Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

Abstract

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or a) locus. HML and HMR, which usually contain copies of a and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIRI encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir-phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.The third chromosome of the budding yeast Saccharomyces cerevisiae ( Fig. 1) contains three loci, all capable of determining cell type by regulating the expression of other cell type-specific genes. Normally, however, only MAT (either MATa or MATa) is expressed, whereas HML and HMR remain unexpressed. The role of MAT in determining cell type in S. cerevisiae has been reviewed extensively (11,15,30,37,48). Briefly, haploids and diploids homozygous at MAT mate efficiently with cells of opposite mating type, but cells of the same mating type rarely mate. MATa/MATTa diploids do not mate but can sporulate when starved for nitrogen. Heterothallic (ho) strains possess a stable mating ability. Homothallic (HO) strains interconvert the MAT alleles at high frequency during mitotic growth and rapidly give rise to a population containing cells of both mating types, which can mate to form MATa/MATTa diploids (16,49). Mating type interconversion is the result of the transposition of a copy of sequences from HML or HMR (normally containing a and a sequences, respectively) to MAT (17,24,25,27,32,39,51). Thus, the two silent loci serve as donors and are said to contain cassettes of information.The two silent HM loci contain complete copies of mating type information (17, 39, 51) but are kept transcriptionally inactive by the products of four genes identified by mutational analysis and known as MAR (mating type regulator; 26), SIR (silent information regulator; J. Rine, Ph.D. thesis, University of Oregon, Eugene, 1979; 42), or CMT (change of mating type; 12). The four SIR genes are unlinked to the HM loci and encode gene products which act in tran...

The Conserved Core of a HumanSIR2Homologue Functions in Yeast Silencing

“…Arguably, silencing is best understood in molecular detail in S. cerevisiae, so the recent discovery of widespread homologues of the classically defined yeast SIR2 gene fueled the prediction that silencing, like transcriptional activation, might have conserved molecular mechanisms. SIR2's silencing-related activities include repression of transcription at the silent mating-type loci, telomeres, and within the rDNA arrays (Shore et al, 1984;Ivy et al, 1986;Aparicio et al, 1991;Bryk et al, 1997;Fritze and Esposito, 1997;Smith and Boeke, 1997), suppression of rDNA recombination (Gottlieb and Esposito, 1989), and modulation of histone (de)acetylation (Braunstein et al, 1993). We previously showed that three of four yeast homologues can function in silencing (Brachmann et al, 1995).…”

“…Among the four SIR (Silent Information Regulator) genes, SIR2 is unique because it is required for silencing and suppression of recombination within the rDNA, as well as silent mating-type (HM) and telomeric silencing (Shore et al, 1984;Ivy et al, 1986;Rine and Herskowitz, 1987;Gottlieb and Esposito, 1989;Aparicio et al, 1991;Bryk et al, 1997;Fritze and Esposito, 1997;Smith and Boeke, 1997;Smith et al, 1998). A sir2⌬ mutant strain exhibits complete derepression at these loci.…”

Transcription Factor UAF, Expansion and Contraction of Ribosomal DNA (rDNA) Repeats, and RNA Polymerase Switch in Transcription of Yeast rDNA

“…For disruption of SIR3, a DNA fragment covering the SIR3 chromosome region, containing a 2.5-kb BglII-XhoI deletion within the SIR3 coding region replaced by the 2.5-kb BglII-SalI fragment containing LEU2, was used. For disruption of SIR4, a DNA fragment covering the SIR4 chromosome region, containing a 330-bp BglII-BamHI deletion inactivating SIR4 function (15) and replaced by the 3.1-kb BglII-BglII fragment containing LEU2, was used. Disruption of SIR2, SIR3, and SIR4 genes was confirmed by the nonmating phenotype of the strains constructed.…”

Sir Antagonist 1 (San1) Is a Ubiquitin Ligase

“…Establishment of silencing at HM loci is under cell cycle control and requires the Sir1 protein (11,12). Sir2, Sir3, and Sir4 are required for both the establishment and the maintenance of the silenced state (13). Telomeric silencing is also influenced by the cell cycle such that it is stronger during G 1 /S and weaker during G 2 /M (14).…”