TTAG Summary: The transcriptional program of sporulation in budding yeast.

S. Chu, J. DeRisi, M. Eisen, J. Mulholland, D. Botstein, P. O. Brown, I. Herskowitz. 1998. The Transcriptional Program of Sporulation in Budding Yeast. Science, 282, 699-705.

Science 1998 Nov 20;282(5393):1421.

Here's my review on the paper.

So under starvation conditions, diploid Saccharomyces cerevisiae produce stress-resistant haploid spores in a process called sporulation. In order to successfully sporulate, diploid cells must undergo meiosis to reduce the ploidy of cells, and spore morphogenesis to package the genome in a prospore membrane, then the protective spore wall. The complexity of sporulation requires regulated changes in gene expression in order for the stages to progress in a coordinated, sequential manner.

The authors of the article are investigating every gene involved in sporulation and how different regulatory sites will affect when these genes are induced. The authors intend to categorize these genes based on their time of induction then link each category’s unique temporal profile to changes within the cell during the course of sporulation. Thus, they are able to hypothesize the functions of genes with previously unknown functions based on the time of their induction during sporulation.

To induce sporulation, cells were put on nitrogen-deficient medium. Sporulation is initiated by deficiencies in nitrogen and fermentable carbon sources, inducing a signal transduction pathway and arresting the cell at G1. At 7 different time points after sporulation initiation, they did a microarray analysis of 97% of all yeast genes to measure the changing abundance of mRNA transcripts of each gene over time compared to vegetative cells. The 7 different time points were based on the time of induction of DMC1, SPS1, DIT1, and SPS100, which represent the expression patterns for early, middle, mid-late, and late genes, respectively. Northern and microarray analysis were used to confirm the time of induction and relative transcript abundance of each of the four genes. Results from the microarray showed that, over the course of sporulation, 500 genes were induced and about 500 were repressed. The induced genes were categorized into seven groups (Metabolic, Early I, Early II, Early Middle, Middle, Late-Middle, and Late) based on the time of initial induction.

The metabolic group is induced first, and functions to acclimatize the cells to nitrogen-starved conditions. They have different temporal expression profiles, despite having the same URS1 sequence, and so must be regulated by other means. The majority of genes with the early I pattern have a URS1 site or a core URS1 site. They are induced after .5 hours and play a role in synapsis and homologous recombination. Genes with the early II pattern are less likely than early I genes to have putative URS1 sites, which may explain why their induction is slightly delayed. Early-middle genes are induced at 2 hours and continue to increase even after7 hours. Some genes that display this pattern have a role in spindle pole body dynamics. Middle genes, induced from 2-5 hours, are regulated primarily by Ndt80. Ectopic expression induced many genes with an MSE site in vegetative cells. Genes in vegetative cells that remain uninduced are likely to require additional factors. In addition to expressing Ndt80 in non-sporulating cells, the authors inactivated Ndt80 expression in sporulating cells. Many genes were expressed at a third the level of wild-type cells, resulting in arrest during prophase. However, other genes, induced independently of Ndt80, required additional input to be induced. The mid-late class of genes is induced after 5-7 hours, and the genes usually have an NRE (negative regulatory element) site in addition to the MSE site that delays their induction compared to middle genes. This class includes genes that contribute to prospore membrane formation. The late class of genes is induced after 7 hours and includes genes involved in spore maturation. Each class of genes has a common regulatory sequence in the promoter region targeted by a specific transcription factor. Binding of the transcription factor to its target site induces the gene’s transcription. Ume6/Ime1 recognizes the URS1 (Ume cognate cis-acting regulatory sequence) found upstream of early genes. Genes with MSE sites (middle gene sporulation element) induced by Ndt80 protein. Proteins that induce mid-late and late genes have not been discovered at the time of publication. Temporal expression patterns during sporulation are possible due to sequences such as URS1 and MSE, allowing induction of an entire set of genes at different stages of meiosis and spore morphogenesis when they are required. By linking what a gene does to how it is regulated by different transcription factors, the authors are able describe the general program of sporulation.

They needed to show that gene induction of the different classes was happened concurrently with physiological changes in cell, so the cells were assessed cytologically. At various time points, DAPI (4’-6’diaminidino-2-phenylindole) was used to stain the nuclei of sporulating cells to determine how many nuclei were present in the cells and measure the rate of meiosis. At around 5 hours, after the middle genes have been induced, mononucleate cells become binucleate. By 9 hours, tetranucleate cells outnumber mononucleate cells. Electron microscopy was used to determine the progress of spore maturation. From 5 to 9 hours, the rate of spore formation rose dramatically: from 0 percent to under 40. By 11.5 hours, the percent of mature spores had risen to equal that of immature spores. These results support the role of mid-late genes in prospore membrane formation and late genes in spore maturation because the cytological changes coincided with the gene’s induction.

The authors’ results show that in sporulation the time of induction of a gene is strongly correlated to its function and the mechanism of regulation. This correlation makes genes with unknown functions candidates for different processes based on their time of induction. The authors use this correlation as part of their strategy to propose the roles many genes such as Spo69, Spo70, and Spo71. The authors also compare homologous proteins with known functions in Drosophila and Xenopus and Caenorhabditis elegans to Spo70, a sporulation-specific protein, to determine whether the protein had the same function in yeast. In conclusion, the authors used temporal induction patterns of every gene in S. cerevisiae to propose gene functions for many previously uncharacterized genes. Genes each class had characteristic regulatory sequences that determined the time of induction. Time of induction during sporulation is tied with gene function as transcripts are induced when they have a role to play in the specific stages of the program. Thus, regulatory sites such as MSE and URS1 can be used to determine gene function.