HM Loci Research Articles

The pheromone response pathway activates transcription of Ty5 retrotransposons located within silent chromatin of Saccharomyces cerevisiae.

The Saccharomyces retrotransposon Ty5 integrates preferentially into transcriptionally inactive regions (silent chromatin) at the HM loci and telomeres. We found that silent chromatin represses basal Ty5 transcription, indicating that these elements are encompassed by silent chromatin in their native genomic context. Because transcription is a requirement for transposition, integration into silent chromatin would appear to prevent subsequent rounds of replication. Using plasmid-borne Ty5-lacZ constructs, we found that Ty5 expression is haploid specific and is repressed 10-fold in diploid strains. Ty5 transcription is also regulated by the pheromone response pathway and is induced approximately 20-fold upon pheromone treatment. Deletion analysis of the Ty5 LTR promoter revealed that a 33 bp region with three perfect matches to the pheromone response element is responsible for both mating pheromone and cell-type regulation. Transcriptional repression of Ty5 by silent chromatin can be reversed by pheromone treatment, which leads to transcription and transposition. Ty5 replication, therefore, is normally repressed by silent chromatin and appears to be induced during mating. This is the first example of transcriptional activation of a gene that naturally resides within silent chromatin.

Identification of a member of a DNA-dependent ATPase family that causes interference with silencing.

DNA in eukaryotic cells is packed in tandem repeats of nucleosomes or higher-order chromatin structures, which present obstacles to many cellular processes that require protein-DNA interactions, such as transcription, DNA repair, and recombination. To find proteins that are involved in increasing the accessibility of specific DNA regions in yeast, we used a genetic approach that exploited transcriptional silencing normally occurring at HML and HMR loci. The silencing is mediated by cis-acting silencer elements and is thought to require the formation of a special chromatin structure that prevents accessibility to the silenced DNA. A previously uncharacterized gene, termed DIS1, was isolated from a screen for genes that interfere with silencing when overexpressed. DIS1 encodes a protein with conserved motifs that are present in a family of DNA-dependent ATPases, the SWI2/SNF2-like proteins. Overproduction of N-terminal half of DIS1 protein interfered specifically with ectopic silencing used in the screen as well as HMR E silencing. Two-hybrid studies revealed a specific interaction between the N terminus of DIS1 and the C-terminal half of SIR4, a protein essential for silencing. Cells with a dis1 knockout mutation had significantly lower mating-type switching rate. These results suggest that DIS1 may contribute to making the silenced DNA template at HM loci more accessible during the mating-type switching process.

Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae.

DNA double-strand breaks are repaired by homologous recombination or DNA end-joining, but the latter process often causes legitimate recombination and chromosome rearrangements. One of the factors involved in the end-joining process is Hdf1, a yeast homologue of Ku protein. We used the yeast two-hybrid assay to show that Hdf1 interacts with Sir4, which is involved in transcriptional silencing at telomeres and HM loci. Analyses of sir4 mutants showed that Sir4 is required for deletion by illegitimate recombination and DNA end-joining in the pathway involving Hdf1. Sir2 and Sir3, but not Sir1, were also found to participate in these processes. Furthermore, mutations of the SIR2, SIR3 and SIR4 genes conferred increased sensitivity to gamma-radiation in a genetic background with a mutation of the RAD52 gene, which is essential for double-strand break repair mediated by homologous recombination. These results indicate that Sir proteins are involved in double-strand break repair mediated by end-joining. We propose that Sir proteins act with Hdf1 to alter broken DNA ends to create an inactivated chromatin structure that is essential for the rejoining of DNA ends.

Nuclear organization and transcriptional silencing in yeast.

