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PubMed1995

Cloning and sequence analysis of cDNA for a possible DNA-binding protein 5E5 in the nervous system.

Monoclonal antibody 5E5 recognized an intranuclear antigen of neurons in the rat. We isolated 5E5cDNA and determined its nucleotide and deduced amino acid sequences. The 5E5cDNA had an open reading frame of 825 amino acids and its amino acid sequence showed no significant homology to any protein or to any DNA binding motif so far known. 5E5 protein had an abundance of basic amino acids, especially arginine, and included a glycine-rich region and a proline cluster. Monoclonal antibody 12H raised against 5E5cDNA fusion protein recognized an intranuclear substance in rat brain sections and a single protein band of about 98 kDa in the brain nuclear extract fraction on immunoblotting. DNA-cellulose column chromatography indicated that 5E5 protein might have DNA-binding ability. Transfection studies indicated that 5E5 protein expressed in COS-1 is localized in cell nuclei. These results suggest that 5E5 protein is a possible DNA-binding protein which is expressed especially in neurons.

Amino Acid Sequence↗
PubMed2002

Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association.

The amino-terminal histone tails are subject to covalent post-translational modifications such as acetylation, methylation, and phosphorylation. In the histone code hypothesis, these exposed and unstructured histone tails are accessible to a repertoire of regulatory factors that specifically recognize the various modified histones, thereby generating altered chromatin structures that mediate specific biological responses. Here, we report that lysine (Lys) 79 of histone H3, which resides in the globular domain, is methylated in eukaryotic organisms. In the yeast Saccharomyces cerevisiae, Lys 79 of histone H3 is methylated by Dot1, a protein shown previously to play a role in telomeric silencing. Mutations of Lys 79 of histone H3 and mutations that abolish the catalytic activity of Dot1 impair telomeric silencing, suggesting that Dot1 mediates telomeric silencing largely through methylation of Lys 79. This defect in telomeric silencing might reflect an interaction between Sir proteins and Lys 79, because dot1 and Lys 79 mutations weaken the interaction of Sir2 and Sir3 with the telomeric region in vivo. Our results indicate that histone modifications in the core globular domain have important biological functions.

Amino Acid Sequence↗
PubMed1997

SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.

Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.

Blotting, Western↗
PubMed1997

An unusual form of transcriptional silencing in yeast ribosomal DNA.

Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcriptional silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Ty1 retrotransposons marked with a weakened URA3 gene (Ty1-mURA3) efficiently integrated into rDNA. The mURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. MET15 and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and MET15 silencing, but deletion of SIR1 or SIR3 did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.

Chromatin↗
PubMed1997

Transcriptional silencing of Ty1 elements in the RDN1 locus of yeast.

We demonstrate that in Saccharomyces cerevisiae, the tandem array of ribosomal RNA genes (RDN1) is a target for integration of the Ty1 retrotransposon that results in silencing of Ty1 transcription and transposition. Ty1 elements transpose into random rDNA repeat units and are mitotically stable. In addition, we have found that mutation of several putative modifiers of RDN1 chromatin structure abolishes silencing of Ty1 elements in the rDNA array. Disruption of SIR2, which elevates recombination in RDN1, or TOP1, which increases psoralen accessibility in rDNA, or HTA1-HTB1, which reduces histone H2A-H2B levels and causes localized chromatin perturbations, abolishes transcriptional silencing of Ty1 elements in RDN1. Furthermore, deletion of the gene for the ubiquitin conjugating enzyme Ubc2p, which ubiquitinates histones in vitro, derepresses not only Ty1 transcription but also mitotic recombination in RDN1. On the basis of these results, we propose that a specialized chromatin structure exists in RDN1 that silences transcription of the Ty1 retrotransposon.

Base Sequence↗
PubMed2003

Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing.

