Strains and Media used in the Study

Strains used in this study are listed in Table 1. Yeast cells were grown in either synthetic glucose (SD) medium or yeast extract/peptone/dextrose (YEPD) at 30°C with aeration. Recipes for media used were described in detail previously [5]. Where indicated, we have replaced the S. cerevisiae URA3 gene with the S. pombe URA4 gene for recombination-mediated gene disruptions and epitope tag fusions. URA4 encodes a protein with the same function (orotidine-5′-phosphate decarboxylase), however there is limited or no recombination with the endogenous URA3 locus.

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Table 1. Strains used in this study.

https://doi.org/10.1371/journal.pone.0056793.t001

Synthetic Genetic Array (SGA)

SGA was conducted as described previously [28], [29].

Genetic Interactions

We constructed null mutants of snf5Δ and snf6Δ in NOY396 using established methods for yeast transformation [30]. Then snf5Δ and snf6Δ were mated with rpa135(D784G) or rpa49Δ in YEPD media. Diploid strains were selected on SD-ura-leu for the combination of rpa49Δ with snf5Δ or snf6Δ; combination of rpa135(D784G) with snf5Δ or snf6Δ was selected on SD-ura+neurseothricin. Following sporulation, tetrads were dissected onto YEPD plates using a Zeiss Axioskop 40 Tetrad Microscope. 50–60 tetrads of each genetic background were dissected. The dissection plates were incubated at 30°C >7 days; genotypes of individual segregants were tested by screening for nutritional markers linked to the deletion mutation.

Chromatin Immunoprecipitation (ChIP)

The procedure was described previously [4] except cells were treated with formaldehyde for 1hr to successfully crosslink SWI/SNF to the rDNA [31]. The detailed ChIP procedure and sequences for primers used in real-time analysis are described in Supplementary Data. ChIP data were quantified from at least 2 10-fold dilutions per sample from duplicate cultures.

Measurement of rRNA Synthesis

[methyl-³H]methionine incorporation assays were performed as described previously [2]. The rationale of this assay is more thoroughly explained in Anderson et al. 2011 [32]. Briefly, we cultured cells in SD-met to exponential phase and pulse-labeled cells for 5 min with 20µCi/ml [methyl-³H]methionine followed by a 5 min chase with 500 µg/ml of cold methionine. RNA was extracted, run on a denaturing gel, transferred to a membrane, and detected by autoradiography.

rDNA Copy Number Determination

rDNA copy number was measured as described previously [4]. Briefly, chromosomes were separated by CHEF, stained with ethidium bromide, transferred to a membrane, hybridized with a ³²P-dCTP labeled 600 bp rDNA probe [primers used to amplify the probe were provided in [4]], and detected by autoradiography.

EM Analysis

Miller chromatin spreads and EM analyses were performed as described [33]. Polymerase occupancy measurements are described in El Hage et al. 2010 [34].

Statistical Analysis

Statistical analyses were performed on all the data from ChIP and EM assays. For EM data, Kolmogorov-Smirnov (K-S) tests were conducted to test the hypothesis that distributions of Pol I on active genes are comparable for snf6Δ and WT. The results indicate that the hypothesis can be rejected. For ChIP data, two-way ANOVA tests were performed to test the following hypotheses: i) within a given strain there is little variation across locations, ii) at given locations there is little variation between strains, and iii) there is no interaction between strain and location. The results indicate that all three hypotheses could be rejected. As a follow-up, TukeyHSD tests were performed to test the hypothesis that binding signals are the same between different strains at the same locations. Rejections of this hypothesis were documented, and p-value was provided in this study.

Genetic Interactions between snf5Δ or snf6Δ and rrn3-ts, rpa49Δ or rpa135(D784G)

We have previously identified a mutation in the gene encoding the second largest subunit of Pol I that renders the enzyme impaired for transcription elongation (rpa135(D784G); [6]). Thus, mutations in genes that interact with rpa135(D784G) would be candidates to influence Pol I transcription elongation. To identify synthetic lethal partners of rpa135(D784G), we performed a synthetic genetic array (SGA) screen [29]. To increase the reliability of our screen, we constructed Mat a and Mat α strains carrying the rpa135(D784G) mutation (DAS483 and DAS484) and screened using both mutant libraries for synthetic lethal interactions. We identified and confirmed 17 synthetic lethal interactions. Of particular interest, we found deletion of SNF6 to be synthetic lethal with rpa135(D784G).