Transcriptional repression at the yeast silent mating type loci requires the formation of a nucleoprotein complex at specific cis-acting elements called silencers, which in turn promotes the binding of a histone-associated Sir-protein complex to adjacent chromatin. A similar mechanism of long-range transcriptional repression appears to function near telomeric repeat sequences, where it has been demonstrated that Sir3p is a limiting factor for the propagation of silencing. A combined immunofluorescence/in situ hybridization method for budding yeast was developed that maintains the three-dimensional structure of the nucleus. In wild-type cells the immunostaining of Sir3p, Sir4p and Rap1 colocalizes with Y' subtelomeric sequences detected by in situ hybridization. All three antigens and the subtelomeric in situ hybridization signals are clustered in foci, which are often adjacent to, but not coincident with, nuclear pores. This colocalization of Rap1, Sir3p and Sir4p with telomeres is lost in sir mutants, and also when Sir4p is overexpressed. To test whether the natural positioning of the two HM loci, located roughly 10 and 25 kb from the ends of chromosome III, is important for silencer function, a reporter gene flanked by wild-type silencer elements was integrated at various internal sites on other yeast chromosomes. We find that integration at internal loci situated far from telomeres abrogates the ability of silencers to repress the reporter gene. Silencing can be restored by creation of a telomere at 13 kb from the reporter construct, or by insertion of 340 bp of yeast telomeric repeat sequence at this site without chromosomal truncation. Elevation of the internal nuclear pools of Sir1p, Sir3p and Sir4p can relieve the lack of repression at the LYS2 locus in an additive manner, suggesting that in wild-type cells silencer function is facilitated by its juxtaposition to a pool of highly concentrated Sir proteins, such as those created by telomere clustering.

Spreading of transcriptional repressor SIR3 from telomeric heterochromatin.

Telomeric genes and the HM loci in saccharomyces cerevisiae are transcriptionally repressed and adopt a heterochromatin-like structure. The trans-acting factors RAP1, SIR3 and SIR4 are required for telomeric and HM silencing, and are thought to be chromosomal, but how they contribute to histone-dependent repression of adjacent chromatin is unclear. SIR3 suppresses silencing defects in histones, is limiting for silencing adjacent to telomeres, and interacts with the H3 and H4 amino termini in vitro. Here we show that SIR3 co-immunoprecipitates SIR4, RAP1 and histones from cellular extracts, suggesting the presence of large chromatin-associated protein complexes. Crosslinking experiments show that SIR3 is present at HMRa, HMLalpha and telomeres in vivo, and that is spreads from telomeric regions into adjacent chromatin when overexpressed. Thus SIR3 is a structural component of yeast heterochromatin, repressing adjacent genes as it spreads along the chromosome.

SUM1-1, a dominant suppressor of SIR mutations in Saccharomyces cerevisiae, increases transcriptional silencing at telomeres and HM mating-type loci and decreases chromosome stability.

Transcriptional silencing in the yeast Saccharomyces cerevisiae occurs at HML and HMR mating-type loci and telomeres and requires the products of the silent information regulator (SIR) genes. Recent evidence suggests that the silencer- and telomere-binding protein Rap1p initiates silencing by recruiting a complex of Sir proteins to the chromosome, where they act in some way to modify chromatin structure or accessibility. A single allele of the SUM1gene (SUM1-1) which restores silencing at HM loci in strains mutant for any of the four SIR genes was identified a number of years ago. However, conflicting genetic results and the lack of other alleles of SUM1 made it difficult to surmise the wild-type function of SUM1 or the manner in which the SUM1-1 mutation restores silencing in sir mutant strains. Here we report the cloning and characterization of the SUM1 gene and the SUM1-1 mutant allele. Our results indicate that SUM1-1 is an unusual altered-function mutation that can bypass the need for SIR function in HM silencing and increase repression at telomeres. A sum1 deletion mutation has only minor effects on silencing in SIR strains and does not restore silencing in sir mutants. In addition to its effect on transcriptional silencing, the SUM1-1 mutation (but not a sum1 deletion) increases the rate of chromosome loss and cell death. We suggest several speculative models for the action of SUM1-1 in silencing based on these and other data.

