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.

Sir2 represses endogenous polymerase II transcription units in the ribosomal DNA nontranscribed spacer.

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 sir2Delta 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.

Isw1 acts independently of the Isw1a and Isw1b complexes in regulating transcriptional silencing at the ribosomal DNA locus in Saccharomyces cerevisiae.

Transcriptional silencing of Pol II-transcribed genes in Saccharomyces cerevisiae occurs at the HM loci, telomeres and ribosomal DNA (rDNA) locus. Gene silencing at these loci requires histone-modifying enzymes as well as factors that regulate local chromatin structure. Previous work has shown that the ATP-dependent chromatin remodeling protein Isw1 is required for silencing of a marker gene inserted at the HMR locus, but not at telomeres. Here we show that Isw1 is required for transcriptional silencing of Pol II-transcribed genes in the ribosomal DNA locus. Our results indicate that Isw1 associates with the rDNA and that this interaction is not altered in cells lacking other members of the Isw1a and Isw1b chromatin remodeling complexes. Further, the association of Isw1 with the rDNA is not altered in cells lacking the histone deacetylase Sir2 or the histone methyltransferase Set1, two factors that are required for gene silencing at the rDNA. Notably, the loss of transcriptional silencing at the rDNA in cells lacking Isw1 is correlated with a change in rDNA chromatin structure. Together, our data support a model in which Isw1 acts independently of the previously characterized Isw1a and Isw1b complexes to maintain a heterochromatin-like structure at the rDNA that is required for gene silencing.

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.

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.

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.

Isw2 regulates gene silencing at the ribosomal DNA locus in Saccharomyces cerevisiae.

Three heterochromatin-like domains have been identified in Saccharomyces cerevisiae that are refractory to transcription by Pol II, the silent mating-type loci, telomeres and the ribosomal DNA. Previous work has shown that chromatin remodelers can regulate silent chromatin. Here, we report the findings of an investigation into the role of ISW2 in transcriptional silencing at the rDNA. We show that the levels of retrotransposition and mRNA from a genetically marked Ty1 element located in the rDNA were increased significantly in isw2Delta cells, while transcript levels from Ty1 elements outside of the rDNA were not increased in cells lacking ISW2. Additionally, we show that Isw2 is not required for silencing at a telomere. Our findings demonstrate that Isw2 is required for transcriptional silencing at the rDNA and emphasize the differences in the regulation of transcriptional silencing at silent loci in S. cerevisiae.