To confirm this synthetic lethal interaction, we deleted SNF6 in a different strain background [W303-1A (NOY 388)] and tested for interaction with the rpa135 mutation using tetrad dissection. Of 48 total double mutant segregants [snf6Δ rpa135(D784G)], none germinated even after 7 days of incubation at 30°C (Figure 1A), confirming SWI/SNF function is required when Pol I transcription elongation is impaired.

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Figure 1. The SWI/SNF complex interacts genetically with Pol I.

(A) Deletion mutants of SNF6 (DAS647) were mated with rpa135(D784G) (NOY2172) or rpa49Δ (DAS50). Diploids were selected, sporulated, and resulting tetrads were dissected. Images of haploid colonies were made after 2 days incubation at 30°C. Segregant genotype is labeled below each image. (B) Genetic interactions between indicated strains are listed. Non-lethal interactions were confirmed to have predicted growth rates, taking into account the defects in growth present in parental haploid strains. “N.D.” indicates genetic combinations that were not analyzed.

https://doi.org/10.1371/journal.pone.0056793.g001

To determine if this genetic interaction was specific to the rpa135(D784G) allele or a more general characteristic of impaired Pol I transcription elongation, we mated a snf6Δ strain to a strain carrying a deletion of RPA49. The A49 subunit of Pol I (also referred to as Rpa49p) is not essential for growth; however, A49 forms a heterodimer with the A34.5 subunit (also referred to as Rpa34p) that acts as an intrinsic transcription elongation factor [35]. Therefore, genetic interactions between snf6Δ and rpa49Δ would further support the model that SWI/SNF plays a role in the elongation step of Pol I transcription.

Tetrad analysis revealed that the combination of snf6Δ with rpa49Δ was also lethal. None of 55 haploid double mutants grew even after long term incubation (Figure 1A). Since the A49 subunit and the rpa135(D784G) allele influence transcription elongation by Pol I, these interactions could support a role for SNF6 in the elongation step of Pol I transcription. However, we also performed tetrad analysis of a cross between snf6Δ and rrn3-ts. Rrn3p is a well characterized transcription initiation factor for Pol I. We found that snf6Δ is lethal with rrn3-ts (Figure 1B). Thus, mutations that substantially impair transcription initiation or elongation by Pol I are synthetic lethal with snf6Δ. These data suggest a direct or indirect role for SWI/SNF in transcription by Pol I, but cannot implicate any single step in the transcription cycle. We note that not all genes whose products influence rRNA metabolism interacted with the snf6 deletion. Neither hmo1Δ nor top1Δ showed significant interactions with snf6Δ.

To further investigate whether the genetic interaction with Pol I is unique to SNF6 or a general property of the SWI/SNF complex, we included Snf5p [a core subunit of SWI/SNF] in our analysis. Similarly, we found that snf5Δ was lethal in combination with rrn3-ts, rpa135(D784G) or rpa49Δ (Figure 1B). These genetic interactions demonstrate that the SWI/SNF complex plays an important role in Pol I transcription, influencing transcription initiation and/or elongation.

Subunits of SWI/SNF Associate with rDNA

To investigate the direct relevance of SWI/SNF in Pol I transcription, chromatin immunoprecipitation (ChIP) assays followed by real-time PCR were designed to detect association of Snf2p, Snf5p and Snf6p with rDNA using 5–8 pairs of primers located throughout the rDNA repeats (Figure 2A and File S1). We tagged Snf2 and Snf5p with a 7his-3-hemagglutinin (HA) epitope on the C-terminus and tagged Snf6p with a 13-Myc epitope on the C-terminus in WT background (Table 1). Neither tag affected growth rate. Rpa135-(his)₇-(HA)₃ and Rrn7-(Myc)₁₃ were used as positive controls. The untagged parental strain was used as a negative control. Snf6-(Myc)₁₃ ChIP to GAL1 served as a positive control for interaction with a Pol II transcribed gene (Figure S1 in File S1).

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Figure 2. SWI/SNF associates with rDNA.