Loss of Transcriptional Silencing Causes Sterility in Old Mother Cells of S. cerevisiae

We show that sterility is an aging-specific phenotype in S. cerevisiae and, by genetic and physical means, demonstrate that this phenotype results from a loss of silencing in most old cells by the SIR complex at the HM loci. This loss of silencing is specific because transcription of genes, such as MEI4 and DCM1, normally induced by sporulation, is not observed, while transcription of HMRa is observed. These findings pinpoint the molecular cause of an aging-specific phenotype in yeast. Further, they provide direct evidence for a breakdown of silencing in old cells, as predicted from earlier findings that SIR4 is a determinant of life span in this organism.

Chromatin-mediated transcription repression in yeast

Chromatin is linked to two modes of transcriptional repression in yeast: silencing at the HM loci and telomeres, and repression by the global regulator complex Ssn6/Tup1. In both cases, unique chromatin configurations appear to be orchestrated by multiprotein complexes targeted to particular regions through interactions with specific DNA-binding proteins, such as RAP1, the origin-recognition complex, and α2.

Mutation in the silencing gene S/R4 can delay aging in S. cerevisiae

Aging in S. cerevisiae is exemplified by the fixed number of cell divisions that mother cells undergo (termed their life span). We have exploited a correlation between life span and stress resistance to identify mutations in four genes that extend life span. One of these, SIR4, encodes a component of the silencing apparatus at HM loci and telomeres. The sir4-42 mutation extends life span by more than 30% and is semidominant. Our findings suggest that sir4-42 extends life span by preventing recruitment of the SIR proteins to HM loci and telomeres, thereby increasing their concentration at other chromosomal regions. Maintaining silencing at these other regions maybe critical in preventing aging. Consistent with this view, expression of only the carboxyl terminus of SIR4 interferes with silencing at HM loci and telomeres, which also extends life span. Possible links among silencing, telomere maintenance, and aging in other organisms are discussed.

The yeast GAL11 protein is involved in regulation of the structure and the position effect of telomeres.

GAL11 is an auxiliary transcription factor that functions either positively or negatively, depending on the structure of the target promoters and the combination of DNA-bound activators. In this report, we demonstrate that a gal11 delta mutation caused a decrease in the length of the telomere C1-3A tract, a derepression of URA3 when it is placed next to telomere, and an increase in accessibility of the telomeric region to dam methylase, indicating that GAL11 is involved in the regulation of the structure and the position effect of telomeres. The defective position effect in a gal11 delta strain was suppressed by overproduction of SIR3, whereas overexpression of GAL11 failed to restore the telomere position effect in a sir3 delta strain. Hyperproduced GAL11 could partially suppress the defect in silencing at HMR in a sir1 delta mutant but not that in a sir3 delta mutant, suggesting that GAL11 can replace SIR1 function partly in the silencing of HMR. Overproduced SIR3 also could restore silencing at HMR in sir1 delta cells. In contrast, SIR1 in a multicopy plasmid relieved the telomere position effect, especially in a gal11 delta mutant. Since chromatin structure is thought to play a major role in the silencing at both the HM loci and telomeres, GAL11 is likely to participate in the regional regulation of transcription by the HM loci and telomeres, GAL11 is likely to participate in the regional regulation of transcription by modulating the chromatin structure.

The yeast GAL11 protein is involved in regulation of the structure and the position effect of telomeres.

GAL11 is an auxiliary transcription factor that functions either positively or negatively, depending on the structure of the target promoters and the combination of DNA-bound activators. In this report, we demonstrate that a gal11 delta mutation caused a decrease in the length of the telomere C1-3A tract, a derepression of URA3 when it is placed next to telomere, and an increase in accessibility of the telomeric region to dam methylase, indicating that GAL11 is involved in the regulation of the structure and the position effect of telomeres. The defective position effect in a gal11 delta strain was suppressed by overproduction of SIR3, whereas overexpression of GAL11 failed to restore the telomere position effect in a sir3 delta strain. Hyperproduced GAL11 could partially suppress the defect in silencing at HMR in a sir1 delta mutant but not that in a sir3 delta mutant, suggesting that GAL11 can replace SIR1 function partly in the silencing of HMR. Overproduced SIR3 also could restore silencing at HMR in sir1 delta cells. In contrast, SIR1 in a multicopy plasmid relieved the telomere position effect, especially in a gal11 delta mutant. Since chromatin structure is thought to play a major role in the silencing at both the HM loci and telomeres, GAL11 is likely to participate in the regional regulation of transcription by the HM loci and telomeres, GAL11 is likely to participate in the regional regulation of transcription by modulating the chromatin structure.

