Mating-type Switching In Fission Yeast Research Articles

Euchromatin factors HULC and Set1C affect heterochromatin organization and mating-type switching in fission yeast Schizosaccharomyces pombe.

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

RNA insertion in DNA as the imprint moiety: the fission yeast paradigm.

This review elaborates on the findings of a new report which possibly resolves the biochemical nature of a novel type of DNA imprint as ribonucleotide and the mechanism of its formation during cell differentiation in fission yeast. The process of mating-type switching in fission yeast, Schizosaccharomyces pombe, displays characteristics of a typical mammalian stem cell lineage, wherein a cell divides to produce an identical cell and a differentiated cell after every two cell divisions. This developmental asymmetry has been ascribed to play a role in generation of a DNA strand-specific imprint at the mat1 locus during lagging strand synthesis and its segregation to one of the two daughter cells by the process of asymmetric, semi-conservative DNA replication. The nature of this imprint and mechanisms of its generation have been a subject of research and debate. A recent report by Singh et al. (Nucleic Acids Res 47:3422-3433. https://doi.org/10.1093/nar/gkz092 , 2019) provides compelling evidence in support of a ribonucleotide as the imprint moiety within the mat1 DNA and demonstrates the role of Mcm10/Cdc23, an important, evolutionarily conserved component of DNA replication machinery in eukaryotes, in installing the imprint through a non-canonical primase activity and interaction with DNA Polα and Swi1. The high degree of conservation of DNA replication machinery, especially the presence of the T7 gene 4 helicase/primase domain in the mammalian orthologs of Mcm10 suggests that similar mechanisms of DNA imprinting may play a role during cell differentiation in metazoans.

Two Portable Recombination Enhancers Direct Donor Choice in Fission Yeast Heterochromatin

Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality.

Molecular and Cellular Dissection of Mating-Type Switching Steps in Schizosaccharomyces pombe

A strand-specific imprint (break) controls mating-type switching in fission yeast. By introducing a thiamine repressible promoter upstream of the mat1 locus, we can force transcription through the imprinted region, erasing the imprint and inhibiting further mating-type switching, in a reversible manner. Starting from a synchronized, virgin M-cell population, we show that the site- and strand-specific break is formed when DNA replication intermediates appear at mat1 during the first S phase. The formation of the break is concomitant with a replication fork pause and binding of the Swi1 protein at mat1 until early G(2) and then rapidly disappears. Upon its formation, the break remains stable throughout the cell cycle and triggers mating-type switching during the second S phase. Finally, we have recreated the mating-type switching pedigree at the molecular and single-cell levels, allowing for the first time separation between the establishment of imprinting and its developmental fate.

DNA Replication: Stalling a Fork for Imprinting and Switching

Mating-type switching in fission yeast has long been known to be directed by a DNA 'imprint'. This imprint has now been firmly characterized as a protected site-specific and strand-specific nick. New work also links the widely conserved Swi1-Swi3 complex to the protection of stalled replication forks in general.

RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching.

Mating-type switching in fission yeast depends on an imprint at the mat1 locus. Previous data showed that the imprint is made in the DNA strand replicated as lagging. We now identify this imprint as an RNase-sensitive modification and suggest that it consists of one or two RNA residues incorporated into the mat1 DNA. Formation of the imprint requires swi1- and swi3-dependent pausing of the replication fork. Interestingly, swi1 and swi3 mutations that abolish pausing do not affect the use of lagging-strand priming site during replication. We show that the pausing of replication and subsequent formation of the imprint occur after the leading-strand replication complex has passed the site of the imprint and after lagging-strand synthesis has initiated at this proximal priming site. We propose a model in which a swi1- and swi3-dependent signal during lagging-strand synthesis leads to pausing of leading-strand replication and the introduction of the imprint.

The smt-0 mutation which abolishes mating-type switching in fission yeast is a deletion.

Mating-type switching in the fission yeast, S. pombe, is initiated by a DNA double-strand break (DSB) between the mat1 cassette and the H1 homology box. The mat1-cis-acting mutant, smt-0, abolishes mating-type switching and is shown here to be a 263-bp deletion. This deletion starts in the middle of the H1 homology box, 31 bp from the site of the DSB, and extends into the flanking region distal to mat1. The sequence of the region distal to H1 in the wild-type is also presented. In this region we observe a bias in the distribution of purine residues between the two DNA strands.

DNA polymerase-alpha is essential for mating-type switching in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the double-stranded chromosomal break (DSB) at the mating-type locus (mat1) initiates recombination during mating-type switching. A constant DSB level is maintained throughout the cell-cycle. In the strand-segregation model for mating-type switching, it was postulated that if the DSB is generated during or soon after mat1 replication, one of the chromatids could be repaired and switched during replication in the next cell cycle, while the other chromatid inherits the break. Here we report a molecular characterization of swi7, one of the genes required for DSB formation. Surprisingly, a gene complementing the swi7 mutation maps to chromosome I and encodes S. pombe DNA polymerase-alpha. Disruption of this gene is lethal in both switching and non-switching strains, as expected. S. pombe DNA polymerase-alpha must therefore play a role in generating the DSB at mat1, suggesting that DSB formation is coupled with DNA replication.

Genes required for initiation and resolution steps of mating-type switching in fission yeast.

The fission yeast Schizosaccharomyces pombe switches mating type by transposition of a copy of DNA derived from either of the two storage cassettes, mat2 -P and mat3 -M, into the expression locus, mat1 . The recombinational event of switching is initiated by a double-stranded DNA break present in approximately 20% of the molecules at mat1 . Fifty-three mutants defective in switching of mating type have been isolated previously, and each has been assigned to 1 of 10 linkage groups. One group consists of cis-acting mutations at mat1 , which reduce the amount of the DNA double-strand cut. The remaining nine groups are mutations in genes that are unlinked to the mating-type locus and are studied here. Three ( swi1 , -3, -7) are required for formation of the double-strand cut, whereas the others are not. Mutants of three genes ( swi4 , -8, -9) undergo high-frequency rearrangement of the mating-type locus indicative of errors of resolution of recombinational intermediates. The remaining three ( swi2 , -5, -6) have normal levels of cut, do not make errors of resolution, and possibly are required either for efficient utilization of the cut or determining the directionality of switching. The data suggest that the switching process can be dissected into genetically distinguishable steps.