Yeast cells were cultured, crosslinked with formaldehyde, lysed, sonicated and immunoprecipitated with anti-Myc 9E10 or anti-HA 12CA5 antibody. (A) Positions of 8 pairs of primers used in real-time PCR analyses are indicated by horizontal bars under the schematic of an rDNA repeat. (B) Positive control [Rrn7-(MYC)₁₃ (DAS303)] and negative control [WT (NOY396)] ChIP results. Y-axis represents the amount of rDNA immunoprecipitated relative to total rDNA in input samples. (C) Snf6p (DAS649) ChIP signal compared to negative control. (D) A135-(his)₇-(HA)₃ (DAS477) associates with the promoter and the entire coding region of rDNA. (E) Snf2p (DAS750) associates with the coding region of the rDNA. Data were quantified from at least 2 10-fold dilutions per sample from duplicate cultures. Error = ±1standard deviation.

https://doi.org/10.1371/journal.pone.0056793.g002

RRN7 encodes a subunit of core factor, an essential transcription initiation factor for Pol I. As expected, Rrn7p specifically bound rDNA at the promoter (Figure 2B). Residual signal in the 5′ external transcribed spacer (ETS) region is often observed for transcription initiation factors, due to the proximity of this region to the promoter. Statistical analysis, using TukeyHSD tests, confirms that the binding signals at the promoter and 5′ ETS are significantly higher than in the untagged control (WT) (with p-value <0.00005). In contrast, Rpa135-(his)₇-(HA)₃ bound to the transcribed region of the rDNA with statistical significance (p-value <0.00005) but not to the non-transcribed spacer (NTS) regions (Figure 2D) [4]. As expected, Snf6p binds to Pol II transcribed GAL1 (Figure S1 in File S1). As seen in Figures 2C, Snf6p specifically bind rDNA in the 5′ ETS. Although the signal for Snf6p in the 5′ ETS is lower than for positive controls, it is at least 3-fold above the background signal (untagged WT). Statistical analysis confirms that the association of Snf6p with 5′ ETS is significantly higher than WT (TukeyHSD Test with p-value <0.00005). Unlike Snf6p, Snf2p detectably associates with the entire rDNA from the promoter through the rDNA coding regions (Figure 2E). We also tagged Snf5 and observed a small peak in the 5′ ETS, however, though qualitatively consistent with our Snf6 data (Figure 2C) this association was not statistically significant (Figure S2 in File S1). In summary, SWI/SNF associates with the rDNA and is poised to affect Pol I transcription.

Snf6p and Snf5p also associate with the NTS regions of the rDNA. Several transcription factors have been reported to bind the NTS regions including Spt4/Spt5 [7], Chd1p and Isw1p [36], and Paf1C [4]. These positive ChIP signals are thought to be due to the association of transcription factors with Pol II transcription units within the spacers. Although the functional consequence of SWI/SNF association with the NTS region is not yet clear, its association with the rRNA coding region is consistent with a model whereby SWI/SNF could directly influence Pol I transcription.

We note that the ChIP signal for Snf2p, Snf5p and Snf6p is substantially lower than is observed for proteins that associate with Pol II-transcribed genes. This observation is consistent with many previous observations from our lab and others. One potential explanation for this observation is that the dense organization or assembly of pre-ribosomes on the nascent rRNA may influence IP efficiency under standard ChIP conditions. Furthermore, since epitope availability is certainly different between subunits of the complex and can vary in response to nearby protein density, the difference in ChIP signal between Snf6 and Snf2 does not necessarily indicate any biologically relevant difference in the complex composition or occupancy. From all of these data (Figures 2 and S2) we simply conclude that SWI/SNF is in close proximity to the rDNA, rendering it capable of influencing Pol I.

Deletion of SNF6 Decreases Pol I Transcription Rate in vivo

To assess whether snf6Δ decreases Pol I transcription rate, we pulse labeled yeast cells with [methyl-³H]methionine [2]. Because rRNA in yeast is co-transcriptionally methylated and the pool of methionine within living cells is low, the [methyl-³H]methionine incorporation assay is a reliable method to quantify rRNA synthesis rates [37]. As shown in Figure 3A, the rRNA synthesis rate in the snf6Δ mutant was 40% of the WT rate.

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Figure 3. rRNA synthesis rate of snf6Δ is only 40% of WT despite similar Pol I occupancy.

