effects of deleting or overexpressing proteins involved in propagating and modulating the signal. While data from these experiments sheds some light on the impact of varying protein levels, it does not represent a systematic exploration of the importance of transcriptional regulation of pathway genes in response to pheromone. Here, we describe work that explicitly addresses the question whether specific instances of pheromone-dependent transcriptional feedback loops are necessary for proper pathway function. Specifically, we are working to replace the promoters of pheromone-inducible pathway genes, both singly and in combination, with constitutive and exogenously regulatable promoters. These custom promoters will allow us to explore pathway response in the absence of transcriptional feedback, via regulatable expression of some of the major contributors to signal transmission and control. The synthetic pathway will be characterized to determine the effects of these modifications and to extend our understanding of pathway function. Assaying such characteristics as mating efficiency, recovery from pheromone-induced cell-cycle arrest and genome-wide transcription levels will allow us to understand the contributions made by wild-type transcriptional regulation to pathway function and mating program induction. Re-engineering transcriptional control in the yeast pheromone response pathway Alex Mallet 1 & Drew Endy 2 MIT Computational and Systems Biology 1 & Biological Engineering 2 Replacement Promoters Determining promoter sequences to replace Design rules: Get rid of all annotated 7 upstream transcription factor binding sites Actually remove the sequence, don’t just insert the new promoter Get rid of as many potential (i.e. based only on motif match) transcription factor regulation sites as possible But: Avoid disrupting transcription initiation or termination of upstream genes STE12-mediated transcriptional feedback The transcription factor STE12 is activated in response to pheromone and increases transcription of several proteins involved in the pathway over their basal expression levels. These proteins, in turn, exert regulatory control on each other, as depicted below. STE12 thus acts as a key component contributing to a set of interlocking positive and negative feedback loops. STE2 SST2 FUS3 FAR1 STE12 MSG5 GPA1 Transcriptional upregulation Post-translational downregulation Signal transduction Pathway overview (K. Benjamin, Molecular Sciences Institute, unpublished) Conclusions We are interested in understanding the contribution to proper function of the yeast pheromone response pathway made by pheromone-mediated transcriptional induction of pathway components and regulators. In this poster, we describe our motivating questions and our approach to answering these questions, namely placing pathway genes under controllable promoters and characterizing the pathway’s response to removal of one or more transcriptional feedback loops. At present, we have constructed strains containing the STE2 and FAR1 genes under the control of constitutive promoters and are in the process of characterizing these strains. References and Acknowledgements We would like to thank members of the Endy and Knight labs, Dr. E. Fraenkel and Dr. A. van Oudenaarden at MIT for providing valuable feedback. Discussions with Dr. F. Winston of Harvard and Drs. K. Benjamin, A. Colman-Lerner, R. Yu and G. Pesce of the Molecular Sciences Institute also provided key insights. This work was funded by the Computational and Systems Biology Initiative at MIT. (1) Roberts CJ et al, 2000. Science 287:873. (2) T. Thomson, unpublished (3) K. Benjamin, personal communication. (4) Alper H, Fischer C, Nevoigt E, Stephanopoulos G, 2005. Proc Natl Acad Sci USA 102:12678 (5) Urlinger S et al, 2000. Proc Natl Acad Sci USA 97:7963 (6) Becskei A, Kaufmann BB, van Oudenaarden A, 2005. Nat Genet 435:937 (7) Harbison CT et al, 2004. Nature 431:99 Yeast can exist as haploid cells, in two “mating types” termed a and alpha, with mating between cells of opposite mating types resulting in a single diploid yeast cell. Mating between yeast cells is mediated by the pheromone response pathway, which is activated when a haploid cell senses the presence of pheromones emitted by cells of the opposite mating type. Activation of the pathway results in transient differentiation of the cell via cell cycle arrest in G1, the formation of a mating projection and differential transcription of several hundred genes 1. If mating does not occur, or the stimulus is removed, the cells re-enter and proceed through the rest of the mitotic cell cycle. In addition to mediating the response to pheromone, the pathway also shares some molecular machinery with pathways activated under different