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Vol. 17, Issue 9, 3848-3859, September 2006

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Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843

Submitted March 16, 2006; Revised June 19, 2006; Accepted June 20, 2006
Monitoring Editor: Wendy Bickmore

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 sir2 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Modified histones at silent genomic domains contribute to a chromatin environment that is refractory to gene expression and genetic recombination (reviewed in Strahl and Allis, 2000; Turner, 2000; Jenuwein and Allis, 2001). In Saccharomyces cerevisiae, chromatin at the homothallic mating-type loci HML and HMR, telomeres, and the ribosomal DNA locus (rDNA) silences genetic recombination and expression of native and ectopic genes transcribed by RNA polymerase (Pol) II. Silencing at the HM loci and telomeres has been studied extensively, whereas the mechanisms of Pol II silencing at the rDNA are not well characterized (reviewed in Moazed, 2001; Rusche et al., 2003). Increasing our understanding of the factors and mechanisms that regulate silencing at the rDNA will provide insight into the pathways that regulate gene expression and genome stability.

In S. cerevisiae, the rDNA contains 150–200 tandem copies of a 9.1-kilobase (kb) repeat, with each repeat containing a Pol I-transcribed 35S rRNA gene and a nontranscribed spacer (NTS) that is subdivided into NTS1 and NTS2 by the Pol III-transcribed 5S rRNA gene (reviewed in Warner, 1999; Figure 1). Despite high levels of transcription by Pol I and Pol III in the rDNA locus, Pol II-transcribed genes integrated into the rDNA are silenced (referred to as rDNA silencing) (Bryk et al., 1997; Fritze et al., 1997; Smith and Boeke, 1997). Additionally, silent chromatin at the rDNA is essential for repression of genetic recombination (Gottlieb and Esposito, 1989; Davis et al., 2000; Kobayashi et al., 2004) and extension of replicative life span (reviewed in Guarente, 2000).

Chromatin-associated proteins and modified histones regulate silencing of Pol II transcription at the rDNA (Bryk et al., 1997; Fritze et al., 1997; Smith and Boeke, 1997; Smith et al., 1999; Straight et al., 1999; Hoppe et al., 2002; Park et al., 2002; Dror and Winston, 2004; Kobayashi et al., 2004; Kuzuhara and Horikoshi, 2004; Machin et al., 2004; Ye et al., 2005). Net1 and the NAD-dependent histone deacetylase Sir2 are members of the RENT complex, which functions in silencing at the rDNA (Shou et al., 1999; Straight et al., 1999). The RENT complex is the functional equivalent of the Sir2–3-4 complex necessary for silencing at the HM loci and telomeres. In addition to being required for the association of Sir2 with the rDNA, Net1 interacts with Pol I and regulates the structure of the nucleolus (Straight et al., 1999; Shou et al., 2001). The histones H3 and H4 are substrates for the deacetylase activity of Sir2. At the HM loci and telomeres, hypoacetylated H3 and H4 promote the interaction of Sir3 and Sir4 with nucleosomes, thereby facilitating the formation and spread of silent chromatin (Braunstein et al., 1993, 1996; Hecht et al., 1995; Wu and Grunstein, 2000; Carmen et al., 2002; Liou et al., 2005). Hypoacetylated histones are present at the rDNA (Bryk et al., 2002; Buck et al., 2002; Sandmeier et al., 2002; Huang and Moazed, 2003), although how they contribute to rDNA silencing remains unclear.

Cells lacking K4-methylated histone H3 exhibit defects in transcriptional silencing at the rDNA and telomeres (Nislow et al., 1997; Briggs et al., 2001; Bryk et al., 2002; Nagy et al., 2002; Mueller et al., 2006). Set1 is the catalytic subunit of the COMPASS complex that is required for mono-, di-, and trimethylation of histone H3 on K4. In set1 cells, the lack of K4-methylated histone H3 is associated with the aberrant spreading of Sir proteins from telomeres into adjacent sequences. Reduced concentration of Sir complexes at telomeres in cells lacking Set1 is hypothesized to cause the loss-of-telomeric–silencing defect in set1 cells (Ng et al., 2002, 2003a; Meneghini et al., 2003; Martin et al., 2004; Katan-Khaykovich and Struhl, 2005). In contrast, at the rDNA, the levels of the silencing factors Sir2 and Net1 are equivalent in set1 and wild-type cells (Bryk et al., 2002). Moreover, in set1 cells, the levels of acetylated histone H3 and H4 remain low, indicating that Sir2 maintains its deacetylase function at the rDNA in the absence of K4-methylated H3. Thus, the mechanism by which silencing is lost at the rDNA in set1 cells is not equivalent to that at telomeres and has yet to be discovered.

