Specific repression of the yeast silent mating locus HMR by an adjacent telomere.

The yeast silent mating loci HML and HMR are located at opposite ends of chromosome III adjacent to the telomeres. Mutations in the N terminus of histone H4 have been previously found to derepress the yeast silent mating locus HML to a much greater extent than HMR. Although differences in the a and alpha mating-type regulatory genes and in the cis-acting silencer elements do not appear to strongly influence the level of derepression at HMR, we have found that the differential between the two silent cassettes is largely due to the position of the HMR cassette relative to the telomere on chromosome III. While HML is derepressed to roughly the same extent by mutations in histone H4 regardless of its chromosomal location, HMR is affected to different extends depending upon its chromosomal positioning. We have found that HMR is more severely derepressed by histone H4 mutations when positioned far from the telomere (cdc14 locus on chromosome VI) but is only minimally affected by the same mutations when integrated immediately adjacent to another telomere (ADH4 locus on chromosome VII). These data indicate that the degree of silencing at HMR is regulated in part by its neighboring telomere over a distance of at least 23 kb and that this form of regulation is unique for HMR and not present at HML. These data also indicate that histone H4 plays an important role in regulating the silenced state at both HML and HMR.     

Epigenetic inheritance of transcriptional states in S. cerevisiae

SIR1, one of several genes required for repression of yeast silent mating type loci, has a unique role in repression of the HML alpha locus. Single-cell assays revealed that cells with mutant alleles of SIR1, including presumptive null alleles, existed as populations of genetically identical cells whose members were in one of two different regulatory states. A minority of cells had a repressed HML alpha locus whereas the majority had a derepressed HML alpha locus. The two states were mitotically stable, although rare changes in state were observed during mitotic growth, possibly reflecting heritable changes to the HML alpha locus at or before replication. Analysis of changes in state suggests that SIR1 protein has a role in the establishment but not the maintenance of repression of silent mating type genes, whereas SIR2, SIR3, and SIR4 are required for maintenance.     

Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae.

The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.     

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 α2 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 α2 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. Received: 31 October 1996; in revised form: 6 March 1997 / Accepted: 25 March 1997     

Four Genes Responsible for a Position Effect on Expression from HML and HMR in Saccharomyces cerevisiae

Mating type interconversion in Saccharomyces cerevisiae occurs by transposition of copies of the a or alpha mating type cassettes from inactive loci, HML and HMR, to an active locus, MAT. The lack of expression of the a and alpha genes at the silent loci results from repression by trans-acting regulators encoded by SIR (Silent Information Regulator) genes. In this paper we present evidence for the existence of four SIR genes. Inactivation of any of these genes leads to expression of cassettes at both HML and HMR. Unusual complementation properties are observed for a number of sir mutations. Specifically, some recessive mutations in different genes fail to complement. The correspondence between SIR1, SIR2, SIR3, SIR4 and other genes with similar roles (MAR, CMT, STE8 and STE9) is presented.     

The DNA-binding polycomb group protein pleiohomeotic mediates silencing of a Drosophila homeotic gene.

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements. Currently, these silencers are ill-defined sequences and it is not known how PcG proteins associate with DNA. Here, we show that the Drosophila PcG protein Pleiohomeotic binds to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers.     

Transposition of yeast mating type genes from two translocations of the left arm of chromosome III.

In the yeast Saccharomyces cerevisiae, the HIS4C gene lies on the left arm of chromosome III. We analyzed two chromosomal rearrangements that have HIS4C translocated either to chromosome XII or to a new translocation chromosome. Using the cmt mutation that allows expression of the normally silent copies of mating type genes, we found that both of these translocations also carried HML alpha, more than 30 map units distal to HIS4C which normally lies on chromosome III. In the case of the translocation chromosome (designated T3), we also found an exchange event between HML alpha on the translocation chromosome and HMLa on chromosome III. In diploids containing two T3 chromosomes (one carrying HML alpha and the carrying HMLa), we found that HML was 32 centimorgans from HIS4C, which was 10 centimorgans from an unknown centromere. In homothallic strains carrying HMLa MATa HMRa on chromosome III, switching from MATa to MAT alpha could occur by using the HML alpha on the translocation as the sole donor of alpha information. Transposition from HML alpha on chromosome T3 was about 20 to 40% as efficient as transposition from intact chromosome III. In contrast, transposition from the HML alpha inserted into chromosome XII was reduced about 100-fold. This reduced efficiency did not appear to be caused by an alteration in the sequences immediately surrounding HML alpha in the translocation. The translocated HML alpha sequence was located in the same size (29-kilobase) SalI fragment as was found in chromosome III, and the same EcoRI, HindIII, and BglII restriction sites were also found. Furthermore, HML alpha was still under the control of the CMT gene, which maintains HML as a silent copy of mating type information. These results suggested that the position of the HML alpha sequence plays an important role in the efficiency of mating type switching.