Genomic dissection of the cell-type-specification circuit in Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae has three cell types (a cells, ␣ cells, and a͞␣ cells), each of which is specified by a unique combination of transcriptional regulators. This transcriptional circuit has served as an important model for understanding basic features of the combinatorial control of transcription and the specification of cell type. Here, using genome-wide chromatin immunoprecipitation, transcriptional profiling, and phylogenetic comparisons, we describe the complete cell-type-specification circuit for S. cerevisiae. We believe this work represents a complete description of cell-type specification in a eukaryote.chromatin immunoprecipitation ͉ mating ͉ transcriptional circuit A problem of central importance in understanding multicellular organisms is how different cell types are stably maintained. Typically, cell-type specification is based on a transcriptional circuit in which combinations of regulatory proteins determine the final pattern of gene expression that is appropriate to a given cell type. Although unicellular, the yeast Saccharomyces cerevisiae has three distinct types of cells, and the cell-specification circuit is combinatorial (refs. 1-3 and Fig. 1). The a and ␣ cell types are typically haploid in DNA content and mate with each other in an elaborate ritual that culminates in cellular and nuclear fusion. These events produce the third type of cell, the a͞␣ cell type, which is typically diploid. This cell type cannot mate but, when environmental conditions are appropriate, can undergo meiosis and sporulation, producing two a and two ␣ cell types. The patterns of cell-type-specific gene expression are set up by a few sequence-specific DNA-binding proteins acting in various combinations. Three critical proteins (␣1, ␣2, and a1) are encoded by the mating-type (MAT) locus. A fourth key sequence-specific DNA-binding protein (Mcm1) is encoded elsewhere in the genome. In this article we use the term ''cell-type-specification circuit'' to refer to the regulatory scheme diagrammed in Fig. 1, because each component and branch of this scheme is necessary and sufficient to establish and maintain three cell types.In this article we apply three methods [genome-wide chromatin immunoprecipitation (ChIP), genome-wide transcriptional profiling, and phylogenetic comparisons] in an attempt to completely determine the cell-type-specification circuit in S. cerevisiae (4-8). The use of three different techniques generated considerably more data than are needed to reconstruct the circuit, and because it is overdetermined, we believe our circuit description to be very accurate, containing at most only a few false negatives or positives. Figs. 3, 5 A and B, and 6. EG123, yDG208, and yDG240 are all derivatives of S288C. For the salt-sensitivity experiment, the a1-␣2 site in the endogenous HOG1 gene promoter was replaced by integration of Kluyveromyces lactis URA3, which was subsequently replaced by the integration of an oligonucleotidegenerated construct to restore the HOG1 promoter with a modified a1-...

Genetically Engineered Transvestites Reveal Novel Mating Genes in Budding Yeast

“…YLR040C has also been shown to be translated by ribosome profiling (Brar et al 2012) and localized to the cell wall (Hamada et al 1999;Giaever et al 2002). Deletion of YLR040C was reported as having no effect on mating (Galgoczy et al 2004). We found that in unstimulated cells the gene is expressed 11-fold more strongly in MATa-playing-a than in MATa cells and that its transcription is not significantly induced when MATa-playing-a cells are exposed to pheromone (see Table S2).…”

“…Ten genes fit this criterion and of these, only one, YLR040C, is annotated as encoding a secreted protein that is not already known to be important in mating (Yeast Genome Database at http://www.yeastgenome.org) (see Table S2). (Galgoczy et al 2004), which induces expression of a-specific genes (Strathern et al 1981). YLR040C has also been shown to be translated by ribosome profiling (Brar et al 2012) and localized to the cell wall (Hamada et al 1999;Giaever et al 2002).…”

“…Mixing experiments suggested that MATa-playing-a cells produced an extracellular factor that interfered with the action of a-factor, prompting us to look for the secreted product of a MATa-specific gene. This computational sieve produced a single gene, YLR040C, which had previously been identified as a gene regulated by the MATa-specific transcription factor, Mata1 (Galgoczy et al 2004). Removing YLR040C increased a-factor production from MATa-playing-a cells and the mating efficiency of MATa-playing-a cells, leading us to rename YLR040C AFB1 for a-factor barrier.…”

The Chromatin and Transcriptional Landscape of NativeSaccharomyces cerevisiaeTelomeres and Subtelomeric Domains

“…Thirty-five genes decreased in expression in sir mutants relative to WT strains. We compared this list to known haploid-specific genes as found by chromatin immunoprecipitation of a2 in a/a diploids followed by hybridization of immunoprecipitated DNA to a genome-wide array (Galgoczy et al 2004). That study found 20 haploid-specific genes, all of which were reproduced in our data set (Table 2, genes without footnote markers).…”

“…Historically, elucidation of transcriptional regulatory circuits of S. cerevisiae has relied on microarray-based technologies, which are limited in sensitivity and dynamic range (Galgoczy et al 2004). The sensitivity of RNA-Seq and the "pseudodiploid" state of sir mutants allowed us to evaluate the completeness of the identification of cell-type-regulated genes, particularly genes that are potential targets of a1/ a2 and a2/Mcm1 regulation.…”