In wild-type cells, silent chromatin at the rDNA contains low levels of acetylated H3 and H4 and low levels of K4-methylated histone H3. In cells lacking Set1 where rDNA silencing is impaired, histones at the rDNA remain hypoacetylated but are unmethylated. In contrast, in cells lacking Sir2, the levels of acetylated histone H3 and H4 are increased at several positions in the rDNA repeat and the level of K4-dimethylated H3 is increased at the NTS (Bryk et al., 2002). Together, these observations indicate that rDNA silencing is controlled by a specific combination of hypoacetylated and hypomethylated histones and that perturbations that alter the profile of modified histones disrupt rDNA silencing. To gain insight into the relationship between Sir2 and K4-methylated histone H3, we used chromatin immunoprecipitation (ChIP) and real-time PCR analysis to generate high-density profiles of K4-methylated histone H3 and Pol II across the rDNA repeat in wild-type and sir2 cells. Here, we show that novel changes occur at the rDNA in sir2 cells that reveal the central role of deacetylase activity of Sir2 in preventing aberrant Pol II transcription and protecting the structure and function of the unique chromatin domain present at the rDNA in S. cerevisiae. Furthermore, our findings provide mechanistic insight into the recent observation that high levels of transcription in the rDNA NTS are associated with amplification of rDNA repeats (Kobayashi and Ganley, 2005).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Yeast Strains, Plasmids, and Media
Standard media recipes were used (Rose et al., 1990). YPADT is YPD medium supplemented with adenine sulfate (40 mg/l) and L-tryptophan (20 mg/l). Yeast strains MBY1198 (wild type), MBY1217 (set1), and MBY1238 (sir2) have been described previously (Briggs et al., 2001; Bryk et al., 2002). The XhoI–NotI fragment containing SIR2 or sir2H364Y from plasmids pAR455 (SIR2 HIS3 CEN) or pAR456 (sir2H364Y HIS3 CEN) (Rudner et al., 2005) were subcloned into pRS415 (LEU2 CEN; Stratagene) by using standard techniques. The resulting plasmids, MBB406 (pSIR2 LEU2 CEN) and MBB407 (psir2H364Y LEU2 CEN), were verified by DNA sequencing. pSIR2 and psir2H364Y were transformed into MBY1238 (Bryk et al., 2002). pSIR2 was also transformed into MBY1249 (MAT his3200 ade2::hisG leu20 trp163 ura30 met150 Ty1his3AI-236 Ty1ade2AI-515 set11:: TRP1 sir2::KANMX4). The integrity of the pSIR2 and psir2H364Y plasmids was verified in silencing assays and by measuring the level of K9, K14-acetylated histone H3 at the rDNA by ChIP (our unpublished data). ChIP experiments (Supplemental Figure S3) using an anti-Sir2 antibody showed that the level of Sir2 protein at the rDNA in sir2 cells containing psir2H364Y was 0.5- to 1.6-fold of wild type, consistent with previously published reports (Tanny et al., 1999; Hoppe et al., 2002). Constructs for NTS-specific RNA probes were made by cloning PCR-generated NTS1 or NTS2 fragments into pSP70 (Promega, Madison, WI) to make MBB419 (pSP70-NTS1) or MBB413 (pSP70-NTS2). In vitro transcription reactions with SP6 or T7 RNA polymerase and linearized plasmid in the presence of [-³²P]CTP were performed to generate strand-specific RNA probes. Yeast strains with wild-type (wt) or temperature-sensitive (ts) Pol II used in temperature-shift experiments were made by genetic crosses: MBY1987 [MAT his3200 leu20 ura30 lys2-128 rpb1187::HIS3 (pRP114 RPB1 LEU2 CEN)]; MBY1988 [his3200 leu20 ura3-52 lys2-128 rpb1187::HIS3 sir2::KANMX4) (pRP114 RPB1 LEU2 CEN)]; MBY1989 [MAT his3200 leu20 ura30 rpb1187::HIS3 (pRP1-1 rpb1-1 URA3 CEN)]; and MBY1992 [his3200 leu20 lys2-128 met150 trp163 ura3-52 sir2::KANMX4 rpb1187::HIS3 (pRP1-1 rpb1-1 URA3 CEN)].