(A) Yeast cells were cultured in SD-met media to an A₆₀₀ ∼ 0.3. Then [methy-³H]methionine was incorporated into cells for 5 min followed by a 5 min chase with excess (500 µg/ml) cold methionine. RNA was isolated, run on an agarose gel, transferred to nitrocellulose membrane, and detected by autoradiography. Pol I transcription in snf6Δ was normalized to WT. Data were analyzed from 2 independent [methy-³H]methionine incorporation assays. Only one dataset is shown here. (B) Pol I occupancy of rDNA in snf6Δ resembles that of WT by ChIP except for a 2-fold increase of Pol I occupancy in snf6Δ at the 5′ end of the 18S gene (asterisk). An anti-A190 polyclonal antibody was used to immunoprecipitate Pol I complexes. The 8 primer pairs described for Figure 2 were used in real-time PCR to check the association of Pol I complex with rDNA. A ninth primer pair measured Pol I association with 5S rDNA. Data were quantified from at least 2 10-fold dilutions per sample from duplicate cultures. Error = ±1standard deviation.

https://doi.org/10.1371/journal.pone.0056793.g003

In our experience, the [methyl-³H]methionine incorporation assay is generally not influenced by alteration in isotope uptake or fluctuations in the cellular methionine pool between strains. Isotopic labeling with uridine, for example, is prone to these indirect effects. However, it is formally possible that snf6Δ-dependent changes in methionine pools or activity of methyltransferases could be incorrectly interpreted as defects in rRNA synthesis. To control for this possibility, we calculated the predicted rRNA synthesis rate by considering the cellular growth rate and total RNA accumulation of WT and snf6Δ cells in liquid culture. This prediction assumes minimal or no rRNA degradation. The snf6Δ mutant grew only 50% as fast as WT. Furthermore, the total RNA level in snf6Δ was only about 75% of WT (data not shown). Based on these values, one would predict an rRNA synthesis rate of 37.5% of WT in snf6Δ cells [0.5×0.75 = 0.375; Figure 3A; see Supplementary data for additional explanation of this calculation]. This predicted rate is very close to the rate determined directly by [methyl-³H]methionine. Thus, our measurement of rRNA synthesis rate is reliable and accurate. These data support the model that SWI/SNF affects one or more rate limiting steps in Pol I transcription.

As an additional control we measured total RNA synthesis in WT and snf6Δ cells using ³H-uridine labeling. Uridine incorporation detected a large defect in transcription in the snf6Δ cells compared to WT (∼25-fold; Figure S3 in File S1). Such a severe defect in rRNA synthesis would not support growth, especially not at the rates observed (Figure 3A). Since fluctuations in the cellular uridine pool can dramatically affect this experimental approach, the magnitude of this effect is not reliable; however, these data provide additional qualitative support that mutation of SNF6 affects rRNA synthesis.

Reduced accumulation of rRNA might also result from an enhanced decay rate rather than a reduction in transcription rate. To test whether accelerated degradation of RNA in snf6Δ mutant cells contributed to the observed decrease in labeled 18S and 25S rRNA, we constructed a snf6Δ rrp6Δ double mutant and reexamined rRNA synthesis using [methyl-³H]methionine. Rrp6p is a subunit of the nuclear exosome and is required for efficient degradation of excess and aberrant rRNAs and mRNAs [38], [39], [40]. We have shown previously that mutation of RRP6 leads to accumulation of unstable rRNA precursors and degradation products [6]. We postulated that if increased exosome-dependent rRNA degradation rate was accounting for decreased rRNA, deletion of RRP6 would restore rRNA levels in the mutant cells. However, the amount of rRNA in snf6Δ rrp6Δ double mutant was not higher than in snf6Δ alone (Figure S4 in File S1), thus we concluded that rRNA degradation did not contribute significantly to the decrease in the rRNA synthesis observed in the snf6Δ strain. These data argue against involvement of exosome-dependent RNA degradation in influencing rRNA levels in snf6Δ cells. Rather, we conclude that SWI/SNF promotes Pol I transcription.