conditions, such as the High Osmolarity Glycerol (HOG) pathway. Proper control of the pathway thus has several facets: A level of basal activity that prevents inappropriate differentiation Efficient induction of the cellular program required for mating in response to pheromone Adaptation to downregulate pathway activity appropriately Resistance to cell cycle arrest when exposed to pheromone outside G1 Maintenance of specificity e.g. avoiding inappropriate activation of the filamentous growth pathway Characterizing the modified pathway Yeast cells react to pheromone on a wide range of timescales, from a response time on the order of seconds by the signaling cascade component of the pheromone response pathway, to the actual mating program, which can take several hours to complete. The pheromone response also produces a wide variety of phenotypes, from morphological changes such as the formation of a mating projection and cell-cycle arrest to the differential transcription of several hundred genes. The functioning of the re-engineered pathway thus needs to be examined at multiple levels. We will examine pathway function by: Monitoring the expression of a chromosomally-integrated copy of YFP driven off the pheromone-responsive PRM1 promoter, which will provide insight on the timecourse of signaling through the pathway Generating dose-response curves of pheromone sensitivity, to characterize the impact on basal signaling Examining the efficiency of recovery from cell-cycle arrest upon removal of pheromone, thereby measuring the extent to which pathway desensitization is affected Generating genome-wide transcript measurements, to obtain a comprehensive picture of the molecular response induced by the altered pathway connectivity Assaying mating efficiency, as a means of gauging the overall impact of the changes to the pathway Are transcriptional feedback loops necessary for pathway function ? Previous work has shown that constitutive overexpression of the genes upregulated by STE12 does not, in the majority of cases, lead to constitutive pathway activation or total loss of signaling upon exposure to pheromone. This argues that pathway function is robust in the face of above-basal constitutive levels of expression of single genes and single changes to the transcriptional architecture of the system (i.e. removal of one transcriptional feedback loop). In addition, a detailed computational model of the pathway (developed in our laboratory 2 ) predicts pathway dynamics that are largely independent of transcriptional induction of certain genes, as illustrated below for the receptor STE2, a gene that is upregulated by a factor of five on exposure to pherome 1. This naturally leads to two questions: 1. Over what range of constitutive (i.e. not mediated by STE12) expression of single proteins does the pathway retain proper function ? 2. How robust is the pathway to the removal of multiple transcriptional feedback loops ? We will answer these questions by removing the STE12-mediated transcriptional feedback loops, both singly and in combination. Pathway genes that are STE12-responsive in the wild-type pathway will be placed under the control of promoters that allow us to vary their mRNA levels across a large range, and the response of the altered pathway will be characterized. The results of these experiments will also allow us to refine our computational model. A terminator built into the new promoter will cause proper transcription termination of the upstream gene, so we can replace as much as we need to of the upstream region. Case 1: Upstream gene in same orientation as target gene Will decide how much to replace on instance-by-instance basis, taking into account factors like relevance of upstream gene to mating, higher stringency for putative regulatory sites based purely on motif matching etc Case 2: Upstream gene in opposite orientation as target gene Intergenic region ATG …Target gene  Upstream gene  GTA TF binding sites ATG …Target gene  New promoter Upstream gene  GTA? Intergenic region ATG …Target gene  Upstream gene … STOP ATG …Target gene  New promoter  Upstream gene … STOP TF binding sites Feedback in the pathwayOpening the feedback loops Regulatable promoter Combination of artificial promoter based on Tetracycline repressor and artificial transcription factor rtTA(S2) 5 rtTA(S2) binds DNA in the presence of doxycycline, allowing induction of transcription by addition of doxycycline Allows varying expression level over 3 orders of magnitude, with unimodal cell response and low noise levels 6 Constitutive promoters Library of TEF promoters 4 Allow varying expression level over 2 orders of magnitude GPD, TEF, ADH1, BMH2, ACT1, CYC Capable of producing ~1000 – 90,000 molecules/cell, depending on protein 3