Oligonucleotides for ChIP
For analysis of histone H3 at HMR and TEL-VIR by real-time PCR, oligonucleotides were designed to amplify 250- to 300-base pair products (sequences available upon request). The primers used to analyze the rDNA (Huang and Moazed, 2003), RPS16A, and the intergenic region on chr VIII (Ng et al., 2003c) have been described previously. For analysis of the promoter and 5' end of the rDNA-Ty1 element by quantitative radioactive PCR, the primers annealed to the Ty1A region and downstream of the 5S rRNA gene (Bryk et al., 2002).

Chromatin Immunoprecipitation
Cells were grown and lysates prepared as described previously (Strahl-Bolsinger et al., 1997; Bryk et al., 2002) with the following modifications. Strains containing pSIR2 or psir2H364Y were grown in synthetic complete media lacking leucine with 2% glucose. Chromatin solutions were sonicated 12 times for 20 s each at 4°C by using a Branson Sonifier 250 at power setting 1.5 and 100% duty cycle to shear the chromatin to an average length of <500 base pairs. For each ChIP, 200 µl of sheared chromatin was incubated in a volume of 500 µl with specific antibodies for 12 h at 4°C. The antisera used were anti-histone H3, 5 µl (ab1791; Abcam, Cambridge, MA); anti-K4monoMeH3, 6 µl (ab8895; Abcam); anti-K4diMeH3, 15 µl (07-030; Upstate Biotechnology, Lake Placid, NY); anti-K4triMeH3, 5 µl (ab8580; Abcam); anti-Sir2, 1 µl (gift from Danesh Moazed, Department of Cell Biology, Harvard Medical School, Boston, MA); and anti-RNA Pol II carboxy-terminal domain (CTD) (4H8), 10 µl (ab5408; Abcam). The specificity of the antisera for K4-methylated H3 was verified by peptide blots by using unmodified and K4-methylated H3 peptides (our unpublished data).

Analysis of ChIPs
Quantitative radioactive PCRs were performed as described previously (Bryk et al., 2002) to determine the level of K4-di- and -trimethylated H3 at the rDNA-Ty1 element in wild-type (MBY1198), sir2 (MBY1238), and set1 (MBY1217) cells. ChIP experiments shown in Figures 1, 2, 4, and 5 and Supplemental Figures S1–S3 were analyzed by quantitative real-time PCR (qPCR). Duplicate reactions containing 1 µl of input DNA (1:100) or immunoprecipitated DNA (1:10) were amplified in 20 µl containing 1.25 µM of each oligonucleotide and 1x SYBR Green Dynamo Hot Start PCR mix (Finnzymes, Espoo, Finland) by using an iCycler Iq real-time PCR machine (Bio-Rad, Hercules, CA). The PCR parameters were 1 cycle of 95°C, 15 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s; 1 cycle at 95°C, 1 min; 1 cycle at 55°C, 1 min; and 80 cycles starting from 55°C with an increasing gradient of 0.5°C for 10 s each cycle to acquire a melting curve for each primer pair. The threshold cycle value was detected at the 72°C elongation step. Percent age of immunoprecipitation (%IP) was calculated by dividing the signal from IP DNA by the signal from input DNA. The %IP values from wild-type and sir2 cells analyzed by ChIP for K4-monomethylated H3, K4-dimethylated H3, and K4-trimethylated H3 were corrected for background by subtracting the %IP value obtained for set1 cells. For the 33 pairs of rDNA oligonucleotides, the range of average %IP values obtained from set1 cells were 0.031-0.128 for K4-monomethylated H3, 0.086-0.384 for K4-dimethylated H3, and 0.012-0.207 for K4-trimethylated H3. Graphs showing %IP data obtained for K4-methylated forms of histone H3 at the rDNA and two control loci before normalization to total histone H3 levels are shown in Supplemental Figure S2. The %IP values of K4-di- and -trimethylated H3 from cells containing pSIR2 or psir2H364Y were corrected for background by subtracting the %IP values obtained from set1 sir2 cells (MBY1249) containing pSIR2. The %IP values from wild-type and sir2 cells analyzed by ChIP for RNA Pol II and total histone H3 were corrected for background by subtracting the %IP value from a no-antibody control.

