Mating in yeast Stressed diploid yeast undergoes meiosis Haploid daughters are either… “a” cells or “a” cells A-cells and a-cells mate Only a-cell with a-cell Attracted by pheromones Produce new diploid cell From http://en.wikipedia.org/wiki/Image:Yeast_Mating.png. Yeast come in three varieties: diploid cells, haploid a-cells, and haploid alpha-cells. A diploid yeast cell is able to reproduce by mitosis; however, when it is exposed to stressful conditions (e.g. lack of nutrients), it can undergo meiosis to produce four haploid cells, two a-cells and two alpha-cells. These haploid cells are able to mate by fusing to create a new diploid cell, but a-cells can only mate with alpha-cells and vice versa. The schematic in the slide illustrates this process. A-cells release pheromones (circles in the diagram) that attract alpha-cells, but have no effect on diploid cells or other a-cells. Similarly, alpha-cells release pheromones (squares) that attract a-cells, but have no effect on other yeast cell types. Alpha-cells and a-cells extend projections in response to the pheromones of their mate until they fuse together as shown at the bottom of the slide. The MAT locus determines whether a yeast haploid cell will be of the a or alpha type. The diploid cell contains copies of both the MATa and MATalpha alleles. Haploid cells that inherit an active MATa allele become a-cells and haploid cells that inherit an active MATalpha allele become alpha cells. The MAT loci encode DNA-binding proteins that trigger a regulatory cascade allowing each haploid cell to differentiate into its mating type. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

The regulation of mating type Distantly related yeast exhibit same differentiation of haploid mating types But use different regulatory elements From Figure 1 in Rokas, A. (2006) “Different paths to the same end” Nature 443: 401-402. Saccharomyces cerevisiae (commonly known as baker’s yeast) and Candida albicans (a human pathogen) are two species of yeast whose lineages diverged between 200 and 800 million years ago. Despite the very long periods of time during which these two organisms evolved independently, they still retain the same mating process. In 2006, a group of researchers from the University of California, San Francisco studied the regulatory networks that determine sex phenotype in a group of phylogenetically diverse yeast. They discovered that although the phenotypic process of sex determination was identical in all yeast species that were examined, the regulatory elements that governed the process were substantially different. The figure in the slide illustrates how the regulatory elements of the transcriptional networks that determine mating type have changed over the last several hundred million years. In the a-cells of C. albicans, the a-specific genes (asgs) that are necessary for a-cell differentiation are transcribed when an a2 protein and the Mcm1 regulatory factor bind to a region of DNA that is upstream from the asgs. These two regulatory factors are absent in C. albicans alpha-cells, and so the asgs are not transcribed. This is shown in the first column in the figure. In the distantly related S. cerevisiae, only the Mcm1 regulatory factor is needed to activate asgs in a-cells, but asgs are actively repressed in alpha-cells by one Mcm1 regulatory factor and two alpha-2 proteins. This is shown in the third column of the figure. Kluyveromyces lactis is a yeast species that is more closely related to S. cerevisiae than to C. albicans. The regulation of mating type in K. lactis has some features in common with S. cerevisiae and others with C. albicans. For example, asgs in K. lactis are transcribed in the presence of the a2 and Mcm1 regulatory factors as in C. albicans ; however, the asgs are actively repressed in alpha-cells by the presence of Mcm1 and a single alpha-2 protein. Given the centrality of mating type differentiation to yeast reproduction, one is prompted to ask how such significant changes in the underlying regulatory networks were possible without any negative impact on mating type phenotype. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Mutations responsible for regulatory network differences Small number of mutations can have big impact on gene regulation Example: Mcm1 – a2 protein interaction Mutations in small region of a2 protein account for absence of repression of asgs in some yeast species From Figure 5 in Tsong, A. et al. (2006) “Evolution of alternative transcriptional circuits with identical logic” Nature 443: 415-420. The yeast study was able to align the sequences of several genes to determine the likely mutations that were responsible for the changes in the regulatory networks underlying mating type determination. For the purpose of brevity, we focus on the mutations that account for differences in Mcm1 – alpha-2 protein interactions in different species of yeast. The alignments shown in the slide illustrate how a handful of mutations can be responsible for significant changes in transcriptional regulation. Part “a” shows Mcm1 sequences from 12 different yeast species. By building computational models of Mcm1 – alpha-2 protein interaction, researchers were able to show how a small number of mutations can manifest themselves as large changes in regulatory networks, by allowing or disallowing interactions between Mcm1 and alpha-2 that are responsible for the repression of asgs in some yeast species. The arrows in part “a” of the figure show amino acids in Mcm1 that contact the alpha-2 protein in S. cerevisiae. Part “b” is an alignment of alpha-2 protein sequence across 13 yeast species with arrows indicating amino acid residues that are required for Mcm1 – alpha-2 protein interaction and active repression of the asgs in S. cerevisiae. It is obvious from the alignment that the alpha-2 sequence in C. albicans, which does not actively repress asgs in alpha-cells, has an alpha-2 protein sequence that is very different from S. cerevisiae and K. lactis, which both have active repression of asgs in alpha-cells via an interaction between Mcm1 and alpha-2 proteins. Part “c” of the figure shows a model of alpha-2 and Mcm1 interaction in S. cerevisiae (left) and K. lactis (right). The model suggests that the interaction is likely to be weaker in K. lactis. This is consistent with the alignment in “b” that shows some similarities and some differences in the alpha-2 protein sequences of the two yeasts. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Evolutionary transitions in the regulatory network Comparison of regulatory networks governing yeast mating type suggests… big changes in genotype without… concurrent changes in phenotype However it is possible that there was selective advantage in different regulatory networks despite identical mating type outcome Traditional evolutionary theory argues that selection acts on phenotypes. Those phenotypes that survive in an organism bring with them the genotype that made them possible. Do the studies described here suggest that this is not always the case? This is a difficult question to answer for a variety of reasons. The ancestral organisms that first acquired differences in their mating type regulatory networks no longer exist and can not therefore be studied. It is possible that those organisms enjoyed significant selective advantages due to changes in their regulatory networks in the context of their ancient environment. Even in the present, it is difficult to say how changes in these regulatory networks impact different yeast species in their contemporary environments without doing extensive experimentation. Nonetheless, the finding that a small number of genotypic changes can greatly alter the regulatory networks governing an essential process like mating without impacting mating behavior is fascinating in and of itself. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458