Yeast Mating-type Interconversion Research Articles

The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer.

Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATα cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.

Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast

Abstract Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Homology modeling and mutational analysis of Ho endonuclease of yeast.

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Alpha2p controls donor preference during mating type interconversion in yeast by inactivating a recombinational enhancer of chromosome III.

Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.

Directionality of fission yeast mating-type interconversion is controlled by the location of the donor loci.

Cells of homothallic strains of Schizosaccharomyces pombe efficiently switch between two mating types called P and M. The phenotypic switches are due to conversion of the expressed mating-type locus (mat1) by two closely linked silent loci, mat2-P and mat3-M, that contain unexpressed information for the P and M mating types, respectively. In this process, switching-competent cells switch to the opposite mating type in 72-90% of the cell divisions. Hence, mat2-P is a preferred donor of information to mat1 in M cells, whereas mat3-M is a preferred donor in P cells. We investigated the reason for the donor preference by constructing a strain in which the genetic contents of the donor loci were swapped. We found that switching to the opposite mating type was very inefficient in that strain. This shows that the location of the silent cassettes in the chromosome, rather than their content, is the deciding factor for recognition of the donor for each cell type. We propose a model in which switching is achieved by regulating accessibility of the donor loci, perhaps by changing the chromatin structure in the mating-type region, thus promoting an intrachromosomal folding of mat2 or mat3 onto mat1 in a cell type-specific fashion. We also present evidence for the involvement of the Swi6 and Swi6-mod trans-acting factors in the donor-choice mechanism. We suggest that these factors participate in forming the proposed folded structure.

The mechanism of fission yeast mating-type interconversion: evidence for two types of epigenetically inherited chromosomal imprinted events.

Mating types of the fission yeast S. pombe interchange such that only one cell among four granddaughters of a cell ever switches, and the sister of the newly switched cell switches efficiently in consecutive cell divisions, thereby producing chains of recurrent switching. The developmental program is known to be mediated through specific parental chromosomal DNA-strand inheritance at the mating-type locus (mat1). A heritable DNA strand- and sequence-specific imprinting event was previously postulated to be required to cleave the chromosome at mat1 to initiate recombination required for switching. The pedigree analysis presented here demonstrates that swi1, swi3, and swi7 genes are required for the DNA cleavage step directly, and not for the imprinting function, since mutations in these genes reduce the efficiency of initial (i.e., switching of one in four granddaughters) and recurrent switching equally. However, when the mat1 gene sequence is placed adjacent to the indigenous mat1 locus, apparently it is imprinted inefficiently, since recurrent switches of the inserted locus occur nearly five times more often than the initial switches. This "runaway switching" is the first evidence formally demonstrating both the existence of and the requirement for an imprinted mat1 DNA, making it cleavable and consequently swichable. Second, the double-stranded break constitutes another imprinted event as it is evidenced to be inherited from the parental cell.

Regulation of fission yeast mating-type interconversion by chromosome imprinting

Mating types of the fission yeast Schizosaccharomyces pombe interchange nonrandomly in a cell lineage so that only one cell among four granddaughters of a cell ever switches, and the sister of the newly switched cell switches efficiently in consecutive cell divisions, thereby producing chains of recurrent switching. The programme of cellular differentiation is mediated by inheritance of parental DNA chains at the mating type locus (mat1) by progeny cells. This review summarizes recent results suggesting that two types of imprinting events at the mat1 locus are required to generate the specific pattern of switching in a cell lineage. One of those is a site- and strand- specific event that is required before the mat1 locus can be cleaved in vivo. The other is a double-stranded break at mat1 that is healed by gene conversion in the progeny cells resulting in switching the mat1 locus.

Differentiated parental DNA strands confer developmental asymmetry on daughter cells in fission yeast.

The two strands of the DNA molecule are complementary but not identical. Hence, upon semiconservative replication, different parental DNA strands are segregated to daughter cells. A molecular analysis suggests that the process of fission yeast mating-type interconversion uses asymmetry of the DNA strands to generate a regular lineage of cellular differentiation.

Gene Conversion in Trypanosome Antigenic Variation

Publisher Summary This chapter discusses gene conversion in African trypunosome antigenic variation. Antigenic variation allows African trypanosomes' to escape the immune defenses of their hosts. This occurs through differential surface antigen gene activation, with only one antigen gene being expressed at any one time among a large repertoire of different sequences. The differential gene activation can be achieved by gene conversion among antigen-specific sequences, taking place in a telomeric gene expression site. The analysis of several gene conversion endpoints and comparison with the mechanism for mating type interconversion in yeast suggests that the process could be triggered by a cut upstream from the antigen gene and then resolved by a crossing-over in a region of homology downstream from this point. This crossing-over could take place anywhere within the regions of homology, but only the recombinations leading to the successful generation of new antigen-coding sequences would be selected. Based on this gene conversion model, it is possible to explain the generation of mini-chromosomes harboring antigen-specific sequences. Translocation of such haploid telomeric sequences to the other end of other chromosomes would lead to the internalization of formerly telomeric genes and a clustering of haploid antigen-specific sequences near the chromosome termini. Studies show that the orientation of the conversion could be determined by the chromatin structure around the antigen gene.

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and β-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes. the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.

Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus.

The ultimate product of yeast mating-type interconversion is a stable a/alpha diploid cell. A haploid cell carrying the HO gene gives rise to a diploid cell in a two-step process: first, the cell switches mating type as a result of genetic rearrangement (cassette substitution) catalyzed by HO; then, cells of opposite type mate to form a/alpha diploids. Mating-type interconversion does not occur in a/alpha diploids despite the presence of the HO gene. We have identified a plasmid carrying the HO gene by screening a yeast clone bank (constructed in vector YEp13) for plasmids that allow mating-type switching by ho cells. The yeast segment responsible for mating-type interconversion integrates by homology at the ho locus, thus confirming that it carries HO. Using the HO gene as a probe, we find that strains with an active mating-type interconversion system produce HO RNA, whereas a/alpha HO/HO cells do not and that this inhibition requires products of both the MATa1 and MATa2 genes. Thus, mating-type interconversion does not occur in a/alpha HO/HO cells because the HO gene product is not synthesized. These results demonstrate the following: (i) The mating-type locus, proposed on genetic grounds to be a regulatory locus, controls expression of an unlinked gene (HO) at the level of RNA production. (ii) The HO gene is under negative feedback control: its expression is inhibited after successful completion of diploidization (formation of a/alpha diploids).

Directionality of yeast mating-type interconversion

The mating-type a and α alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus ( MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HMLα HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMRα arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches—that is, MATa to MATa and MATα to MATα—and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that a cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HMLα HMRa strains generate predominantly heterologous while HMLa HMRα strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the Inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.

Gene conversion between duplicated genetic elements in yeast.

The mitotic recombination behaviour of a duplication of the his4 region on chromosome III in the yeast Saccharomyces cerevisiae was studied. The major recombination event between the duplicated segments is gene conversion unassociated with reciprocal recombination. The rad52-1 mutation preferentially decreases mitotic gene conversion. These results suggest that mitotic gene conversion may occur by a different pathway from that occurring in meiosis. This mitotic gene conversion may be important in yeast mating type interconversion and the maintenance of sequence homogeneity in families of repeated eukaryotic genes.

Pattern of switching and fate of the replaced cassette in yeast mating-type interconversion.

Pattern of switching and fate of the replaced cassette in yeast mating-type interconversion.