Northern Analysis
Total RNA was isolated from yeast cells as described previously (Bryk et al., 1997). Northern analyses were performed as described previously (Swanson et al., 1991). Strand-specific ³²P-labeled RNA probes were used to detect NTS transcripts and PYK1 RNA. An ACT1 (+564 to +1200) probe and 35S rRNA probe were made by PCR amplification of yeast genomic DNA and then purified from an agarose gel and labeled with [-³²P]dATP by random priming (Ausubel et al., 1988). Growth conditions for the Pol II shut-off experiments (Figure 7) were as described previously (Herrick et al., 1990). Briefly, for each strain (MBY1987, MBY1988, MBY1989, and MBY1992), a 50-ml culture in YPADT was grown at 24°C to 1–2 x 10⁷ cells/ml before being split into two 25-ml cultures. Twenty-five milliliters of fresh YPADT at 24°C was added to one culture, and growth was continued at 24°C for 30 min. To the other culture, 25 ml of YPADT at 48°C was added, and the culture was incubated at 36°C for 30 min. For time-course experiments, the volumes of the initial culture and fresh YPADT added were increased so that RNA was isolated from 50 ml of culture at each time point (0, 15, 30, and 60 min). Northern blots were quantified on a GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) Storm 860 PhosphorImager by using ImageQuant software.

Primer Extension
Total RNA (30–50 µg) from MBY1198, MBY1217, and MBY1238 was annealed to 1 pmol of ³²P-labeled oligonucleotide (OM454; 5'-GTTGGTTTTGGTTTCGGTTG-3') at 58°C for 20 min. Extension reactions were performed using the Primer Extension System-AMV Reverse Transcriptase kit (Promega) according to the manufacturer’s protocol. Sequencing reactions with oligonucleotide OM454 and double-stranded DNA template were performed using Sequenase Version 2.0 (USB, Cleveland, OH). Primer extension products and sequencing ladders were separated on 8 M urea, 8% polyacrylamide gels in 1x Tris borate-EDTA. Dried gels were visualized using a GE Healthcare Storm 860 PhosphorImager with ImageQuant software and autoradiography.

Analysis of Chromatin Structure with Micrococcal Nuclease
Yeast cells MBY1198, MBY1217, and MBY1238 were grown in 100 ml of YPADT medium to 1.2 x 10⁷/ml and harvested by centrifugation. Preparation of spheroplasts and digestion with micrococcal nuclease (MNase; Worthington, Biochemicals, Freehold, NJ) was performed as described previously with slight modifications (Kent and Mellor, 1995). Spheroplasts (1.7 x 10⁸) were incubated with MNase (0, 2.15, 4.3, 6.45, or 8.6 U/ml) for 4 min at 37°C and then the DNA was purified. Purified DNA that had not been treated with MNase was digested with 0.86 U of MNase for 2 min at 37°C to control for MNase sequence preferences. All DNAs were digested to completion with EcoRI or PvuII, separated on 1.5% agarose gels, transferred to nylon membranes, and analyzed by indirect end labeling. Blots of DNA digested with EcoRI were hybridized with a ³²P-labeled probe that anneals to +2229 to +2496 of the rDNA locus (beginning of NTS1 is +1), and blots with DNA digested with PvuII were hybridized with a ³²P-labeled probe that anneals to +1015 to +1268 of the rDNA. Positions of MNase cleavages and nucleosomes were calculated using restriction fragments as a reference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sir2 Excludes K4-Methylated Histone H3 from Silent Chromatin
Cells lacking Sir2 have high levels of acetylated histones at the HM loci and telomeres (Braunstein et al., 1993, 1996) and the rDNA (Bryk et al., 2002; Buck et al., 2002; Sandmeier et al., 2002; Huang and Moazed, 2003). We observed that K4-dimethylated histone H3 was also increased at the rDNA NTS in sir2 cells (Bryk et al., 2002), suggesting that Sir2 regulates the levels of K4-methylated histone H3 at the rDNA. Specific antisera against three forms of K4-methylated H3, K4-mono-, di-, and -trimethylated histone H3 have been used in numerous studies investigating the association of K4-methylated H3 with genes that are actively transcribed by RNA Pol II. These studies, in S. cerevisiae, have shown that K4-dimethylated H3 is enriched at euchromatin and that K4-trimethylated H3 is present at high levels at genes transcribed by Pol II (Bernstein et al., 2002; Santos-Rosa et al., 2002, 2004; Krogan et al., 2003; Ng et al., 2003b, c). Therefore, high levels of K4-methylated H3 at the rDNA NTS were unexpected due to the lack of Pol II-transcribed genes at the rDNA.

