The significance of regulatory evolution has been recognized since the 1960s, but technical difficulties in obtaining suitable data have inhibited the study of regulatory evolution. Advances in molecular biology (specifically, gene regulation), and recent advances in genomics have made it possible to study not only the regulation of single genes, but also the co-regulation of many genes, even going so far as to investigate regulatory evolution in eukaryotes and the evolution of regulatory modules. 

The purpose of Li’s project on Evolution of Gene Regulation and Regulatory Modules in Yeast, is to study how the regulation of yeast genes has evolved over time. However, instead of looking at one gene at one time, Li’s group has looked at a group of genes, or regulatory module, that are subject to the same or similar regulation at the same time. The fruit of this research is the following two publications:

An on-going project: Evolution of Yeast Non-Fermentative Regulatory Network

Although yeast much prefers sugars, especially glucose, it can also use non-fermentable substrates such as ethanol, lactate or glycerol for the generation of energy and cellular biomass. That is, when fermentable substrates become limiting or depleted, yeast will shift from fermentative to non-fermentative growth. However, unlike fermentative metabolism, non-fermentative metabolism has not been well studied. This is particularly so with respect to the question of “What are the key factors responsible for the transition from fermentative to non-fermentative growth?” We have therefore chosen to study the evolution of yeast non-fermentative regulatory pathways. For this purpose, we have chosen the lab and a wild strain because the lab strain has been maintained on glucose media for decades, so that it might have undergone many genetic changes. Indeed, two recent studies have found expression differences in more than 1500 genes between the two strains. However, these studies were done only at the rapid phase of growth under fermentative metabolism, so it provides no information on non-fermentative growth.

Our purposes are (1) to identify the major genes that control the transition from fermentative to non-fermentative growth (i.e., key regulators) and also their downstream genes, so that we can understand the regulatory pathways involved, and (2) to study how these genes and their regulatory pathways have evolved between the two strains, that is, is the evolution of gene expression between the two strains mainly due to cis element changes or due to changes in trans-acting factors.

This project is rather ambitious and requires the use of modern technology such as DNA microarrays, real-time PCR, and chromatin-immunoprecipitation (ChIP). These experimental components are mainly conducted by the young postdoc Ya-Wen Chang because she is an expert in yeast genetics and molecular biology. A second postdoc Robert Liu has recently joined the project.

We have made substantial progress. First, using traditional methods we have found that the two strains show quite different rates of glucose consumption and that the transition from fermentative to non-fermentative occurs approximately one to two hours earlier in the wild strain. These observations show that the choice of the two strains is suitable for our purpose. Second, using microarrays and real-time PCR, we have found many important genes that show different expression patterns between the two strains. That is, they are turned on earlier in the wild strain than in the lab strain. Such differences will help us identify the major genes that cause the differences in growth pattern and in gene expression between the two strains, though it will take much more effort. Third, we have succeeded in establishing a method of gene-swapping, that is, to swap a gene from one strain to the other strain. This is a key technique in our project because using this technique we can swap genes between the two strains and see how a swapped gene affects the growth pattern and the expression of downstream genes. Such experiments can identify which genes are the key genes that cause the differences in growth or gene expression pattern between the two strains. Fourth, we have recently found that the pyrosequencing technique is highly suitable for assessing the relative expression levels of alleles in the same media or in hybrid diploids.

Using the above procedures or techniques we have now obtained data for the Cat8 pathway, which is the gluconeogenesis pathway, an important pathway in the transition from fermentative to non-fermentative metabolism. Our data indicate: (1) the upstream gene (MIG1) that regulates the expression of the Cat8 is turned on earlier (~1 to 2 hours) in the wild strain than in the lab strain and for this reason the Cat8 pathway is induced 1 to 2 hours earlier in the wild strain than in the lab strain and (2) the difference in expression profile in the MIG1 gene between the two strains is mainly due to changes in cis elements in the MIG1 gene whereas the expression differences in Cat8 and other genes in the Cat8 pathway are mainly due to changes in trans-acting factors. We are preparing a manuscript for this study.