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.     

The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region

Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.     

Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.     

Chromodomain protein Swi6-mediated role of DNA polymerase alpha in establishment of silencing in fission Yeast

Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase alpha. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase alpha in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol alpha and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase alpha in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.     

Complex mechanism of site-specific DNA replication termination in fission yeast

A site-specific replication terminator, RTS1, is present at the Schizosaccharomyces pombe mating-type locus mat1. RTS1 regulates the direction of replication at mat1, optimizing mating-type switching that occurs as a replication-coupled recombination event. Here we show that RTS1 contains two cis-acting sequences that cooperate for efficient replication termination. First, a sequence of approximately 450 bp containing four repeated 55 bp motifs is essential for function. Secondly, a purine-rich sequence of approximately 60 bp without intrinsic activity, located proximal to the repeats, acts cooperatively to increase barrier activity 4-fold. Our data suggest that the trans-acting factors rtf1p and rtf2p act through the repeated motifs and the purine-rich element, respectively. Thus, efficient site-specific replication termination at RTS1 occurs by a complex mechanism involving several cis-acting sequences and trans-acting factors. Interestingly, RTS1 displays similarities to mammalian rDNA replication barriers.     

Identification of Uhp1, a ubiquitinated histone-like protein,as a target/mediator of Rhp6 in mating-type silencing in fission yeast

Mating-type silencing in Schizosaccharomyces pombe is brought about by cooperative interactions between cis-acting DNA sequences flanking mat2P and mat3M and the trans-acting factors, namely Swi6, Clr1-Clr4, Clr6, and Rik1. In addition, DNA repair gene rhp6, which plays a role in post-replication DNA repair and ubiquitination of proteins including histones, is also involved in silencing, albeit in a unique way; its effect on silencing and chromatin structure of the donor loci is dependent on their switching competence. Earlier, we hypothesized the existence of a mediator of Rhp6 that plays a role in reestablishment of the chromatin structure coincidentally with DNA replication associated with mating-type switching. Here we report the identification of a 22-kDa protein as an in vivo target and mediator of Rhp6 in mating-type silencing. The level of this protein is greatly elevated in sng1-1/rhp6(-) mutant and rhp6Delta as compared with wild type strain. Both the deletion and overexpression of the gene encoding this protein elicit switching-dependent loss of silencing. Furthermore, the 22-kDa protein undergoes Rhp6-dependent multiubiquitination and associates with mat2 locus during S phase in wild type cells. Interestingly, it contains a histone-fold motif similar to that of histone H2A, and like histone H2A, it interacts strongly with histone H2B in vitro. These results indicate that the 22-kDa protein, renamed as the ubiquitinated histone-like protein Uhp1, is an in vivo target/mediator of Rhp6 in silencing. Thus, regulation of association of Uhp1 with chromatin and ubiquitination followed by degradation may play a role in reestablishment of inactive chromatin structure at the silent mating-type loci.     

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression. Here we describe results that indicate that homologous recombination between transferred and chromosomal Ig genes can be used to map Ig mutations by marker rescue.