Deletion of SNF6 does not Affect Pol I Occupancy of rDNA

We postulated that if Snf6p primarily affects transcription initiation by Pol I, we would observe a dramatic decrease in Pol I occupancy of rDNA in snf6Δ mutant cells relative to WT. To assess the effect of Snf6p on Pol I transcription initiation, we measured Pol I occupancy of the rDNA using ChIP. Our data showed that Pol I occupancy of the rDNA repeat did not decrease in snf6Δ compared to WT (Figure 3B). Furthermore, there was a significant elevation of Pol I signal at one location, position 18S#1 in the snf6Δ mutant compared to WT (TukeyHSD test with p-value <0.0005) (Figure 3B, asterisk). This observation suggests that Pol I may transiently stall or arrest in the 5′ end of rDNA in snf6Δ mutant cells. Together, these data demonstrate that impaired transcription initiation alone cannot account for the defect in rRNA synthesis observed in the snf6Δ strain, since impaired initiation alone would result in lower Pol I occupancy of rDNA (see discussion below).

Deletion of SNF6 does not Affect rDNA Copy Number

Eukaryotic rDNA exits as tandem repeats that can rapidly and efficiently recombine to alter the number of repeats present in any given cell [41]. Thus, one must control for mutation-induced alteration in the rDNA copy number. We measured relative rDNA copy numbers in snf6Δ mutant cells compared to WT by contour-clamped homogenous field electrophoresis (CHEF) followed by Southern blot hybridization as described previously [4] (Figure S5 in File S1). We included control strains with known alterations of rDNA copy number. There was no reduction in the rDNA copy number in snf6Δ mutant compared to WT (Figure S5 in File S1). Thus, decreased Pol I transcription in the snf6Δ mutant is not due to alteration of the rDNA copy number.

Mutation of SNF6 impairs rRNA synthesis; however, Pol I occupancy of rDNA is unchanged, RNA degradation by the exosome is normal, and there is no alteration in the rDNA copy number. The simplest explanation for these data is that SWI/SNF affects transcription elongation by Pol I.

Mutation of SNF6 does not Alter the Number of Polymerases Engaged in Transcription

To further test our model that Snf6p acts in Pol I transcription elongation, we used electron microscopy of Miller chromatin spreads (EM analysis) to visualize Pol I transcription in vivo. EM analysis permits quantification of Pol I density per gene, percentage of actively transcribed genes, and Pol I positioning over the gene.

Representative rDNA repeats are shown in Figure 4 for WT and snf6Δ cells. We analyzed >60 genes per strain (>2500 polymerases per strain) to quantify Pol I occupancy of the rDNA. We concluded from quantification of the EM data (Figure 5A and 5B), that Pol I occupancy of the rDNA was not increased or decreased significantly in the snf6Δ strain compared to WT, consistent with our ChIP data (Figure 3B). Since impaired transcription elongation without any decrease in initiation rate would result in increased Pol I occupancy, these data indicate that both Pol I initiation and elongation rate are impaired, but that initiation rate was not inhibited more severely than elongation in the mutant cells.

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Figure 4. Miller chromatin spreads of WT and snf6Δ suggest similar Pol I occupancy of rRNA genes.

Direction of transcription is from left to right. rRNA transcripts per rDNA gene were quantified for these representative and many additional genes. For the genes shown, the number of rRNA transcripts was 51 for WT (DAS648), and was 44, 39, and 60 for the three snf6Δ (DAS647) genes (top to bottom).

https://doi.org/10.1371/journal.pone.0056793.g004

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Figure 5. A small accumulation of Pol I complexes in the 5′ end of rDNA in snf6Δ.

(A) Distribution frequency for the number of polymerases per gene was revealed by EM analysis of Miller chromatin spreads in snf6Δ (DAS647) and WT (DAS648). (B) More than 100 rDNA genes from Miller chromatin spreads were analyzed in WT and snf6Δ. Pol I density and percentage of actively transcribed genes in snf6Δ and WT are similar. (C) Polymerase occupancy as a function of position within the transcribed region of rDNA in snf6Δ and WT cells. A small peak of Pol I occupancy in the 5′ end of rDNA in snf6Δ is indicated by an asterisk. All mappable genes in the dataset were analyzed, corresponding to >60 genes per strain and >2500 polymerases per strain. Schematic below the X-axis represents the Pol I transcribed region of the rDNA. (D) Same as C but an additional two WT control strains (NOY388 and BY4741) are plotted. BY4741 data are from El Hage et al. 2010 [34].

https://doi.org/10.1371/journal.pone.0056793.g005

Not all rDNA repeats are actively transcribed at any one moment. Consequently, the percentage of actively transcribed rDNA repeats can affect overall rRNA synthesis rate. However, because EM data showed that ∼60% of rDNA repeats are transcribed in both snf6Δ and WT cells (Figure 5B), mutation of SWI/SNF does not influence the “on-off” status of rDNA repeats.

