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 ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.     

Yeast silencers create domains of nuclease-resistant chromatin in an SIR4-dependent manner

Previous analysis of the repression of the silent mating type loci in Saccharomyces cerevisiae has linked the mechanism of silencing to the formation of a chromatin domain at the silenced loci. In this study, a TRP1 reporter gene was used to examine changes in chromatin structure in a neutral environment. This enabled the chromatin structure organized by yeast silencers to be compared directly with changes effected by the yeast alpha2 repressor. It was found that silencers mediate the formation of lengthy nuclease-resistant domains on the DNA, rather than specifically positioning nucleosomes over promoter regions as the alpha2 repressor does. Silencing at the TRP1 reporter gene closely resembled silencing at the HMR and HML loci. Repression of the test gene was optimal when two silencers flanking the reporter gene were used, mimicking the situation at the silent loci. In addition, both repression of the reporter gene and the formation of nuclease-resistant chromatin domains was SIR4 dependent.     

Sir- and silencer-independent disruption of silencing in Saccharomyces by Sas10p

A promoter fusion library of Saccharomyces cerevisiae genes was used to exploit phenotypes associated with altered protein dosage. We identified a novel gene, SAS10, by the ability of Sas10p, when overproduced, to disrupt silencing. The predicted Sas10p was 70,200 kD and strikingly rich in charged amino acids. Sas10p was exclusively nuclear in all stages of the cell cycle. Overproduction of Sas10p caused derepression of mating type genes at both HML and HMR, as well as of URA3, TRP1, and ADE2 when inserted near a telomere or at HMR or the rDNA locus. Repressed genes not associated with silenced chromatin were unaffected. Sas10p was essential for viability, and the termination point following Sas10p depletion was as large budded cells. Remarkably, Sas10p overproduction disrupted silencing even under conditions that bypassed the requirement for Sir proteins, ORC, and Rap1p in silencing. These data implied that Sas10p function was intimately connected with the structure of silenced chromatin.     

Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I

Saccharomyces cerevisiae carries approximately 150 copies of rDNA in tandem repeats. It was found that the absence of an essential subunit of RNA polymerase I (Pol I) in rpa135 deletion mutants triggers a gradual decrease in rDNA repeat number to about one-half the normal level. Reintroduction of the missing RPA135 gene induced a gradual increase in repeat number back to the normal level. Gene FOB1 was shown to be essential for both the decrease and increase of rDNA repeats. FOB1 was shown previously to be required for replication fork blocking (RFB) activity at RFB site in rDNA and for recombination hot-spot (HOT1) activity. Thus, DNA replication fork blockage appears to stimulate recombination and play an essential role in rDNA expansion/contraction and sequence homogenization, and possibly, in the instability of repeated sequences in general. RNA Pol I, on the other hand, appears to control repeat numbers, perhaps by stabilizing rDNA with the normal repeat numbers as a stable nucleolar structure.     

A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Rap1 protein binds with high affinity to sites within the poly(C(1-3)A) tracts at telomeres, where it plays a role in both telomere length regulation and the initiation of telomeric silencing. Rap1p initiates silencing at telomeres by interacting through its carboxy-terminal domain with Sir3p and Sir4p, both of which are required for repression. This same domain of Rap1p also negatively regulates telomere elongation, through an unknown mechanism. We have identified a new Rap1-interacting factor (Rif2p) that plays a role in telomere length regulation. Rif2p has considerable functional similarities with a Rap1p-interacting factor (Rif1p) identified previously. Mutations in RIF1 or RIF2 (unlike mutations in the silencing genes SIR3 and SIR4) result in moderate telomere elongation and improved telomeric silencing. However, deletion of both RIF1 and RIF2 in the same cell results in a dramatic increase in telomere length, similar to that seen with a carboxy-terminal truncation of Rap1p. In addition, overexpression of either RIF1 or RIF2 decreases telomere length, and co-overexpression of these proteins can reverse the telomere elongation effect of overexpression of the Rap1p carboxyl terminus. Finally, we show that Rif1p and Rif2p can interact with each other in vivo. These results suggest that telomere length regulation is mediated by a protein complex consisting of Rif1p and Rif2p, each of which has distinct regulatory functions. One role of Rap1p in telomere length regulation is to recruit these proteins to the telomeres.     

Consequences of the photodynamic treatment of resting and activated peripheral T lymphocytes

Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.