Silencing within the yeast rDNA repeats inhibits hyperrecombination, represses transcription from foreign promoters, and extends replicative life span. rDNA silencing is mediated by a Sir2-containing complex called RENT (regulator of nucleolar silencing and telophase exit). We show that the Net1 (also called Cfi1) and Sir2 subunits of RENT localize primarily to two distinct regions within rDNA: in one of the nontranscribed spacers (NTS1) and around the Pol I promoter, extending into the 35S rRNA coding region. Binding to NTS1 overlaps the recombination hotspot and replication fork barrier elements, which have been shown previously to require the Fob1 protein for their activities. In cells lacking Fob1, silencing and the association of RENT subunits are abolished specifically at NTS1, while silencing and association at the Pol I promoter region are unaffected or increased. We find that Net1 and Sir2 are physically associated with Fob1 and subunits of RNA polymerase I. Together with the localization data, these results suggest the existence of two distinct modes for the recruitment of the RENT complex to rDNA and reveal a role for Fob1 in rDNA silencing and in the recruitment of the RENT complex. Furthermore, the Fob1-dependent associations of Net1 and Sir2 with the recombination hotspot region strongly suggest that Sir2 acts directly at this region to carry out its inhibitory effect on rDNA recombination and accelerated aging.

Cell Cycle Proteins↗
PubMed2004

Local chromatin structure at the ribosomal DNA causes replication fork pausing and genome instability in the absence of the S. cerevisiae DNA helicase Rrm3p.

Lack of the yeast Rrm3p DNA helicase causes replication defects at multiple sites within ribosomal DNA (rDNA), including at the replication fork barrier (RFB). These defects were unaltered in rrm3 sir2 cells. When the RFB binding Fob1p was deleted, rrm3-generated defects at the RFB were eliminated, but defects at other rDNA sites were not affected. Thus, specific protein-DNA complexes make replication Rrm3p-dependent. Because rrm3-induced increases in recombination and cell cycle length were only partially suppressed in rrm3 fob1 cells, which still required checkpoint and fork restart activities for viability, non-RFB rrm3-induced defects contribute to rDNA fragility and genome instability.

Binding Sites↗
PubMed2004

The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication.

The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.

Cell Cycle Proteins↗
PubMed1999

The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms.

The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.

DNA, Fungal↗
PubMed2006

SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis.

The mammalian cytoplasmic protein SirT2 is a member of the Sir2 family of NAD+-dependent protein deacetylases involved in caloric restriction-dependent life span extension. We found that SirT2 and its yeast counterpart Hst2 have a strong preference for histone H4K16Ac in their deacetylation activity in vitro and in vivo. We have pinpointed the decrease in global levels of H4K16Ac during the mammalian cell cycle to the G2/M transition that coincides with SirT2 localization on chromatin. Mouse embryonic fibroblasts (MEFs) deficient for SirT2 show higher levels of H4K16Ac in mitosis, in contrast to the normal levels exhibited by SirT1-deficient MEFs. The enzymatic conversion of H4K16Ac to its deacetylated form may be pivotal to the formation of condensed chromatin. Thus, SirT2 is a major contributor to this enzymatic conversion at the time in the cell's life cycle when condensed chromatin must be generated anew.

Animals↗
PubMed2001

The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

Amino Acid Sequence↗
PubMed2002

Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus.

How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of approximately 150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing in sir2 Delta mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.

DNA Replication↗
PubMed2005

Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing.

We show that in budding yeast large rDNA deletions arise frequently and cause an increase in telomeric and mating-type gene silencing proportional to repeat loss. Paradoxically, this increase in silencing is correlated with a highly specific down-regulation of SIR2, which encodes a deacetylase enzyme required for silencing. These apparently conflicting observations suggest that a large nucleolar pool of Sir2 is released upon rDNA loss and made available for telomeric and HM silencing, as well as down-regulation of SIR2 itself. Indeed, we present evidence for a reduction in the fraction of Sir2 colocalizing with the nucleolar marker Nop1, and for SIR2 autoregulation. Despite a decrease in the fraction of nucleolar Sir2, and in overall Sir2 protein levels, short rDNA strains display normal rDNA silencing and a lifespan indistinguishable from wild type. These observations reveal an unexpectedly large clonal variation in rDNA cluster size and point to the existence of a novel regulatory circuit, sensitive to rDNA copy number, that balances nucleolar and nonnucleolar pools of Sir2 protein.

