Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Citation: Teytelman L, Eisen MB, Rine J ( Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts Leonid Teytelman 0 1 Michael B. Eisen 0 1 Jasper Rine 0 1 Gregory S. Barsh, Stanford University School of Medicine, United States of America 0 Funding: This work was supported by National Institutes of Health grants GM31105 to JR and R01-HG002779 to MBE, and by an NSF predoctoral fellowship to LT 1 1 Department of Molecular & Cell Biology, University of California Berkeley, Berkeley, California, United States of America, 2 California Institute for Quantitative Biosciences, Berkeley, California, United States of America, 3 Center for Integrative Genomics, University of California Berkeley , Berkeley, California , United States of America Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair. - The ends of chromosomes in yeasts, vertebrates, Drosophila, and eukaryotic pathogens such as Plasmodim falciparum diverge more rapidly than the rest of their genomes [1]. In budding yeasts of the genus Saccharomyces, chromosome ends contain a high density of repeated sequences and relatively few genes; they are more diverged between species than any other portions of the genomes, and are highly variable within species [2,3]. The accelerated diversification of subtelomeric DNA is commonly attributed to the presence of transposons and the repetitive nature of these regions, as both contribute to recombination between different chromosome ends [4,5]. However, subtelomeric regions in yeasts are also silenced, analogously to metozoan heterochromatin [6], raising the possibility that the formation and maintenance of a silenced chromatin state contribute to the observed rapid evolution. In S. cerevisiae, the best characterized silenced regions are the HML and HMR transcriptionally inactive mating loci of chromosome III. They contain non-expressed copies of the MATa and MATa mating-type genes. During mating type interconversion, HML or HMR is copied into the MAT locus, also on chromosome III, where the resident allele is transcribed. Since haploid cells that express both MATa and MATa behave as nonmating diploids, it is crucial that HML and HMR are silenced. This is achieved through the E and I silencers that flank both of the silenced loci (Figure 1) and recruit Silent Information Regulator (Sir) proteins which then spread throughout the regions. The Sir proteins bind to and deacetylate the tails of histones H3 and H4, leading to silencing of HML and HMR [7]. The Sir2/Sir3/Sir4 protein complex that is responsible for HML and HMR silencing also binds to subtelomeric regions of S. cerevisiae chromosomes [8]. In contrast to the strong and robust silencing of HML and HMR, subtelomeric silencing is weaker [9]. Nevertheless, native telomere-proximal genes and reporter genes inserted near telomeres are reliably silenced [1013]. The Saccharomyces sensu stricto species (S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus) genome sequences are sufficiently closely related to allow identification of conserved regulatory sequences [14]. Essentially all S. cerevisiae protein-coding genes are found in these other species, and most orthologous intergenic regions in the sensu stricto yeasts can be readily aligned [2,15]. However, in analyzing the evolution of the HML and HMR silencers, we discovered a surprising lack of DNA conservation in all four flanking regions, motivating an in-depth exploration of the evolution of silenced regions within and between these yeast species. Our observations suggested an additional force in the shaping of these regions. Lack of Cross-Species Conservation in Sequences Flanking HML and HMR To identify the E and I silencers in the sensu stricto species, we searched for peaks of conservation in multiple sequence alignments. For both of the S. cerevisiae HML and HMR, we identified contigs in the sequenced sensu stricto species that contained a part of the locus and the adjacent gene. The right side of HMR was misassembled in S. paradoxus with two disjointed contigs with incorrect inverted ends, so we resequenced and assembled the region (GenBank EU597267). HML and HMR were conserved across all five species with clearly conserved orthologs of the neighboring genes (Table S1). However, unlike most intergenic sequences in the genome, the regions around HML and HMR were too diverged to allow multiple alignments. Moreover, local Many plants, fungi, pathogens, and animals have chromosome regions that are silenced. Special proteins change the chromosome structure in these domains, turning genes off or lowering their expression levels. We found an increased frequency of DNA mutations in these silenced regions of closely related yeasts. This increase is likely due to silencing proteins interfering with DNA repair or replication. Accurate replication of genetic information with minimal mutations is usually critical for the survival and fitness of an organism; however, there are examples where a high mutation rate is beneficial. The silenced regions of chromosomes are often associated with viruslike transposable elements, and with genes that are important in responding to environmental changes. Hence, it is possible that elevated DNA mutations in silenced regions contribute to genome defense against transposable elements or increased genetic diversity to cope with variation in surrounding conditions. pairwise alignments of these flanking sequences between any of the ten species pairs were also unexpectedly dissimilar. The best pairwise alignments were between the two closest species S. cerevisiae and S. paradoxus, but instead of the genome-wide average of 80% identity for orthologous intergenic regions, the percent identities were: 46% left of HML, 55% right of HML, 52% left of HMR, 45% right of HMR. These alignments were almost as dissimilar as if the sequences were unrelated; 1000 random equallength sequences with identical base composition that we generated had an averaged local pairwise similarity of 45%. BLAST-based comparisons also did not reveal matches for the sequences between HML or HMR a (...truncated)