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.     

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.     

RAP-1 factor is necessary for DNA loop formation in vitro at the silent mating type locus HML

DNA fragments containing the silencers that flank the mating type genes at HML alpha are shown to bind specifically to the nuclear scaffold of yeast. The scaffold proteins are solubilized with urea and then renatured to form a soluble extract which allows reconstitution of sequence-specific DNA loops. At the silent mating type locus HML alpha, loops are formed by either silencer-silencer (E-I) interaction or silencer-promoter interactions (E-P and I-P). The nuclear protein RAP-1 fractionates efficiently with the nuclear scaffold, and binds to the E, I, and promoter regions. Affinity purification of RAP-1 and oligonucleotide competition show that RAP-1 is necessary for reconstitution of loops in vitro. These results are consistent with a model in which silencers define a chromatin loop within which occur modifications that maintain the promoter in an inactive state.     

The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, most likely mediated through two cis-acting sites located on opposite sides of the locus. We showed that deletion of either of these two cis-acting sites from the chromosome did not yield any detectable derepression of HML, while deletion of both sites yielded full expression of the locus. In addition, each of these sites was capable of exerting repression of heterologous genes inserted in their vicinity. Thus, HML expression is regulated by two independent silencers, each fully competent for maintaining repression. This situation was distinct from the organization of the other silent locus, HMR, at which a single silencer served as the predominant repressor of expression. Examination of identifiable domains and binding sites within the HML silencers suggested that silencing activity can be achieved by a variety of combinations of various functional domains.     

Identification of a functional domain within the essential core of histone H3 that is required for telomeric and HM silencing in Saccharomyces cerevisiae

Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the alpha1 helix and L1 loop of the essential histone fold. Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter. Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.     

Analysis of Y-Linked Mutations to Male Sterility in DROSOPHILA MELANOGASTER

Mating type in haploid cells of the yeast Saccharomyces cerevisiae is determined by a pair of alleles MATa and MAT alpha. Under various conditions haploid mating types can be interconverted. It has been proposed that transpositions of silent cassettes of mating-type information from HML OR HMR to MAT are the source of mating type conversions. A mutation described in this work, designated AON1, has the following properties. (1) MAT alpha cells carring AON1 are defective in mating. (2) AON1 allows MAT alpha/MAT alpha but not MATa/MATa diploids to sporulate; thus, AON1 mimics the MATa requirement for sporulation. (3) mata-1 cells that carry AON1 are MATa phenocopies, i.e., MAT alpha/mata-1 AON1 diploids behave as standard MAT alpha/MATa cells; therefore, AON1 suppresses the defect of mata-1. (4) AON1 maps at or near HMRa. (5) Same-site revertants from AON1 lose the ability to convert mating type to MATa, indicating that reversion is associated with the loss of a functional HMRa locus. In addition, AON1 is a dominant mutation. We conclude that AON1 is a regulatory mutation, probably cis-acting, that leads to the constitutive expression of silent a mating-type information located at HMRa.     

A rapid technique for the visualization of live immobilized yeast cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also Xu et al., Mol. Cell 2006.     

The structure of transposable yeast mating type loci

A recombinant plasmid containing a MAT alpha mating type locus of Saccharomyces cerevisiae has been isolated by its ability to complement a sterile mat alpha mutation. The plasmid hybridizes to restriction fragments containing both active mating type loci (MATa and MAT alpha) and both silent mating type loci (HMRa and HML alpha). All loci therefore have common sequences. Recombinant lambda clones of the locihave been isolated by plaque hybridization and their structures have been compared by a heteroduplex analysis. At its center, each locus contains one of two apparently nonhomologous sequences. Loci concerned with the alpha phenotype (MAT alpha and HML alpha) contain and 850 bp alpha-specific sequence, whereas loci concerned with the a phenotype (MATa and HMRa) contain a 700 bp a-specific sequence. The a- or alpha-specific sequences are surrounded by DNA sequences that are common to all loci. These homologous sequences extend for 230 bp on the left and 700 bp on the right. They appear to be unrelated to each other. Surprisingly, HML alpha and HMRa differ in their extent of homology to MATa and MAT alpha outside the above regions. HMRa lacks an extensive (700 bp) DNA sequence to the right of the large right-hand homologous region, and possibly also a small (90 bp) sequence to the left of the small left-hand homologous region, both of which are present at HML alpha, MATa and MAT alpha. Hybridization studies have shown that the 700 bp sequence is present at HMLa but absent at HMR alpha alleles. It is therefore characteristic of HML, irrespective of whether it contains a- or alpha-specific sequences. The results imply that mating type interconversion is effected by transposition of DNA sequences from HML or HMR to MAT, as predicted by the controlling element model of Oshima and Takano (1971) and the Cassette model of Hicks, Strathern and Herskowitz (1977).     

Donor Locus Selection during Saccharomyces Cerevisiae Mating Type Interconversion Responds to Distant Regulatory Signals

Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT. Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa. Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion. Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR. We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III. Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other. We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR. This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.     

Mutations Leading to Expression of the Cryptic HMR a Locus in the Yeast SACCHAROMYCES CEREVISIAE

Mutations leading to expression of the silent HMRa information in Saccharomyces cerevisiae result in sporulation proficiency in mata1/MAT alpha diploids. An example of such a mutation is sir5-2, a recessive mutation in the gene SIR5. As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMRa and HML alpha, resulting in the nonmating phenotype of an a/alpha diploid. However, sir5-2/sir5-2 mata1/MAT alpha diploids mate as alpha yet are capable of sporulation. The sir5-2 mutation is unlinked to sir1-1, yet the two mutations do not complement each other: mata1/MAT alpha sir5-2/SIR5 SIR1/sir1-1 diploids are capable of sporulation. In this case, recessive mutations in two unlinked genes form a mutant phenotype, in spite of the presence of the normal wild-type alleles. The PAS1-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMRa. PAS1-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MATa locus to sporulate. It is proposed that PAS1-1 leads to partial expression of the otherwise cryptic a1 information at HMRa.