There is no method for direct measurement of transcription elongation by Pol I in vivo, however, we can estimate the relative elongation rates in different strains based on the data presented. We calculate the number of engaged Pol I complexes using the following equation: engaged Pol I complexes = (pols/gene) * (rDNA copy number) * (percentage of actively transcribed rDNA). We found each parameter in the equation to be approximately equal in WT versus snf6Δ, therefore we conclude that a similar number of Pol I complexes is engaged in transcription in WT and snf6Δ cells. Since the synthesis rate of rRNA in the snf6Δ strain is only 40% of the WT level (based on [methyl-³H]methionine incorporation data) we conclude that the Pol I initiation and elongation rate in snf6Δ must be ∼40% of WT.

Mutation of SNF6 Changes the Distribution of Pol I on Active Genes

ChIP data suggested that there may be a site in the 5′ end of rDNA where Pol I accumulates in snf6Δ mutant cells (Fig. 3B). To further test for such a pause or arrest site, we reexamined the EM data and mapped Pol I distribution along rDNA for the genes in our dataset (>2500 pols mapped per strain). We observed a reproducible increase in polymerase density ∼15–20% into the gene compared to the control strain (Figure 5C). Although this increase in polymerase density is relatively small, it is often noticeable by visual inspection of individual genes from snf6Δ cells (Fig. 4), and it is consistent with the Pol I peak observed by ChIP at position 18S#1 (Figure 3B). Both of these peaks are near the position of the most robust ChIP signal for Snf6p and Snf5p (Figures 2 and S2). To further test whether this peak in polymerase occupancy is significant, we mapped the Pol I distribution in two additional control strains of different genetic backgrounds. Both lacked a peak in Pol I density at this position. Kolmogorov-Smirnov tests indicated that the distribution of Pol I in the snf6Δ strain is statistically different from all the control strains (p-value <0.05) (Figure 5D). Statistical analysis identified a bimodal distribution of Po I in all four strains with one mode in the 15–20% range, and another should appear in the 68–75% range. However, the larger values of the standard deviations when computing the second mode prevent the exact location of that peak. Though it seems Pol I occupancy in ITS and 25S regions is reduced in snf6Δ relative to WT (Figure 5C and 5D), our statistical analysis does not support this. Pol I occupancy measured by ChIP also did not identify such reduction (Figure 3). However, identification of modes in the data confirms that the peak in the 5′ end of rDNA is significant. Taken together, the simplest explanation of these data is that SWI/SNF normally functions to promote transcription elongation by Pol I. In the absence of efficient SWI/SNF function, a transient kinetic barrier to elongation exists in the 5′ end of rDNA. The potential significance of this barrier is discussed below.

Deletion of SNF6 Modestly Affects rRNA Processing

Transcription elongation by Pol I is functionally coupled to efficient rRNA processing. Strains carrying mutations in RPA135, SPT4/5 or PAF1 had reduced rRNA synthesis rates and significant defects in rRNA processing [4], [6]. Since factors that influence transcription elongation also have the ability to affect rRNA processing, we investigated whether SWI/SNF can influence the efficiency of rRNA processing. No obvious differences were seen in co-transcriptional RNA processing events, such as SSU processome formation, as visualized in Miller chromatin spreads (examples in Figure 4). For more quantitative data, we analyzed pre-rRNA and mature rRNA transcripts by Northern blot (Figure S6 in File S1). We prepared total RNA from snf6Δ and WT cells. The ratio of pre-rRNA species compared to mature rRNA was quantified in WT and snf6Δ samples (Figure S6 in File S1).

