Unbiased segregation of fission yeast chromosome 2 strands to daughter cells.

The base complementarity feature (Watson and Crick in Nature 171(4356):737-738, 1953) and the rule of semi-conservative mode of DNA replication (Messelson and Stahl in Proc Natl Acad Sci U S A 44:671-682, 1958) dictate that two identical replicas of the parental chromosome are produced during replication. In principle, the inherent strand sequence differences could generate nonequivalent daughter chromosome replicas if one of the two strands were epigenetically imprinted during replication to effect silencing/expression of developmentally important genes. Indeed, inheritance of such a strand- and site-specific imprint confers developmental asymmetry to fission yeast sister cells by a phenomenon called mating/cell-type switching. Curiously, location of DNA strands with respect to each other at the centromere is fixed, and as a result, their selected segregation to specific sister chromatid copies occurs in eukaryotic cells. The yeast system provides a unique opportunity to determine the significance of such biased strand distribution to sister chromatids. We determined whether the cylindrical-shaped yeast cell distributes the specific chromosomal strand to the same cellular pole in successive cycles of cell division. By observing the pattern of recurrent mating-type switching in progenies of individual cells by microscopic analyses, we found that chromosome 2 strands are distributed by the random mode in successive cell divisions. We also exploited unusual "hotspot" recombination features of this system to investigate whether there is selective segregation of strands such that oldest Watson-containing strands co-segregate in the diploid cell at mitosis. Our data suggests that chromosome 2 strands are segregated independently to those of the homologous chromosome.

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.

Remarkably high rate of DNA amplification promoted by the mating-type switching mechanism in Schizosaccharomyces pombe.

A novel mating-type switching-defective mutant showed a highly unstable rearrangement at the mating-type locus (mat1) in fission yeast. The mutation resulted from local amplification of a 134-bp DNA fragment by the mat1-switching phenomenon. We speculate that the rolling-circle-like replication and homologous recombination might be the general mechanisms for local genome region expansion.

Going in the right direction: mating-type switching of Schizosaccharomyces pombe is controlled by judicious expression of two different swi2 transcripts.

Schizosaccharomyces pombe, the fission yeast, cells alternate between P- and M-mating type, controlled by the alternate alleles of the mating-type locus (mat1). The mat1 switching occurs by replacing mat1 with a copy derived from a silenced "donor locus," mat2P or mat3M. The mechanism of donor choice ensuring that switching occurs primarily and productively to the opposite type, called directionality, is largely unknown. Here we identified the mat1-Mc gene, a mammalian sex-determination gene (SRY) homolog, as the primary gene that dictates directionality in M cells. A previously unrecognized, shorter swi2 mRNA, a truncated form of the swi2, was identified, and its expression requires the mat1-Mc function. We also found that the abp1 gene (human CENPB homolog) controls directionality through swi2 regulation. In addition, we implicated a cis-acting DNA sequence in mat2 utilization. Overall, we showed that switching directionality is controlled by judicious expression of two swi2 transcripts through a cell-type-regulated dual promoter. In this respect, this regulation mechanism resembles that of the Drosophila sex-determination Slx gene.

The Clr7 and Clr8 directionality factors and the Pcu4 cullin mediate heterochromatin formation in the fission yeast Schizosaccharomyces pombe.

Fission yeast heterochromatin is formed at centromeres, telomeres, and in the mating-type region where it mediates the transcriptional silencing of the mat2-P and mat3-M donor loci and the directionality of mating-type switching. We conducted a genetic screen for directionality mutants. This screen revealed the essential role of two previously uncharacterized factors, Clr7 and Clr8, in heterochromatin formation. Clr7 and Clr8 are required for localization of the Swi6 chromodomain protein and for histone H3 lysine 9 methylation, thereby influencing not only mating-type switching but also transcriptional silencing in all previously characterized heterochromatic regions, chromosome segregation, and meiotic recombination in the mating-type region. We present evidence for physical interactions between Clr7 and the mating-type region and between Clr7 and the S. pombe cullin Pcu4, indicating that a complex containing these proteins mediates an early step in heterochromatin formation and implying a role for ubiquitination at this early stage prior to the action of the Clr4 histone methyl-transferase. Like Clr7 and Clr8, Pcu4 is required for histone H3 lysine 9 methylation, and bidirectional centromeric transcripts that are normally processed into siRNA by the RNAi machinery in wild-type cells are easily detected in cells lacking Clr7, Clr8, or Pcu4. Another physical interaction, between the nucleoporin Nup189 and Clr8, suggests that Clr8 might be involved in tethering heterochromatic regions to the nuclear envelope by association with the nuclear-pore complex.