We examined the effect of Sir2 on the association of each of the three forms of K4-methylated H3 across the rDNA repeat. ChIPs using antisera against histone H3 as well as K4-mono-, di-, or -trimethylated histone H3 were analyzed by qPCR by using 33 primer pairs that amplify 260- to 280-base pair regions at intervals of 300 base pairs across the 9.1-kb rDNA repeat and two sets of primers that amplify control loci. Samples from cells lacking Set1 that are devoid of K4-mono-, di-, and -trimethylated histone H3 were included to provide a measurement of background that was subtracted from the signal obtained from wild-type and sir2 cells (see Materials and Methods). To determine whether Sir2 affected the level of total histone H3 at the rDNA, ChIP experiments were performed with antisera that recognize the C-terminal tail of histone H3. The interaction of the anti-histone H3 antisera with histone H3 is independent of modifications present at the N terminus of H3. The results of these experiments revealed that the level of total histone H3 was significantly lower at several positions in the rDNA repeat in sir2 cells compared with wild-type cells (Supplemental Figure S1). To control for differences in the amount of total histone H3 at the rDNA, in Figure 1, we have normalized data examining the levels of K4-methylated histones at the rDNA to the level of total histone H3 in wild-type and sir2 cells.

View larger version (34K): [in this window] [in a new window]   Figure 1. Altered distribution of K4-methylated histone H3 at the rDNA in sir2 cells relative to wild-type cells. Graphical representations of the average ratio of %IP of K4-monomethylated (top; ±SE; n = 2 or 3), K4-dimethylated (middle; ±SE; n = 3), and K4-trimethylated (bottom; ±SE; n = 4) histone H3 in sir2 cells (MBY1238) relative to wild-type cells (MBY1198) at the rDNA, the RPS16A gene, and an intergenic region on chr VIII. Data presented were normalized to total histone H3 to correct for reduced levels of H3 in sir2 cells at some regions of the rDNA repeat (see text and Supplemental Figure S1). The structure of a 9.1-kb rDNA repeat unit is shown below the bottom panel, indicating the location of the Pol I-transcribed 35S rRNA (35S) gene and the NTS that is divided into NTS1 and NTS2 by the Pol III-transcribed 5S rRNA gene (5S). ARS, replication origin; bent line with arrow, transcription start site of the 35S rRNA gene; , location of a silenced Ty1his3AI element present in one repeat, referred to as rDNA-Ty1 in text. Horizontal lines above the rDNA indicate PCR products generated during the analysis of ChIP experiments by qPCR.