DNA, Fungal↗
PubMed1994

Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1.

The maintenance of transcriptional silencing at HM mating-type loci and telomeres in yeast requires the SIR2, SIR3, and SIR4 proteins, none of which appear to be DNA-binding proteins. Here we show that SIR3 and SIR4 interact with a carboxy-terminal domain of the silencer, telomere, and UAS-binding protein RAP1. We identified SIR3 and SIR4 in a two-hybrid screen for RAP1-interacting factors and showed that SIR3 interacts both with itself and with SIR4. The interaction between RAP1 and SIR3 can be observed in vitro in the absence of other yeast proteins. Consistent with the notion that native SIR proteins interact with the RAP1 carboxyl terminus, we show that mutation of the endogenous SIR3 and SIR4 genes increases transcriptional activation by LexA/RAP1 hybrids. To test the importance of the RAP1-SIR3 interaction for silencing, we identified mutations in the RAP1 carboxyl terminus that either diminish or abolish this interaction. When introduced into the native RAP1 protein, these mutations cause corresponding defects in silencing at both HMR and telomeres. We propose that RAP1 acts in the initiation of transcriptional silencing by recruiting a complex of SIR proteins to the chromosome via protein-protein interactions. These data are consistent with a model in which SIR3 and SIR4 play a structural role in the maintenance of silent chromatin and indicate that their action is initiated at the silencer itself.

Bacterial Proteins↗
PubMed2001

Conversion of a gene-specific repressor to a regional silencer.

In Saccharomyces cerevisiae, gene silencing at the HMR and HML loci is normally dependent on Sir2p, Sir3p, and Sir4p, which are structural components of silenced chromatin. Sir2p is a NAD+-dependent histone deacetylase required for silencing. Silencing can be restored in cells lacking Sir proteins by a dominant mutation in SUM1, which normally acts as a mitotic repressor of meiotic genes. This study found that mutant Sum1-1p, but not wild-type Sum1p, associated directly with HM loci. The origin recognition complex (ORC) was required for Sum1-1p-mediated silencing, and mutations in ORC genes reduced association of Sum1-1p with the HM loci. Sum1-1p-mediated silencing also depended on HST1, a paralog of SIR2. Both Sum1-1p and wild-type Sum1p interacted with Hst1p in coimmunoprecipitation experiments. Therefore, the SUM1-1 mutation did not change the affinity of Sum1p for Hst1p, but rather relocalized Sum1p to the HM loci. Sum1-1-Hst1p action led to hypoacetylation of the nucleosomes at HM loci. Thus, Sum1-1p and Hst1p could substitute for Sir proteins to achieve silencing through formation of a compositionally distinct type of heterochromatin.

Cell Division↗
PubMed1995

The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability.

Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.

Amino Acid Sequence↗
PubMed2002

Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast.

In Saccharomyces cerevisiae, heterochromatin-like regions are found near telomeres and at the silent mating-type loci, where they can repress genes in an epigenetic manner. Several proteins are involved in telomeric heterochromatin structure including Rap1, Sir2, Sir3, Sir4, yKu70 (Hdf1), yKu80 (Hdf2), and the N termini of histones H3 and H4. By recognizing cis-acting DNA-binding sites, Rap1 is believed to recruit Sir and other silencing proteins and determine where heterochromatin forms. The integrity of heterochromatin also requires the binding of Sir proteins to histones that may form a scaffold for Sir protein interactions with chromatin. In this study we describe how the heterochromatin complex may form initially and how it differs from the complex that spreads along the chromosome. We found that close to the telomere end, Sir4 can bind Rap1 independently of Sir2, Sir3, yKu70/yKu80, and the intact H4 N terminus. In contrast, Sir4 binding requires all of the silencing factors further along telomeric heterochromatin. These data indicate that Sir4 binding to Rap1 initiates the sequential association of Sir and other proteins, allowing the subsequent spreading of the heterochromatin proteins along the chromosome.

Antigens, Nuclear↗