Mary Bryk

Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent mechanism

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.

The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.

I‐TevI, a double‐strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I‐TevI, all three domains are important for formation of a cleavage‐competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I‐TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I‐TevI recognizes T‐even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.

Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.

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.

The Requirements for COMPASS and Paf1 in Transcriptional Silencing and Methylation of Histone H3 in Saccharomyces cerevisiae

The Set1-containing complex, COMPASS, methylates histone H3 on lysine 4 (K4) in Saccharomyces cerevisiae. Despite the preferential association of K4-trimethylated H3 with regions of the genome that are transcribed by RNA polymerase II, transcriptional silencing is one of the few cases in S. cerevisiae where histone-methylation defects have a clear effect on gene expression. To better understand the role of COMPASS in transcriptional silencing, we have determined which members of COMPASS are required for silencing at the ribosomal DNA locus (rDNA), a telomere, and the silent mating loci (HM) using Northern analyses. Our findings indicate that most members of COMPASS are required for silencing at the rDNA and telomere, while none are required for silencing of endogenous genes at the HM loci. To complement gene-expression analysis, quantitative Western blot experiments were performed to determine the members of COMPASS that are required for methylation of histone H3. While most are required for trimethylation, cells lacking certain COMPASS proteins maintain reduced levels of K4 mono- and dimethylated H3, suggesting that some COMPASS members have redundant function. Finally, we show Paf1 is required for silencing and K4-methylated H3 at the rDNA, suggesting a possible direct role for K4-methylated H3 in gene silencing.

The Sgs1 helicase of Saccharomyces cerevisiae inhibits retrotransposition of Ty1 multimeric arrays.

ABSTRACT Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state. When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination. In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels. Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism. Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32- to 79-fold in sgs1Δ mutants. Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription. Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration. Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1Δmutants, resulting in transposition of heterogeneous Ty1 multimers. Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements. Therefore, Ty1 multimers that transpose at a high frequency in sgs1Δ mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration. Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.

Calorie restriction effects on silencing and recombination at the yeast rDNA.

Aging research has developed rapidly over the past decade, identifying individual genes and molecular mechanisms of the aging process through the use of model organisms and high throughput technologies. Calorie restriction (CR) is the most widely researched environmental manipulation that extends lifespan. Activation of the NAD+‐dependent protein deacetylase Sir2 (Silent Information Regulator 2) has been proposed to mediate the beneficial effects of CR in the budding yeast Saccharomyces cerevisiae, as well as other organisms. Here, we show that in contrast to previous reports, Sir2 is not stimulated by CR to strengthen silencing of multiple reporter genes in the rDNA of S. cerevisiae. CR does modestly reduce the frequency of rDNA recombination, although in a SIR2‐independent manner. CR‐mediated repression of rDNA recombination also does not correlate with the silencing of Pol II‐transcribed noncoding RNAs derived from the rDNA intergenic spacer, suggesting that additional silencing‐independent pathways function in lifespan regulation.

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.

Linker histone H1 represses recombination at the ribosomal DNA locus in the budding yeast Saccharomyces cerevisiae

Several epigenetic phenomena occur at ribosomal DNA loci in eukaryotic cells, including the silencing of Pol I and Pol II transcribed genes, silencing of replication origins and repression of recombination. In Saccharomyces cerevisiae, studies focusing on the silencing of Pol II transcription and genetic recombination at the ribosomal DNA locus (rDNA) have provided insight into the mechanisms through which chromatin and chromatin‐associated factors regulate gene expression and chromosome stability. The core histones, H2A, H2B, H3 and H4, the fundamental building blocks of chromatin, have been shown to regulate silent chromatin at the rDNA; however, the function of the linker histone H1 has not been well characterized. Here, we show that S. cerevisiae histone H1 represses recombination at the rDNA without affecting Pol II gene silencing. The most highly studied repressor of recombination at the rDNA is the Silent information regulator protein Sir2. We find that cells lacking histone H1 do not exhibit a premature‐ageing phenotype nor do they accumulate the rDNA recombination intermediates and products that are found in cells lacking Sir2. These results suggest that histone H1 represses recombination at the rDNA by a mechanism that is independent of the recombination pathways regulated by Sir2.

Catalytic and functional roles of conserved amino acids in the SET domain of the S. cerevisiae lysine methyltransferase Set1.

In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

4 Homing Endonucleases

I. INTRODUCTION Homing endonucleases are a group of enzymes whose catalytic activity results in self-propagation. The sequences that code for these endonucleases usually interrupt genes by localizing as open reading frames in introns or as inframe spacers in protein-coding sequences. The target of a homing endonuclease is its cognate intronless or spacerless allele. The endonuclease initiates a DNA mobility or “homing” event by making a double-strand cut in its target. Repair of the cleaved allele results in the conversion of this gene to the interrupted endonuclease-encoding form. Homing endonucleases are widespread, found in all three kingdoms and in a range of genetic environments, which include mitochondrial, chloroplast, nuclear, and bacteriophage genomes (Table 1). Although the discovery of these endonucleases is recent, genetic consequences attributable to their presence have been observed for some time (see Fig. 1) We review here the history and general properties of homing endonucleases, point out both similarities and differences among the individual enzymes, and address the evolutionary implications of endonuclease gene mobility. II. HISTORIC REVIEW In 1970, the unidirectional transfer of a Saccharomyces cerevisiae genetic marker, termed omega (ω), from ω + to ω − yeast strains, was reported (Coen et al. 1970), sparking a great deal of interest regarding the role of nonreciprocal recombination in yeast mitochondrial genetics. With the discovery of restriction enzymes and the introduction of advanced molecular techniques, the ω locus was mapped to an intron in the mitochondrial large ribosomal RNA gene (L-rRNA gene) (Dujon and Michel 1976; Bos et al. 1978; Heyting...