RNA polymerase I transcription silences noncoding RNAs at the ribosomal DNA locus in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify "ribosomal" Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

DNA protein interactions at the rRNA of Saccharomyces cerevisiae.

The rDNA cluster is the genetic locus encoding the ribosomal RNAs and physically defines where ribosomes begin to be assembled. In the yeast Saccharomyces cerevisiae, the highly repetitive structure of this locus makes it a very interesting target for studies about genome stability, chromatin-mediated transcriptional silencing and progression of aging. In fact, recombination among the repeated units is suppressed in a WT cell. Moreover, when genes transcribed by RNA polymerase II are inserted in the rDNA cluster, their transcription is silenced. Finally, the formation of rDNA minicircles (ERCs) has been shown to be one of the causes of aging in yeast. DNA topoisomerase I have been shown to suppress recombination specifically at the rDNA of S.cerevisiae. Moreover, also the chromatin structure of this locus is affected in a top1 strain, because rDNA specific transcriptional silencing is abolished. Nonetheless, the molecular basis of how this enzyme interferes with these functions is yet unknown. Here are reported results obtained by in vivo studies of DNA protein interactions occurring on the rDNA locus. The analyses include a fine mapping of nucleosome positioning; RNA polymerase I transcription factors and DNA topoisomerase I cleavage sites. Important conclusions can be drawn: i) nucleosome positioning in the Non Transcribed Spacer is not affected by RNA polymerase I transcription; ii) the RNA polymerase I transcription factors bind DNA in vivo with a defined hierarchy; iii) the DNA topoisomerase I cleaves the NTS in very specific sites, but cleavage is not induced by RNA polymerase I transcription. These in vivo studies help to characterize the molecular basis of important phenomena as the transcriptional silencing and genome stability in yeast.

FOB1 affects DNA topoisomerase I in vivo cleavages in the enhancer region of the Saccharomyces cerevisiae ribosomal DNA locus.

In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results reported in this paper, indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the presence of Fob1p and independent of replication and transcription activities. We thus hypothesize that the binding to DNA of Fob1p itself may be the cause of the DNA topoisomerase I activity in the rDNA enhancer.

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis.

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.

Silencing in yeast rDNA chromatin: reciprocal relationship in gene expression between RNA polymerase I and II.

About half of approximately 150 rRNA genes are transcriptionally active in Saccharomyces cerevisiae. Chromatin structures in the inactive, and not the active, copies were previously thought to silence both rRNA genes and reporter Pol II genes. Contrary to this belief, we found that silencing of reporters is much stronger in a mutant with approximately 25 rDNA copies, all of which are transcriptionally active. By integrating reporter gene mURA3 with an inactive rDNA copy missing the Pol I promoter, we found that mURA3 is not silenced in chromosomal rDNA repeats. Together with the demonstration of a requirement for active Pol I in silencing, these results show a reciprocal relationship in gene expression between Pol I and Pol II in rDNA.

In exponentially growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes.

Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes ( approximately 143 in this case) and a strain in which the rRNA gene number was reduced to approximately 42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was approximately 50 +/- 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 +/- 29 polymerases/gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of approximately 60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

Acetylation and accessibility of rDNA chromatin in Saccharomyces cerevisiae in (Delta)top1 and (Delta)sir2 mutants.

The insertion of reporter genes in the ribosomal DNA (rDNA) locus of Saccharomyces cerevisiae causes their transcriptional repression. This kind of transcriptional silencing depends on proteins such as Sir2p and Top1p, and has been shown to be mediated by chromatin. While Sir2p modifies nucleosomes directly through its histone deacetylase activity, little is known about changes in the chromatin structure that occur at the rDNA locus when TOP1 is deleted. Here, we show that the absence of Top1p causes increased histone acetylation at the rDNA locus. Moreover, rDNA chromatin becomes more accessible in a similar manner in both top1 and sir2 mutant strains.