Saccharomyces cerevisiae, one species of budding yeast, is a single-cell eukaryote capable of existing in both a diploid (two homologous copies of each chromosome, or 2n), and a haploid (one copy of each chromosome, or n) stage. Haploid cells exist as one of two possible mating types, MATa or MATα, and mate with haploid cells of the opposite mating type.
Time-lapse video showing yeast as they first bud and then begin to shmoo, or grow projections towards one another, in response to mating pheromones secrete by cells of the opposite mating type. A pheromone is a chemical substance secreted by a species that triggers a social response (in this case, mating) in members of the same species.
Figure 1: Pictorial representation of two haploid yeast cells of opposite mating types. Each cell secretes specific pheromones (1); both cells grow projections (shmoo) (2); and the haploid cells mate to form a diploid (3).
The expression of master regulatory genes on the active MAT locus near the centromere of chromosome III determines whether a cell expresses an a or α mating type. The HO gene is a DNA endonuclease that physically cleaves the DNA at the MAT locus, and this double-stranded break initiates mating-type switching (Fig. 2). Through genetic recombination, haploid cells can switch mating type as often as once per cell cycle.
Figure 2: HO endonuclease cleaves the DNA sequence at the MAT locus. This double-stranded break causes mating-type switching to occur via genetic recombination using one of two donor sequences.
Why is mating-type switching of budding yeast important?
Since wild type haploid yeast cells are capable of switching mating types, colonies will almost always contain both a and α haploid cells. For example, even if all a cells form a colony, some of the cells will switch mating type in order to be able to mate with one another. There is strong evolutionary drive for haploid cells to mate and produce diploid cells, and as a result of mating-type switching, all cell colonies are able to do this.
In addition, S. cerevisiae is a single-celled organism with a short generation time, making it useful as a model organism. Moreover, it is a eukaryote, so it shares the complex internal cell structure of plants and animals without the high percentage of non-coding DNA that can confound research in higher eukaryotes.
Which genes serve as donors for mating-type switching?
When haploid cells undergo mating-type switching, HMLα and HMRa, haploid mating loci located respectively on the left and right arms of chromosome III, serve as genetic donors. MATa haploid cells preferentially choose HMLα as a genetic donor during mating-type switching, and MATα cells prefentially select HMRa.
What determines donor preference during mating-type switching?
- Recombination enhancer (RE)
- Two RE activation pathways
a.) Swi4/Swi6 (SBF) transcription factors
b.) Forkhead proteins (Fkh1p and Fkh2p)
The role of the Recombination Enhancer (RE):
Coic, Richard, & Haber (2006) demonstrated that donor preference depends on activation of the recombination enhancer (RE), which controls recombination along the left arm of chromosome III. Based on this study, researchers showed that both HMRa and the MAT locus are located on the right arm of chromosome III; thus, HMRa serves as the default donor when RE is inactive. HMLα is located on the opposite arm of chromosome III, so activation of RE is required for increased HMLα usage (Fig.3).
Further, when MAT was inserted into the same domain as HMLα, RE played a minimal role in activating HMLα (Fig. 4). This suggests that RE does not remodel the silent chromatin of HMLα, but rather makes the left arm physically accessible to other regions of the genome.
Figure 4: HMLα is the preferred donor when MATa is located on the left arm in RE+ and RE- strains. HMLα usage was quantified on Southern blots. (A) The MAT locus was moved from its natural locus to the HIS4 locus. (B) RE was deleted in a strain bearing MATa on the left arm at the HIS4 locus. (C) RE was reinserted near the right donor. (D) Southern blots showing the products of MAT switching in strains bearing MAT on the left arm at the HIS4 locus.
In addition, when MAT and RE were moved to chromosome V, donor usage was nearly random. Thus, the inaccessibility of HMLα in the absence of RE appears to be dependent on chromosome III-specific sequences, as the architecture of chromosome V did not lead to exclusion of either donor (Coic et al., 2006).
Based on this study, researchers concluded that the architecture of chromosome III is the foundation of donor preference regulation. The two arms of chromosome III constitute independent domains that are inaccessible from each other in the absence of RE. Hence, in MATα cells, RE is inactive, and HMRa serves as the default donor. In MATa cells, RE is active, resulting in increased HMLα selection (Coic et al., 2006).
This study provides convincing evidence for the role of the recombination enhancer in determination of donor preference in mating-type switching, and no weaknesses are evident in the conclusions drawn based on the results presented.
RE Activation pathways:
Two independent RE activation pathways exist.
First, in the G1 phase of the cell cycle, Swi4/Swi6 (SBF) transcription factors bind to an evolutionarily conserved SCB site within the RE. Next, in the G2 phase of the cell cycle, forkhead proteins (Fhk1p and Fhk2p) bind to specific proteins in RE (Coic, Sun, Wu, & Haber, 2006).