From these studies, we conclude that disruption of SNF6 had no effect on the processing of the 18S rRNA. We did not detect any alteration of 23S or 20S pre-rRNA in snf6Δ mutant cells. However, there was a ∼60% increase of 27S pre-rRNA in the mutant compared to WT. We also observed a small increase in the abundance of the unprocessed 35S rRNA species in the mutant compared to WT (Figure S6A in File S1). Thus, there appear to be modest defects in rRNA processing in the absence of Snf6p. Since SWI/SNF affects Pol I transcription elongation, these data are consistent with the model that Pol I elongation and rRNA processing are functionally coupled [6].

We note that a large defect in pre-rRNA processing could lead to a decrease in rRNA accumulation in pulse-chase studies. However, we did not observe accumulation of pre-rRNA when the nuclear exosome was mutated (Figure S4 in File S1) or in pulse-chase isotopic labeling experiments (Figure S7 in File S1). Thus, pre-rRNA processing is not sufficiently defective in snf6Δ cells to account for the observed defects in rRNA synthesis.

Potential Mechanisms by which SWI/SNF may Influence Pol I Transcription

SWI/SNF is directly involved in the mechanisms of transcription initiation and elongation by Pol II [42], [43], [44], [45]. Here, we show that this chromatin remodeler may also directly affect transcription initiation and elongation by Pol I. ChIP analysis identified a peak in association of both Snf5 and Snf6 with the 5′ end of rDNA. EM and Pol I occupancy data revealed a small peak of Pol I accumulation in the 5′ end of the rDNA in the absence of Snf6p, but no enhanced occupancy in other positions. These data suggest that SWI/SNF normally functions to clear one or more pause sites in the 5′ end of the gene.

We do not know the nature of this putative pause. In principle, it could result from DNA sequence specific effects on transcribing polymerases or via a nucleoprotein mediated barrier. Pol I may transiently pause or slow down upon confronting this roadblock. Our data also suggest that there is a general decrease in the elongation rate of Pol I complexes throughout the gene in snf6Δ cells. The Proudfoot lab has shown previously that nucleosomes occupy the entire rDNA, but they lack the regular chromatin structure as in Pol II-transcribed genes [36]. Thus, one explanation for our results is that SWI/SNF participates in clearance of unphased nucleosomes throughout the rDNA repeat. A different study concluded that actively transcribed rDNA repeats lack nucleosomes, but associate with high-mobility group protein Hmo1 [46]. If the latter model is accurate, then SWI/SNF would have a histone-independent role at the rDNA. We have performed micrococcal nuclease mapping experiments in an attempt to identify alterations in the rDNA chromatin in the snf6Δ strain relative to WT. Although minor differences were detected, none of these differences was sufficiently clear to differentiate these potential models (data not shown). It is likely that the repeated nature of the rDNA and the maintenance of active and inactive rDNA repeats limit the utility of existing methods in detection of small changes in active rDNA chromatin. Further characterization of the roles that SWI/SNF and other factors play in rRNA synthesis should lead to a better understanding of rDNA chromatin structure.

It has been suggested that SWI/SNF plays a role in facilitating promoter escape by Pol II [promoter escape is a stage in transcription during which polymerases leave the promoter region and engage in productive transcription [47]]. During the first 30 min of induction of Gcn4 expression, Pol II occupancy is not decreased at the ARG1 promoter in snf2Δ mutant cells, but it is decreased at the 3′ end of the ORF, suggesting a role for SWI/SNF in promoter escape. We found no decrease in Pol I occupancy of the promoter or rDNA coding region in snf6Δ compared to WT, indicating that promoter escape by Pol I is not impaired by mutation of SWI/SNF.

In the snf6Δ strain, polymerase density per gene was very similar to WT. For the polymerase occupancy to remain unchanged in the context of a 2.5-fold decrease in synthesis rate, the initiation rate and the elongation rate must have been reduced by approximately the same magnitude in the two strains. The reduction in initiation could be a consequence of defects in transcription elongation (i.e. slower Pol I clearance from the 5′ end of the gene slows loading of the next polymerase). Alternatively, mutation of SNF6 may have independent effects on transcription elongation and initiation (perhaps by altering expression of one or more Pol I transcription initiation factors). The latter model requires alteration of multiple steps in transcription by the same magnitude.

