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.In Saccharomyces cerevisiae the ribosomal DNA (rDNA) locus coding for rRNAs is represented by a single gene cluster of 150 to 200 units repeated in tandem on chromosome XII (23). Each unit contains the 35S RNA gene transcribed by RNA polymerase I (Pol I) and is separated from the next repeat by a nontranscribed spacer (NTS) (Fig. 1). Despite its name, the latter sequence is highly transcribed by RNA polymerase III, at the 5S gene, and at very low levels by RNA polymerase II (Pol II) at different promoters, generating noncoding RNAs (ncRNAs) (10, 15, 19, 33).Open in a separate windowFig. 1.Map of rDNA, probes, and oligonucleotides used in this study. Schematic map of ribosomal genes in Saccharomyces cerevisiae. Horizontal black arrows represent RNA transcripts. Filled boxes indicate 35S and 5S coding units. Gray boxes represent ncRNA promoters. Ellipses refer to positioned nucleosomes. The boxed areas in the lower part of the figure contain probes used in Southern and Northern blotting experiments (thin horizontal black lines). White arrows indicate oligonucleotides used in RT-PCR and primer extension experiments. The thick black lines represent the positions of amplicons produced in Chr-IP analyses.Recent observations (10, 15, 19, 33) concerning mutant strains overproducing ncRNAs from the NTS region have shown that the presence of these RNA species correlates with the SIR2-dependent transcriptional silencing occurring at the ribosomal locus (19); interestingly, DNA topoisomerase I mutants (top1Δ) show increases of these ncRNA species. Also the mitotic recombination in the rDNA (suppressed in wild-type [WT] strains) is increased when ncRNAs are overproduced (15); this phenomenon has been proposed to be due in part to the Pol II activity that locally may displace cohesins and induce recombination. More recently, alteration of ribosomal gene copy number has also been observed in mutant strains with increased production of ncRNAs (10, 15, 33).Thus, at the rDNA, a connection among transcriptional silencing, recombination between repeated units, and ncRNA production appears to exist.Moreover, it has been discovered (6, 39) that the yeast S. cerevisiae has an inherent ability to produce rRNA by Pol II, but this transcription activity is silenced in normal cells. In mutants lacking the Pol I transcription factor upstream activation factor (UAF), rDNA transcription is due to Pol II activity (the polymerase-switched state [PSW]). The presence of UAF in WT cells appears to stabilize this state, thus showing a powerful silencing of rDNA transcription by Pol I.Hypotheses about the basis of transcriptional silencing at the rDNA locus take into consideration both chromatin organization (2, 21, 27, 28) and Pol I transcription (3, 5). Different observations have identified the elements required for silencing at the rDNA locus: histones and those enzymes capable of histone modifications and/or chromatin remodeling (2, 21, 27, 28). Putative chromatin structures have been reported for silenced Pol II transcription in the rDNA region, particularly at the NTS level (8, 22), when reporter genes are artificially inserted in the rDNA locus (2, 27). It has also been suggested (3, 5) that Pol I activity on this region is involved in setting of transcriptional silencing, creating peculiar structures inhibiting Pol II activity (5).In order to verify whether Pol I and Pol II transcription correlates with ncRNA production and chromatin modifications, we studied several yeast strains differing in Pol I transcription of 35S RNA, in particular the W303 strain (WT) (30), in which about 50% of the units are transcribed; the NOY1071 strain (5), with all transcribed units (ATU), where the strong contraction of the repeats (from 200 to 25) is stably maintained by the lack of the FOB1 gene; and the NOY699 strain (39), carrying a UAF subunit deletion (RRN5), with a strongly reduced 35S RNA transcription by RNA polymerase I and where rRNA synthesis is mainly carried out by RNA polymerase II (PSW). In addition, we used two strains characterized by a complete loss of RNA polymerase I transcription: NOY558, in which the core factor (CF) complex is impaired due to the absence of the RRN7 component (17), and the D128-1d (A43) strain, lacking the A43 subunit of RNA polymerase I (31). A summary of the strain features is given in Table 1.

Table 1.

Features of strains used in this study^a

Name	Copy no. (rDNA)	Pol I transcription production	ncRNA	Acetylation
				Coding	Cryptic promoters
ATU (NOY1071)	25	++++	−	+++	−
WT (W303-1a)	150	+++	+/−	+	+/−
CF (NOY558)	100	−	+	ND	ND
PSW (NOY699-PSW)	300	+/−	+++	−	+++
A43 (D128-1d)	150	−	+	ND	ND

Open in a separate window^aThe reference strain (W303-1a, WT [30]) has about 50% of the units transcribed; in the ATU (NOY1071 [5]) strain all units are transcribed, and the strong contraction of the repeats (from 200 to 25) is stably maintained by the lack of the FOB1 gene; the CF strain (NOY558 [17]) lacks the rrn7 subunit of the CF complex, and thus, Pol I transcription is abolished; the PSW (NOY699 [39]) strain carries a UAF subunit deletion that abolishes 35S RNA transcription; in addition, a polymerase switch (PSW) occurred and transcription of 35S RNA by RNA polymerase I is switched to RNA polymerase II. In the A43 strain (D128-1d [31]) a deletion of the gene of the A43 subunit of Pol I abolishes its transcriptional activity. − to ++++, lowest to highest levels, respectively; ND, not determined.We have measured ncRNA synthesis in the different strains and evaluated chromatin structure and histone acetylation in the NTS region. The efficiency of site-specific cleavage activity of DNA topoisomerase I has also been studied.