Targeting of SIR1 protein establishes transcriptional silencing at HM loci and telomeres in yeast

Previous studies suggest that the yeast SIR1 protein is involved in the establishment of transcriptional silencing at the HM mating-type loci. Here we show that a GAL4 DNA-binding domain-SIR1 hybrid protein (G BD-SIR1), when targeted to an HMR locus containing GAL4-binding sites (UAS G), can establish silencing and bypass the requirement for the silencer element HMR-E. Silencing mediated by G BD-SIR1 requires the trans-acting factors that normally participate in repression, namely, SIR2, SIR3, SIR4, and histone H4. However, G BD hybrids with SIR2, SIR3, or SIR4 cannot establish silencing. Telomeric silencing, which does not require SIR1 and is normally unstable, is greatly improved by tethering G BD-SIR1 to the telomere. These experiments support a model in which native SIR1 protein is brought to the HM loci by proteins bound to the silencers. Telomeres appear to lack the ability to recruit SIR1, and that is why telomeric silencing is unstable.

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.

Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae

Genes placed near telomeres in S. cerevisiae succumb to position-effect variegation. SIR2, SIR3, SIR4, NAT1, ARD1, and HHF2 (histone H4) were identified as modifiers of the position effect at telomeres, since transcriptional repression near telomeres was no longer observed when any of the modifier genes were mutated. These genes, in addition to SIR1, have previously been shown to repress transcription at the silent mating loci, HML and HMR. However, there were differences between transcriptional silencing at telomeres and the HM loci, as demonstrated by suppressor analysis and the lack of involvement of SIR1 in telomeric silencing. These findings provide insights into telomeric structure and function that are likely to apply to many eukaryotes. In addition, the distinctions between telomeres and the HM loci suggest a hierarchy of chromosomal silencing in S. cerevisiae.

Extragenic suppressors of mar2(sir3) mutations in Saccharomyces cerevisiae.

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by four trans-acting MAR (or SIR) loci. We have isolated extragenic suppressors of the mar2-1 mutation which, based on genetic complementation tests, define two additional loci involved in regulating the expression of HML and HMR. A strain with the genotype HMLa MAT alpha HMRa mar2-1 is sterile due to the simultaneous expression of a and alpha information. Two mutants exhibiting an alpha phenotype (which may result from the restoration of MAR/SIR repression) were isolated and genetically characterized. The mutations in these strains: (1) are recessive, (2) are capable of suppressing a mar2-deletion mutation, (3) are unlinked to MAT, (4) complement one another as well as the previously identified sum1-1 mutation, and (5) are not new alleles of the known MAR/SIR loci. We designate these new regulatory loci SUM2 and SUM3 (suppressor of mar). Unlike the sum1-1 mutation, suppression by sum2-1 and sum3-1 is mar2-locus specific. Both sum2-1 and sum3-1 affect the expression of a information at the HM loci. Transcript analysis shows a significant reduction in HMLa and HMRa gene transcription in mar2-1 sum2-1 and mar2-1 sum3-1 cells. Furthermore, we have found genetic evidence to suggest that mar2-1 sum2-1 cells exhibit only partial expression of silent alpha information. We conclude that the SUM2 and SUM3 gene products are required for expression of the HM loci and act downstream of the MAR2 (SIR3) gene function. Possible mechanisms for the action of the SUM gene products are discussed.

SUM1, an apparent positive regulator of the cryptic mating-type loci in Saccharomyces cerevisiae.

The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information. A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (suppressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and alpha information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.

Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae.

