Discovery of the mitotic selective chromatid segregation phenomenon and its implications for vertebrate development.

The asymmetric cell division process is required for cellular differentiation and embryonic development. Recent evidence obtained in Drosophila and C. elegans suggest that this process occurs by non-equivalent distribution of proteins or mRNA (intrinsic factors) to daughter cells, or by their differential exposure to cell extrinsic factors. In contrast, haploid fission yeast sister cells developmentally differ by inheriting sister chromatids that are differentiated by epigenetic means. Specifically, the act of DNA replication at the mating-type locus in yeast switches it's alternate alleles only in one specific member of chromosome 2 sister chromatids in nearly every chromosome replication cycle. To employ this kind of mechanism for cellular differentiation, strictly based on Watson-Crick structure of DNA in diploid organism, selective segregation mechanism is required to coordinate distribution of potentially differentiated sister chromatids to daughter cells. Genetic evidence to this postulate was fortuitously provided by the analysis of mitotic recombinants of chromosome 7 in mouse cells. Remarkably, the biased segregation occurs in some cell types but not in others and the process seems to be chromosome-specific. This review summarizes the discovery of selective chromatid segregation phenomenon and it suggests that such a process of Somatic Sister chromatid Imprinting and Selective chromatid Segregation (SSIS model) might explain development in eukaryotes, such as that of the body axis left-right visceral organs laterality specification in mice.

Rtf1-mediated eukaryotic site-specific replication termination.

The molecular mechanisms mediating eukaryotic replication termination and pausing remain largely unknown. Here we present the molecular characterization of Rtf1 that mediates site-specific replication termination at the polar Schizosaccharomyces pombe barrier RTS1. We show that Rtf1 possesses two chimeric myb/SANT domains: one is able to interact with the repeated motifs encoded by the RTS1 element as well as the elements enhancer region, while the other shows only a weak DNA binding activity. In addition we show that the C-terminal tail of Rtf1 mediates self-interaction, and deletion of this tail has a dominant phenotype. Finally, we identify a point mutation in Rtf1 domain I that converts the RTS1 element into a replication barrier of the opposite polarity. Together our data establish that multiple protein DNA and protein-protein interactions between Rtf1 molecules and both the repeated motifs and the enhancer region of RTS1 are required for site-specific termination at the RTS1 element.

A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S. pombe.

Mating-type switching in Schizosaccharomyces pombe involves a strand-specific, alkali-labile imprint at the mat1 (mating-type) locus. The imprint is synthesized during replication in a swi1, swi3, and polymerase alpha (swi7) dependent manner and is dependent on mat1 being replicated in a specific direction. Here we show that the direction of replication at mat1 is controlled by a cis-acting polar terminator of replication (RTS1). Two-dimensional gel analysis of replication intermediates reveals that RTS1 only terminates replication forks moving in the centromere-distal direction. A genetic analysis shows that RTS1 optimizes the imprinting process. Transposing the RTS1 element to the distal side of mat1 abolishes imprinting of the native mat1 allele but restores imprinting of an otherwise unimprinted inverted mat1 allele. These data provide conclusive evidence for the "direction of replication model" that explains the asymmetrical switching pattern of S. pombe, and identify a DNA replication-arrest element implicated in a developmental process. Such elements could play a more general role during development and differentiation in higher eukaryotes by regulating the direction of DNA replication at key loci.

The histone deacetylase genes HDA1 and RPD3 play distinct roles in regulation of high-frequency phenotypic switching in Candida albicans.

Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2, and HOS3) have been cloned from Candida albicans and characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylases HDA1 and RPD3 is down-regulated in the opaque phase of the white-opaque transition in strain WO-1. HOS3 is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase. HDA1 and RPD3 were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion of HDA1 resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion of RPD3 resulted in an increase in the frequency of switching in both directions. Deletion of HDA1 resulted in reduced white-phase-specific expression of the EFG1 3.2-kb transcript, but had no significant effect on white-phase-specific expression of WH11 or opaque-phase-specific expression of OP4, SAP1, and SAP3. Deletion of RPD3 resulted in reduced opaque-phase-specific expression of OP4, SAP1, and SAP3 and a slight reduction of white-phase-specific expression of WH11 and 3.2-kb EFG1. Deletion of neither HDA1 nor RPD3 affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein gene MCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genes HDA1 and RPD3 play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.