RE is divided into 4 conserved regions, A, B, C, and D. Region E is adjacent to RE and enhances its activity. Regions A, B, D, and E contain binding sites for the Fkh transcription factors, and region C contains binding sites for the Swi4/Swi6 (SBF) transcription factors.
The role of Swi4/Swi6 (SBF) transcription factors:
Coic, Sun, Wu, & Haber (2006) demonstrated that donor preference depends on binding of the Swi4/Swi6 (SBF) transcription factors to an SCB site within the RE. Researchers measured HMLα usage in Swi4 deleted and wild-type strains. MATa usage of HMLα was significantly reduced in strains with Swi4 deleted (30% usage) compared to the wild type strains (70% usage). This indicates that the SBF complex is involved in RE activation in MATa cells.
Further, to confirm the importance of SBF, Coic et al. (2006) tested the effect of swi6∆ (swi6 deleted mutants) on spontaneous recombination on one chromosome. Results indicate that swi6∆ caused a threefold reduction in the rate of spontaneous recombination in MATa cells. Thus, the absence of the SBF complex leads to a reduction of HMLα usage and spontaneous recombination, and SBF plays a crucial role in RE activation. This study has no evident weaknesses.
The role of forkhead proteins (Fhk1p and Fhk2p):
Sun et al. (2002) demonstrated that an Fhk1∆ mutation reduces HMLα usage in MATa cells, though not to the level seen when RE is deleted. Researchers compared RE sequences among 3 yeast species, including S. cerevisiae, S. carlsbergensis, and S. bayanus and confirmed five highly conserved regions (A, B, C, D, and E) within and adjacent to RE.
Proteins in RE regions A, B, D, and E each bind to transcription activator forkhead proteins Fhk1p and Fhk2p to promote selective use of HMLα. Deletion of Fkh1p significantly reduced MATa use of HMLα. Further, deletion of Fkh1/Fkh2 binding sites in region A significantly reduced MATa use of HMLα.
Figure 5: Effect of different combinations of regions A, C, D, and E on the percentage of HMLα usage in MATa cells. Different synthetic RE fragments were introduced into the chromosome III to replace the 1.8-kb sequence containing the RE.
Based on the results of this experiment, researchers concluded that forkhead proteins regulate MATa donor preference via direct interaction with RE. No limitations of this study are presented or evident.
Genetic studies of donor preference have led to new conclusions regarding chromosome architecture, nuclear organization, and RE function. Recent studies rule out the possibility that donor preference occurs via mating type-regulated changes in chromatin structure of donors. Chromosomally tethered fluorescent proteins and deconvolution microscopy show that the left arm is tethered and folded into an isolated domain, so it is not accessible to sequences on the right arm. RE activates HMLα by modifying the architecture and mobility of the left arm of chromosome III. Though the two arms of chromosome III constitute different domains not accessible to each other in MATα cells, the activation of RE in MATa cells suppresses this barrier (Coic et al., 2006).
Overall, the architecture of chromosome III is the foundation of donor preference regulation in yeast. Chromosome III is composed of two independent arms, inaccessible to each other in the absence of the RE. The RE works through the entire left arm of chromosome III, and it is dependent on other sequences of the chromosome.
Recap: Top 10 must-know facts (for quick reference and studying purposes):
- S. cerevisiae can exist in a diploid and a haploid state.
- Haploid cells can be either a or α mating types and can switch mating types as often as every cell cycle.
- MAT locus on chromosome III: Expression of genes on this locus determines mating type preference.
- HO endonuclease: This physically cleaves DNA at the MAT locus to initiate switching.
- HMRa and HMLα: Two possible genetic donors used during mating-type switching.
- MATa cells choose HMLα as a donor, MATα cells choose HMRa
- Recombination Enhancer (RE): This determines donor preference. Activation of RE is required to increase HMLα preference.
- Swi4/Swi6 (SBF) transcription factors and Forkhead proteins (Fkh1p and Fkh2p): These are the two independent RE activation pathways.
- Swi4/Swi6 (SBF) transcription factors: These have binding sites in region C of RE and bind during phase G1.
- Forkhead proteins (Fkh1p and Fkh2p): These have binding sites in region A, B, D, and E of RE and bind during phase G2.
- Coic, E., Richard, G. F., & Haber, J. E. (2006). Saccharomyces cerevisiae donor preference during mating-type switching is dependent on chromosome architecture and organization. [Article]. Genetics, 173(3), 1197-1206.
- Coic, E., Sun, K., Wu, C., & Haber, J. E. (2006). Cell cycle-dependent regulation of Saccharomyces cerevisiae donor preference during mating-type switching by SBF (Swi4/Swi6) and Fkh1. [Article]. Molecular and Cellular Biology, 26(14), 5470-5480.
- Sun, K. M., Coic, E., Zhou, Z. Q., Durrens, P., & Haber, J. E. (2002). Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer. [Article]. Genes & Development, 16(16), 2085-2096.
- Weiss, K., & Simpson, R. T. (1998). High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HML alpha. [Article]. Molecular and Cellular Biology, 18(9), 5392-5403.