Histone methylation is known to be associated with both transcriptionally active and repressive chromatin states. Recent studies have identified SET domain-containing proteins such as SUV39H1 and Clr4 as mediators of H3 lysine 9 (Lys9) methylation and heterochromatin formation. Interestingly, H3 Lys9 methylation is not observed from bulk histones isolated from asynchronous populations of Saccharomyces cerevisiae or Tetrahymena thermophila. In contrast, H3 lysine 4 (Lys4) methylation is a predominant modification in these smaller eukaryotes. To identify the responsible methyltransferase(s) and to gain insight into the function of H3 Lys4 methylation, we have developed a histone H3 Lys4 methyl-specific antiserum. With this antiserum, we show that deletion of SET1, but not of other putative SET domain-containing genes, in S. cerevisiae, results in the complete abolishment of H3 Lys4 methylation in vivo. Furthermore, loss of H3 Lys4 methylation in a set1⌬ strain can be rescued by SET1. Analysis of histone H3 mutations at Lys4 revealed a slow-growth defect similar to a set1⌬ strain. Chromatin immunoprecipitation assays show that H3 Lys4 methylation is present at the rDNA locus and that Set1-mediated H3 Lys4 methylation is required for repression of RNA polymerase II transcription within rDNA. Taken together, these data suggest that Set1-mediated H3 Lys4 methylation is required for normal cell growth and transcriptional silencing.

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

Several types of histone modifications have been shown to control transcription. Recent evidence suggests that specific combinations of these modifications determine particular transcription patterns. The histone modifications most recently shown to play critical roles in transcription are arginine-specific and lysine-specific methylation. Lysine-specific histone methyltransferases all contain a SET domain, a conserved 130 amino acid motif originally identified in polycomb- and trithorax-group proteins from Drosophila. Members of the SU(VAR)3-9 family of SET-domain proteins methylate K9 of histone H3. Methylation of H3 has also been shown to occur at K4. Several studies have suggested a correlation between K4-methylated H3 and active transcription. In this paper, we provide evidence that K4-methylated H3 is required in a negative role, rDNA silencing in Saccharomyces cerevisiae. In a screen for rDNA silencing mutants, we identified a mutation in SET1, previously shown to regulate silencing at telomeres and HML. Recent work has shown that Set1 is a member of a complex and is required for methylation of K4 of H3 at several genomic locations. In addition, we demonstrate that a K4R change in H3, which prevents K4 methylation, impairs rDNA silencing, indicating that Set1 regulates rDNA silencing, directly or indirectly, via H3 methylation. Furthermore, we present several lines of evidence that the role of Set1 in rDNA silencing is distinct from that of the histone deacetylase Sir2. Together, these results suggest that Set1-dependent H3 methylation is required for rDNA silencing in a Sir2-independent fashion.

Silencing at the rDNA, HM loci, and telomeres in Saccharomyces cerevisiae requires histone-modifying enzymes to create chromatin domains that are refractory to recombination and RNA polymerase II transcription machineries. To explore how the silencing factor Sir2 regulates the composition and function of chromatin at the rDNA, the association of histones and RNA polymerase II with the rDNA was measured by chromatin immunoprecipitation. We found that Sir2 regulates not only the levels of K4-methylated histone H3 at the rDNA but also the levels of total histone H3 and RNA polymerase II. Furthermore, our results demonstrate that the ability of Sir2 to limit methylated histones at the rDNA requires its deacetylase activity. In sir2⌬ cells, high levels of K4-trimethylated H3 at the rDNA nontranscribed spacer are associated with the expression of transcription units in the nontranscribed spacer by RNA polymerase II and with previously undetected alterations in chromatin structure. Together, these data suggest a model where the deacetylase activity of Sir2 prevents euchromatinization of the rDNA and silences naturally occurring intergenic transcription units whose expression has been associated with disruption of cohesion complexes and repeat amplification at the rDNA. INTRODUCTIONModified histones at silent genomic domains contribute to a chromatin environment that is refractory to gene expression and genetic recombination (reviewed in Strahl and Allis, 2000;Turner, 2000;Jenuwein and Allis, 2001). In Saccharomyces cerevisiae, chromatin at the homothallic mating-type loci HML and HMR, telomeres, and the ribosomal DNA locus (rDNA) silences genetic recombination and expression of native and ectopic genes transcribed by RNA polymerase (Pol) II. Silencing at the HM loci and telomeres has been studied extensively, whereas the mechanisms of Pol II silencing at the rDNA are not well characterized (reviewed in Moazed, 2001;Rusche et al., 2003). Increasing our understanding of the factors and mechanisms that regulate silencing at the rDNA will provide insight into the pathways that regulate gene expression and genome stability.In S. cerevisiae, the rDNA contains ϳ150 -200 tandem copies of a 9.1-kilobase (kb) repeat, with each repeat containing a Pol I-transcribed 35S rRNA gene and a nontranscribed spacer (NTS) that is subdivided into NTS1 and NTS2 by the Pol III-transcribed 5S rRNA gene (reviewed in Warner, 1999; Figure 1). Despite high levels of transcription by Pol I and Pol III in the rDNA locus, Pol II-transcribed genes integrated into the rDNA are silenced (referred to as rDNA silencing) (Bryk et al., 1997;Fritze et al., 1997;Smith and Boeke, 1997). Additionally, silent chromatin at the rDNA is essential for repression of genetic recombination (Gottlieb and Esposito, 1989;Davis et al., 2000;Kobayashi et al., 2004) and extension of replicative life span (reviewed in Guarente, 2000).Chromatin-associated proteins and modified histones regulate silencing of Pol II transcription at the rDNA (Bryk et al., 1997;Fritze et al., 199...

I‐TevI, the intron‐encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence‐tolerant fashion. We demonstrate here that the 28 kDa I‐TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I‐TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I‐TevI‐DNA complex is proposed to promote top‐strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base‐substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I‐TevI‐induced conformational change. These results, combined with genetic, physical and computer‐modeling studies, form the basis of a model, wherein I‐TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top‐strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine‐containing DNA, as exists in the T‐even phages.