A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans.

Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.

A role for DNA polymerase alpha 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 alpha (pol(alpha)) 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 pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures.

Solution structure, domain features, and structural implications of mutants of the chromo domain from the fission yeast histone methyltransferase Clr4.

The encapsulation of otherwise transcribable loci within transcriptionally inactive heterochromatin is rapidly gaining recognition as an important mechanism of epigenetic gene regulation. In the fission yeast Schizosaccharomyces pombe, heterochromatinization of the mat2/mat3 loci silences the mating-type information encoded within these loci. Here, we present the solution structure of the chromo domain from the cryptic loci regulator protein Clr4. Clr4 is known to regulate silencing and switching at the mating-type loci and to affect chromatin structure at centromeres. Clr4 and its human and Drosophila homologs have been identified as histone H3-specific methyltransferases, further implicating this family of proteins in chromatin remodeling. Our structure highlights a conserved surface that may be involved in chromo domain-ligand interactions. We have also analyzed two chromo domain mutants (W31G and W41G) that previously were shown to affect silencing and switching in full-length Clr4. Both mutants are significantly destabilized relative to wild-type.

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.

swi1 and swi3 perform imprinting, pausing, and termination of DNA replication in S. pombe.

The developmental program of cell-type switching of S. pombe requires a strand-specific imprinting event at the mating-type locus (mat1). Imprinting occurs only when mat1 is replicated in a specific direction and requires several trans-acting factors. This work shows (1) that the factors swi1p and swi3p act by pausing the replication fork at the imprinting site; and (2) that swi1p and swi3p are involved in termination at the mat1-proximal polar-terminator of replication (RTS1). A genetic screen to identify termination factors identified an allele that separated pausing/imprinting and termination functions of swip. These results suggest that swi1p and swi3p promote imprinting in novel ways both by pausing replication at mat1 and by terminating replication at RTS1.

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.

Genetic models for handedness, brain lateralization, schizophrenia, and manic-depression.

There has been a long-standing debate to explain the complex correlation of development of human hand preference with brain lateralization, and occasionally, the correlation of both lateralizations with psychiatric disorders. A major unanswered question in this debate is whether nature (i.e., genetics) or nurture (environment/culture) controls the development of these attributes of human behavior. Simple genetic models have failed to satisfactorily explain the mode of inheritance of psychotic disorders as well as of the handedness trait. This paper advances several hypothetical and testable genetic models to explain the complex inheritance of these traits. In one model, brain lateralization is proposed to result from nonrandom segregation of the 'Watson' and 'Crick' strands of a particular chromosome, causing hemisphere lateralization, and that a gene, designated RGHT (for right), is further proposed to be responsible for the distribution of DNA chains to specific hemispheres. Accordingly, dominant, familially inherited schizophrenia and bipolar disorders are postulated to result from chromosomal rearrangements disrupting strand segregation, while sporadic cases are proposed to occur at increased frequencies in individuals with the recessive handedness genotype. Finally, discordance in monozygotic twins is suggested to occur due to developmental differences in brain lateralization in twins of the recessive genotype. Psychotic disorders are suggested to be due to developmental anomalies of cerebral asymmetry.

Orientation of DNA replication establishes mating-type switching pattern in S. pombe.