We have previously observed Pol I accumulation in unique regions of the rDNA when elongation is impaired [4]. EM analysis revealed frequent large gaps between transcribing Pol I complexes in a strain carrying a deletion of the gene that encodes the Ctr9p subunit of Paf1C (Polymerase-associated factor complex 1) [4]. Often, upstream of the gap we observed increased polymerase density. The data presented here are distinct from previous observations in the ctr9Δ strain. First, the positions of most frequent pausing observed in the ctr9Δ cells do not correlate with the position of enhanced Pol I accumulation observed in snf6Δ cells ([4] and Figure 5). Second, we did not observe a dramatic increase in gaps between transcribing polymerases in the snf6Δ strain. Thus, mutation of genes that encode subunits of Paf1C or the SWI/SNF complex affects Pol I distribution on the rDNA in different ways. Furthermore, these mutations do not interact genetically with one another (data not shown). These observations suggest that there are multiple, discrete mechanisms by which Pol I can be influenced during transcription elongation.

A recent real-time analysis of transcription kinetics at the single cell level reported that transcription elongation is the deterministic step for Pol II transcription whereas initiation is stochastic [48]. Many data demonstrate that transcription initiation by Pol I is regulated; however, several recent studies have implicated transcription elongation factors in the control of Pol I activity [49], [50]. The relative contributions of factors that control transcription initiation, elongation and rRNA decay to the overall regulation of ribosome synthesis remain unclear.

Although our data indicate that SWI/SNF is positioned to directly affect Pol I transcription, we cannot exclude indirect models in which depletion of Snf6p impedes Pol II-dependent expression of genes, which in turn impair Pol I transcription.

Efficient rRNA Processing Requires Efficient Pol I Transcription Elongation

Coupling of Pol I transcription elongation to rRNA processing has been reported multiple times [3], [4], [6], [7]. The data presented here suggest that mutation of SNF6 results in a decrease in the amount of rRNA produced per transcribing RNA polymerase I per unit time. Thus, Pol I transcription elongation rate is apparently reduced. Consistent with the model that elongation is coupled to rRNA processing, we observed modest defects on rRNA processing in the snf6Δ strain. Thus, these data support the functional linkage between rRNA transcription and rRNA processing.

Role of SWI/SNF in rDNA Silencing

Previous studies have shown that Pol II-dependent transcription of reporter genes is epigenetically silenced when the reporters are located in the telomeres, the mating-type locus and the ribosomal DNA [51], [52], [53], [54]. SWI/SNF was shown previously to influence silencing of RNA polymerase II-dependent transcription of reporter genes located in the rDNA [55]. The SUC2 gene normally depends on SWI/SNF for its transcription; however, when it is artificially located in the 25S rDNA coding region, its transcription is instead repressed by SWI/SNF. We suggest that this repression by SWI/SNF is indirect, due to activation of Pol I transcription (leading to occlusion of the Pol II reporter gene). Two additional lines of evidence support this interpretation.

First, the mechanism of SWI/SNF- mediated repression of the reporter is independent of the NAD-dependent histone deacetylase Sir2p or the histone methyltransferase Set1p (36). The snf2Δ mutation (Snf2p is the ATPase of SWI/SNF complex) causes several fold higher induction of SUC2 mRNA than sir2Δ and set1Δ in this construct. Double mutants of snf2Δsir2Δ and snf2Δset1Δ induce similar levels of SUC2 mRNA as in snf2Δ single mutant. Moreover, loss of SNF2 does not affect the levels of histone H3 K4 methylation or the association of Sir2p and Set1p with the rDNA repeat. Thus, SWI/SNF regulates rDNA silencing independently of Sir2p and Set1p.

We did not observe any alteration in the ratio of active versus inactive rDNA repeats in the snf6Δ strain compared to WT. The Nomura lab showed previously that rDNA chromatin structure favored by Pol I represses Pol II reporter gene inserted in the rDNA array. They observed that impairment of Pol I transcription leads to induction of Pol II reporter genes located in the rDNA array [56]. All of these data suggest that WT SWI/SNF functions to enhance Pol I transcription elongation, which in turn leads to inhibition of Pol II reporters located in the rDNA coding region.

In summary, our studies demonstrate that SWI/SNF plays important roles in Pol I transcription. Future studies are needed to further define the mechanisms by which SWI/SNF affects Pol I and Pol II functions differentially.