The yeast S. cerevisiae switches a and alpha cell types by a transposition mechanism that replaces genetic information residing at the mating-type locus (MAT) with information copied from either of the two donor loci, HML and HMR. The donor HML and HMR loci contain the same genetic information as the MATa and MAT alpha alleles, yet they do not switch. Additionally, Strathern et al. (1982) have described an in vivo double-strand DNA break found at subgenomic levels (approximately 2% of MAT DNA) within the MAT locus but not within HML and HMR. We have examined the role of this double-strand DNA break in the switching process. Cell lineage studies show that strains containing deletions of the donor HML and HMR loci produce lethal progeny in the exact pattern described for MAT switching in standard strains. Our interpretation is that the double-strand MAT break in the deletion strains cannot be repaired because of the lack of the donor loci, resulting in cell death. We suggest that the double-strand DNA break is an initiating event for switching and that this event is lethal in the absence of the donor loci. MAT mutants isolated as survivors from this "pedigree of death" define a site required for switching where the double-strand break occurs. We have also examined marl mutant strains in which the donor loci are expressed and observed to switch (Klar et al. 1981a). The double-strand DNA cut appears at the HM loci in these strains. Thus, there is a strong correlation between the presence or absence of the double-strand break at each cassette and its ability or inability to switch as observed at the single cell level.

Efficient production of a ring derivative of chromosome III by the mating-type switching mechanism in Saccharomyces cerevisiae.

The mating-type switches in the yeast Saccharomyces cerevisiae occur by unidirectional transposition of replicas of unexpressed genetic information, residing at HML or HMR, into the mating-type locus (MAT). The source loci, HML and HMR, remain unchanged. Interestingly, when the HM cassettes are expressed, as in marl strains, the HML and HMR cassettes can also efficiently switch, apparently by obtaining genetic information from either of the other two cassettes (Klar et al., Cell 25:517-524, 1981). We have isolated a novel chromosome III rearrangement in heterothallic (marl ho) strains, which is also produced efficiently in marl HO cells, presumably the consequence of a recombination event between HML and HMR. The fusion results in the loss of sequences which are located distal to HML and to HMR and produces a ring derivative of chromosome III. Cells containing such a ring chromosome are viable as haploids; apparently, no essential loci are located distal to the HM loci. The fusion cassette behaves as a standard HM locus with respect to both regulation by the MAR/SIR control and its role in switching MAT.

Efficient production of a ring derivative of chromosome III by the mating-type switching mechanism in Saccharomyces cerevisiae.

The mating-type switches in the yeast Saccharomyces cerevisiae occur by unidirectional transposition of replicas of unexpressed genetic information, residing at HML or HMR, into the mating-type locus (MAT). The source loci, HML and HMR, remain unchanged. Interestingly, when the HM cassettes are expressed, as in marl strains, the HML and HMR cassettes can also efficiently switch, apparently by obtaining genetic information from either of the other two cassettes (Klar et al., Cell 25:517-524, 1981). We have isolated a novel chromosome III rearrangement in heterothallic (marl ho) strains, which is also produced efficiently in marl HO cells, presumably the consequence of a recombination event between HML and HMR. The fusion results in the loss of sequences which are located distal to HML and to HMR and produces a ring derivative of chromosome III. Cells containing such a ring chromosome are viable as haploids; apparently, no essential loci are located distal to the HM loci. The fusion cassette behaves as a standard HM locus with respect to both regulation by the MAR/SIR control and its role in switching MAT.

The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition.

The two yeast mating-type alleles MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters. Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed, except in sir- strains. Since SIR represses not only transcription from a silent copy but also its ability to act as a recipient in a mating-type interconversion, we have investigated whether it might act by regulating the entire chromatin structure of a silent locus. We have therefore compared the profile of DNAase I and micrococcal nuclease cleavage at HML alpha with MAT alpha and HMRa with MATa in sir- and SIR+ strains. We find that SIR is necessary for the maintenance of a different chromatin structure at HM loci from their active counterparts at MAT. One particularly striking change that SIR induces provides a simple explanation for one of its biological properties: control of directionality of switching. SIR causes the disappearance of a DNAase I-hypersensitive site at Y-Z boundary (found at MAT or HM sir-) that is coincident with a double-strand cleavage possibly created by HO in the initiation of a mating-type switch.