Amar J. S. Klar

Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.

Four mating-type genes control sexual differentiation in the fission yeast.

The mating‐type region of fission yeast consists of three components, mat1, mat2‐P and mat3‐M, each separated by 15 kb. Cell‐type is determined by the alternate allele present at mat1, either P in an h+ or M in an h‐ cell. mat2‐P and mat3‐M serve as donors of information that is transposed to mat1 during a switch of mating type. We have determined the nucleotide sequence of each component of mat. The P and M specific regions are 1104 and 1128 bp, respectively, and bounded by sequences common to each mating‐type cassette (H1; 59 bp and H2; 135 bp). A third sequence is present at mat2‐P and mat3‐M but absent at mat1 (H3; 57 bp), and may be involved in transcriptional repression of these cassettes. mat1‐P and mat1‐M each encode two genes (Pc; 118 amino acids, Pi; 159 amino acids, Mc; 181 amino acids and Mi; 42 amino acids). Introduction of opal or frame‐shift mutations into the open‐reading‐frame of each gene revealed that Pc and Mc are necessary and sufficient for mating and confer an h+ or h‐ mating type respectively. All four genes are required for meiotic competence in an h+/h‐ diploid. The transcription of each mat gene is strongly influenced by nutritional conditions and full induction was observed only in nitrogen‐free medium. The predicted product of the Pi gene contains a region of homology with the homeobox sequence, suggesting that this gene encodes a DNA binding protein that directly regulates the expression of other genes.

Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

Chromosomal Inheritance of Epigenetic States in Fission Yeast During Mitosis and Meiosis

Inheritance of the active and inactive states of gene expression by individual cells is crucial for development. In fission yeast, mating-type region consists of three loci called mat1, mat2, and mat3. Transcriptionally silent mat2 and mat3 loci are separated by a 15 kb interval, designated the K-region, and serve as donors of information for transcriptionally active mat1 interconversion. In a strain carrying replacement of 7.5 kb of the K-region with the ura4 gene, we discovered that ura4 silencing and efficiency of mating-type switching were covariegated and were regulated by an epigenetic mechanism. Genetic analyses demonstrated that epigenetic states were remarkably stable not only in mitosis but also in meiosis and were linked to the mating-type region. This study indicates that different epigenetic states are heritable forms of chromatin organization at the mat region.

A Chromodomain Protein, Swi6, Performs Imprinting Functions in Fission Yeast during Mitosis and Meiosis

Inheritance of stable states of gene expression is essential for cellular differentiation. In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known. We show that a chromodomain-containing Swi6 protein is a dosage-critical component involved in imprinting the mat locus. Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state. The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis. Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint. Our analyses suggest that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex.

Regulation of mating-type information in yeast: Negative control requiring sequences both 5′ and 3′ to the regulated region☆

The genome of the yeast Saccharomyces cerevisiae contains three complete copies of the genetic information governing cell mating type. Normally, only the information in one of the copies (the MAT locus) is expressed; the other two copies (HML and HMR) are repressed and serve as donors of mating-type sequences that can be transposed to MAT in cells capable of switching mating type. We have mutagenized the silent HMR locus and have found that the repression of this locus requires two sites, one lying on each side of the mating-type sequences at HMR. The regulatory sites are positioned outside of the sequences that are included in the pair of divergent transcripts coded for by HMR, and lie about 1000 base-pairs to either side of the central promoter region of the locus. Deletion of one of the regulatory sites results phenotypically in complete loss of repression, whereas deletion of the other site gives only partial loss of control. Both of the sites are associated with an autonomous replication activity, though the relationship between this activity and the process of repression is unclear.

Rearrangements of the transposable mating-type cassettes of fission yeast.

The fission yeast, Schizosaccharomyces pombe, switches mating type every few cell divisions. Switching is controlled by the genes of the mating‐type locus, which consists of three components, mat1, mat2‐P and mat3‐M, each separated by approximately 15 kb. Copy transposition of P (Plus) or M (Minus) information from mat2‐P or mat3‐M into the expression locus mat1 mediates cell type switching. The mating‐type locus undergoes events at high frequency (10(‐2)‐10(‐6)) which stabilize one or other mating type. These events are shown to be rearrangements which result in either deletion or insertion of DNA between cassettes.

The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands.

A key feature for development consists of producing sister cells that differ in their potential for cellular differentiation. Following two cell divisions, a haploid Schizosaccharomyces pombe cell produces one cell in four ‘granddaughters’ with a changed mating cell type, implying nonequivalence of sister cells in each of two consecutive cell divisions. The observed pattern of switching is analogous to the mammalian ‘stem cell’ lineage by which a cell produces one daughter like itself while the other daughter is advanced in its developmental program. It is tested here whether sisters differ because of unequal distribution of cytoplasmic and/or nuclear components to them or due to inheriting a specific parental DNA chain at the mating type locus. Only the DNA strand‐segregation model predicts that those cells engineered to contain an inverted tandem duplication of the mating type locus should produce equivalent sisters. Consequently, two ‘cousins’ in four related granddaughter cells should switch. The results verified the prediction, thus establishing that all cells otherwise fully possess the potential to switch. Therefore, the program of cell type change in S.pombe cell lineages is determined by the pattern of DNA strand inheritance at the mating type locus. A specific DNA sequence present at the mating type locus is postulated to be the cause of developmental asymmetry between sister cells. A general model for cellular differentiation is proposed in which the act of DNA replication itself is hypothesized to produce developmentally nonequivalent sister genomes.

Directionality of yeast mating-type interconversion

The mating-type a and alpha 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 HML alpha 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 HMR alpha 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 MAT alpha to MAT alpha--and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that alpha 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, HML alpha HMRa strains generate predominantly heterologous while HMLa HMR alpha 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.

A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating‐type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase α (polα) affects Swi6 localization at the mating‐type region and causes a 45‐fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing‐defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures.