Defining the Epigenetic Mechanism of Asymmetric Cell Division of Schizosaccharomyces japonicus Yeast

A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four “granddaughter” cells switches. The DNA-strand–specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand–specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well.     

Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast.

Homothallic Saccharomyces yeasts efficiently interconvert between two cell types, the mating types a and alpha. These interconversions have been proposed to occur by genetic rearrangement ("cassette" insertion) at the locus controlling cell type (the mating type locus). The pattern of switching from one cell type to the other during growth of a clone of homothallic cells has been followed by direct microscopic observation, and the results have been summarized as "rules" of switching. First, when a cell divides, it produces either two cells with the same mating type as the original cell or two cells that have switched to the other mating type. This observation suggests that the mating type locus is changed early in the cell cycle, in late Gl or during S. Second, the ability to produce cells that have switched mating type is restricted to cells that have previously divided ("experienced cells"). Spores and buds ("inexperienced cells") rarely if ever give rise to cells with changed mating type. A homothallic yeast cell thus exhibits asymmetric segregation of the potential for mating type interconversion--at each cell division, the mother, but not the daughter, is capable of switching cell types in its next division. Homothallic cells also exhibit directionality in switching: experienced cells switch to the opposite cell type in more than 50% of cell divisions. These results show that the process of mating type interconversion is itself controlled during growth of a clone of homothallic cells. By analogy and extension of these results, we propose that multiple cell types can be produced in a specific pattern during development of a higher eucaryote in a model involving sequential cassette insertion.     

Developmental choices in mating-type interconversion in fission yeast.

Fission yeast cells follow a specific pattern of mating (cell) type switching in single cell pedigrees. Asymmetric cell divisions producing sisters of different developmental fates result from inheritance of specific parental DNA strands according to the classical model of semiconservative replication and segregation.     

Fission yeast switches mating type by a replication-recombination coupled process

Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion. Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type. It was shown recently that a site- and strand-specific DNA modification at the mat1 locus precedes mating-type switching. By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating-type switching in S-phase. The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating-type switching occurs through a replication-recombination coupled pathway. Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating-type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage.     

A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe.

The pattern of parental DNA strand inheritance at the mating type locus (mat1) determines the pattern of mat1 switching in a cell lineage by regulating the formation of the site-specific double-stranded break (DSB) required for mating type interconversion in Schizosaccharomyces pombe. To study the molecular basis of this programmable cell type change, we conducted structural and functional analyses of the DNA sequence flanking the DSB at mat1. We have identified and characterized a DNA-binding activity that interacts with a specific sequence located 140 bp from the DSB site. Deletion analysis of DNA sequences located distal to mat1 cassette revealed the presence of at least two switch-activating sites (SAS1 and SAS2), both of which are required for generating an efficient level of DSBs and consequently, for efficient switching. We found that SAS1 overlaps with the target site of the DNA-binding activity called SAP1 (for switch-activating protein). Point mutations generated in the SAS1 element that adversely affect binding of SAP1 protein in vitro were found to reduce the efficiency of switching in vivo, suggesting the requirement of SAP1 for switching. Pedigree analysis revealed that SAS1 is equally required for initial switching (one switch in four grand-daughters of a cell) and for consecutive switching (where the sister of a recently switched cell switches again), indicating that the two developmentally asymmetric cell divisions required to generate a particular pattern of switching share the same molecular control mechanism.     

The Mechanism of Fission Yeast Mating Type Interconversion: Seal/Replicate/Cleave Model of Replication across the Double-Stranded Break Site at Mat1

The interconversion of cell type in the fission yeast, Schizosaccharomyces pombe, is initiated by a double-stranded break (DSB) found at the mating type locus (mat1). A heritable site- and strand-specific DNA "imprinting" event at mat1 was recently hypothesized to be required to make the mat1 locus cleavable, and the DSB was suggested to be produced one generation before the actual switching event. It is known that only one cell among four granddaughters of a cell ever switches, and the sister of the recently switched cell switches efficiently in consecutive cell divisions. The feature of consecutive switching creates a major difficulty of having to replicate chromosomes possessing the DSB. The mat1 cis-acting leaky mutation, called smt-s, reduces the level of the DSB required for switching and is shown here to be a 27-bp deletion located 50 bp away from the cut site. Determination of the pattern and frequency of switching of the mutant allele by cell lineage studies has allowed us to conclude the following: (1) the chromosome with the DSB is sealed and replicated, then one of the specific chromatids is cleaved again to generate switching-competent cells in consecutive cell divisions and (2) the smt-s mutation affects DNA cleavage and not the hypothesized DNA imprinting step.     

lin-17 mutations of Caenorhabditis elegans disrupt certain asymmetric cell divisions

The identification of a gene necessary for the asymmetry of cell division would be an important first step toward understanding how sister cells come to differ in their developmental fates. The lin-17 gene of the nematode Caenorhabditis elegans is an excellent candidate for being such a gene. lin-17 mutations cause several blast cells that normally generate sister cells of two distinct types to generate instead sister cells of the same type. Moreover, lin-17 mutations cause sister cells to be equal in size as well as equivalent in developmental fate, suggesting that lin-17 acts at or prior to the asymmetric cell division. The lin-17 gene product is involved in asymmetric cell divisions in a variety of tissues, indicating that lin-17 functions in a general mechanism for the establishment of cellular asymmetry in parent cells.     

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.     

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.     

Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism

Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.     

Interconversion of Yeast Mating Types I. Direct Observations of the Action of the Homothallism (HO) Gene

The HO gene promotes interconversion between a and α mating types. As a consequence, homothallic diploid cells are formed by mating between siblings descended from a single α HO or a HO spore. In order to determine the frequency and pattern of the mating-type switch, we have used a simple technique by which the mating phenotype can be assayed without losing the cell to the mating process itself. Specifically, we have performed pedigree analysis on descendants of single homothallic spores, testing these cells for sensitivity to α-factor.

The switch from α to a and vice versa is detectable after a minimum of two cell divisions. 50% of the clones tested showed switching by the four-cell stage. Of the four cells descended from a single cell, only the oldest cell and its immediate daughter are observed to change mating type. This pattern suggests that one event in the switching process has occurred in the first cell division cycle. Restriction of the switched mating-type to two particular cells may reflect the action of the homothallism system followed by nonrandom segregation of DNA strands in mitosis.

The mating behavior of cells which have sustained a change in mating type due to the HO gene is indistinguishable from that of heterothallic strains.

     

Does S. pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching?

Typically cell division is envisaged to be symmetrical, with both daughter cells being identical. However, during development and cellular differentiation, asymmetrical cell divisions have a crucial role. In this article, we describe a model of how Schizosaccharomyces pombe exploits the intrinsic asymmetry of DNA replication machinery--the difference between the replication of the leading strand and the lagging strand--to establish an asymmetrical mating-type switching pattern. This is the first system where the direction of DNA replication is involved in the formation of differentiated chromosomes. The discovery raises the possibility that DNA replication might be more generally involved in the establishment of asymmetric cellular differentiation.     

Stress-Induced Switch to Pseudohyphal Growth in S. Pombe

Several genetic mutants of Schizosaccharomyces pombe that form multiple septa and pseudohyphae (i.e. branching growth) have been isolated.1-15 The current understanding of these mutants is that they lack the ability to separate the two sister cells after formation of the septum. Here it is shown that switching to multisepta and pseudohyphal growth can be induced in a reversible manner in wild-type S. pombe cells by changing the growth conditions, thus indicating an inherent cellular switch. Flow cytometry profiles of exponentially growing cultures of both wild-type and mutant cells further support that a bi-stable switch is controlling the morphological state of the cell in a stress-dependent manner.     

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.     

Mating type control in Saccharomyces cerevisiae: a frameshift mutation at the common DNA sequence, X, of the HML alpha locus.

A mutation defective in the homothallic switching of mating type alleles, designated hml alpha-2, has previously been characterized. The mutation occurred in a cell having the HO MATa HML alpha HMRa genotype, and the mutant culture consisted of ca. 10% a mating type cells, 90% nonmater cells of haploid cell size, and 0.1% sporogenous diploid cells. Genetic analyses revealed that nonmater haploid cells have a defect in the alpha 2 cistron at the MAT locus. This defect was probably caused by transposition of a cassette originating from the hml alpha-2 allele by the process of the homothallic mating type switch. That the MAT locus of the nonmater cells is occupied by a DNA fragment indistinguishable from the Y alpha sequence in electrophoretic mobility was demonstrated by Southern hybridization of the EcoRI-HindIII fragment encoding the MAT locus with a cloned HML alpha gene as the probe. The hml alpha-2 mutation was revealed to be a one-base-pair deletion at the ninth base pair in the X region from the X and Y boundary of the HML locus. This mutation gave rise to a shift in the open reading frame of the alpha 2 cistron. A molecular mechanism for the mating type switch associated with the occurrence of sporogenous diploid cells in the mutant culture is discussed.     

Analysis of mutations in the mat1 region of Schizosaccharomyces pombe strain with the deletion of gene rhp55+

DNA double-strand breaks may occur both under the action of various exogenous factors and in the course of cell metabolism processes, in particular, upon mating type switching in yeast. Genes belonging to the epistatic group RAD52 are known to repair such DNA damage. Molecular defects in mating type switching occurring after the deletion of gene rhp55+ encoding the paralog of recombinational protein Rhp51, which is a functional homolog of Escherichia coli RecA, were studied in fission yeast. Analysis of stable nonswitching segregants in h90 rhp55 mutants with unchanged configuration of the mating type switching locus but with a drastically decreased level of double-strand DNA break formation at the mat1 :1 locus demonstrated changes in DNA sequences within the region responsible for the generation of the breaks. These changes might have resulted from incorrect gene conversion upon repair of double-strand DNA breaks in Schizosaccharomyces pombe rhp55 mutants.     

Coconversion of flanking sequences with homothallic switching

Homothallic switching in S. cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR. The MATa and MAT alpha alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR. Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region. Coconversion events occur on both sides of the double-strand cleavage by the HO gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate.     

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.