The fission yeast Schizosaccharomyces pombe normally has haploid cells of two mating types, which differ at the chromosomal locus mat1. After two consecutive asymmetric cell divisions, only one in four 'grand-daughter' cells undergoes a 'mating-type switch', in which genetic information is transferred to mat1 from the mat2-P or mat3-M donor loci. This switching pattern probably results from an imprinting event at mat1 that marks one sister chromatid in a strand-specific manner, and is related to a site-specific, double-stranded DNA break at mat1. Here we show that the genetic imprint is a strand-specific, alkali-labile DNA modification at mat1. The DNA break is an artefact, created from the imprint during DNA purification. We also propose and test the model that mat1 is preferentially replicated by a centromere-distal origin(s), so that the strand-specific imprint occurs only during lagging-strand synthesis. Altering the origin of replication, by inverting mat1 or introducing an origin of replication, affects the imprinting and switching efficiencies in predicted ways. Two-dimensional gel analysis confirmed that mat1 is preferentially replicated by a centromere-distal origin(s). Thus, the DNA replication machinery may confer different developmental potential to sister cells.

Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast.

Position-effect control at the silent mat2-mat3 interval and at centromeres and telomeres in fission yeast is suggested to be mediated through the assembly of heterochromatin-like structures. Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly. Here, we report the identification of an essential gene, clr6 (cryptic loci regulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the mat2-mat3 region and at centromeres and reduces the fidelity of chromosome segregation. Furthermore, we show that the Clr3 protein, when mutated, alleviates recombination block at mat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases. Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities. We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state. This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.

A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast.

Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure.

The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast.

Heritable inactivation of specific regions of the genome is a widespread, possibly universal phenomenon for gene regulation in eukaryotes. Self-perpetuating, clonally inherited chromatin structure has been proposed as the explanation for such phenomena as position-effect variegation (PEV) and control of segment determination and differentiation in flies, X-chromosome inactivation and parental imprinting in mammals, gene silencing by paramutation in maize and silencing of the mating-type loci in yeasts. We have now found that the clr4 gene, which is essential for silencing of centromeres and the mating-type loci in Schizosaccharomyces pombe, encodes a protein with high homology to the product of Su(var)3-9, a gene affecting PEV in Drosophila. Like Su(var)3-9p, Clr4p contains SET and chromo domains, motifs found in proteins that modulate chromatin structure. Site-directed mutations in the conserved residues of the chromo domain confirm that it is required for proper silencing and directional switching of the mating type, like SET domain. Surprisingly, RNA differential display experiments demonstrated that clr4+ can mediate transcriptional activation of certain other loci. These results show that clr4 plays a critical role in silencing at mating-type loci and centromeres through the organization of repressive chromatin structure and demonstrate a new, activator function for Clr4p.

Multiple epigenetic events regulate mating-type switching of fission yeast.

Two epigenetic events at mat1, one of which is DNA strand specific, are required to initiate recombination during mating-type switching. The third, a chromosomally borne imprinted event at the mat2/3 interval regulates silencing and directionality of switching, and prohibits interchromosomal recombination. We speculate that the unit of inheritance in the mat2/3 interval is both DNA plus its associated chromatin structure. Such a control is likely to be essential in maintaining particular states of gene expression during development.

A recombinationally repressed region between mat2 and mat3 loci shares homology to centromeric repeats and regulates directionality of mating-type switching in fission yeast.

Cells of the fission yeast Schizosaccharomyces pombe switch mating type by replacing genetic information at the transcriptionally active mat1 locus with sequences copied from one of two closely linked silent loci, mat2-P or mat3-M. By a process referred to as directionality of switching, cells predominantly switch to the opposite mat1 allele; the mat1-P allele preferentially recombines with mat3, while mat1-M selects the mat2. In contrast to efficient recombination at mat1, recombination within the adjoining mat2-mat3 interval is undetectable. We defined the role of sequences between mat2 and mat3, designated the K-region, in directionality as well as recombinational suppression. Cloning and sequencing analysis revealed that a part of the K-region is homologous to repeat sequences present at centromeres, which also display transcriptional and recombinational suppression. Replacement of 7.5 kb of the K-region with the ura4+ gene affected directionality in a variegated manner. Analysis of the swi6-mod locus, which was previously shown to affect directionality, in K delta::ura4+ strains suggested the existence of at least two overlapping directionality mechanisms. Our work furthers the model that directionality is regulated by cell-type-specific organization of the heterochromatin-like structure in the mating-type region and provides evidence that the K-region contributes to silencing of the mat2-mat3 interval.