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To understand how Sir2 affected the distribution of K4-methylated histone H3 at other silent loci, we performed ChIP experiments using wild-type, sir2, and set1 cells to obtain profiles of K4-methylated H3 at the HMR locus and near the telomere on the right arm of chr VI (TEL-VIR). Experiments measuring histone H3 at HMR and TEL-VIR revealed that the average ratio of histone H3 in sir2 to wild-type cells was between 0.9 and 1.2, indicating that the level of histone H3 was similar in wild-type and sir2 cells (our unpublished data). In contrast to the rDNA where we observed distinct profiles of K4-methylated H3, the levels of K4-di- and -trimethylated H3 were increased similarly across 4 kb of the HMR locus and up to 2.8 kb from the right end of chr VI (Figure 2, A and B). At HMR, the highest levels of K4 di- and -trimethylated histone H3 were present at a region containing the Pol II-transcribed a1 and a2 genes (Figure 2A, primer pair X/Ya). The region of HMR where we detect changes in the association of K4-methylated histone H3 falls within the boundaries of the silent domain that were mapped in previous studies (Donze et al., 1999; Donze and Kamakaka, 2001). At TEL-VIR, the highest levels of K4 di- and -trimethylated histone H3 were present 1.0–1.5 kb from the end of the chromosome. This region of chr VI contains the promoter and 5' end of the Pol II-transcribed gene YFR057W, whose expression has been shown to be increased in cells lacking Sir2 or Ubp10 (Wyrick et al., 1999; Emre et al., 2005). In contrast, we did not observe an effect of Sir2 on the levels of K4-methylated H3 at YFR055W a Pol II-transcribed gene located 4.7 kb from the end of chr VI. In summary, in cells lacking Sir2, the rDNA, HMR, and TEL-VIR are exposed to factors that are required for the methylation of histone H3 on K4.

View larger version (27K): [in this window] [in a new window]   Figure 2. The levels of K4 di- and trimethylated histone H3 were increased at HMR and TEL-VIR in sir2 cells. (A) Diagram of HMR located on chr III with the positions of silencers (E and I), cis-sequences (X and Z1), Ty long terminal repeats (LTR), and the genes HMRa1 and HMRa2. Short lines labeled B, C, D, X/Ya, E, Z, F, and H indicate the location of the PCR products used in ChIP analysis. The graph shows the average ratio of %IP of K4-dimethylated H3 (black bars) and K4-trimethylated H3 (hatched bars) at HMR, the RPS16A gene, and an intergenic region on chr VIII in sir2 cells relative to wild-type cells. (B) Diagram of TEL-VIR (right telomere on chr VI) with the positions of ORFs (YFR057W and YFR055W) and telomeric repeats indicated. Short lines labeled 0.35, 0.6, 1.0, 1.5, 2.8, and 4.7 indicate regions analyzed by qPCR. The numbers indicate the distance (in kb) from the right end of the PCR product to the right end of chr VI. Graph shows the average ratio of %IP of K4-di- and -trimethylated H3 at TEL-VIR in sir2 cells relative to wild-type cells. For both panels, the average ratio of % IP ± SE from sir2 cells to wild-type cells from three independent experiments is plotted.

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View larger version (28K): [in this window] [in a new window]   Figure 3. sir2 cells have higher levels of K4-trimethylated H3 at the rDNA-Ty1 element. ChIP experiments were analyzed by quantitative radioactive PCR to measure K4-di- and -trimethylated H3 at the promoter region of the silent Ty1his3AI element in the rDNA in wild-type and sir2 cells. Immunoprecipitations using set1 cells were performed to provide a measure of background for the ChIP experiments. The average %IP ± SE of K4-dimethylated H3 at the rDNA Ty1 promoter region from three independent experiments in wild-type cells was 5.0 ± 1.1, in sir2 cells is 7.4 ± 1.4, and in set1 cells is 0.08 ± 0.02. The average %IP ± SE of K4-trimethylated H3 at the rDNA-Ty1 promoter region from four independent experiments in wild-type cells was 3.4 ± 0.5, in sir2 cells is 6.1 ± 1.2, and in set1 cells is 0.03 ± 0.02. Slanted triangles indicate a twofold increase in the amount of template DNA used in the PCR reactions.

To distinguish between these two modes of regulation by Sir2, we used a catalytically inactive allele of SIR2, sir2H364Y, which encodes a mutant Sir2 protein that retains the ability to bind to the rDNA but lacks histone deacetylase activity (Tanny et al., 1999; Hoppe et al., 2002). ChIP experiments were performed using antisera against K4-di- and -trimethylated H3 and cross-linked chromatin from sir2 cells containing a plasmid with either a wild-type copy of SIR2 (pSIR2) or the mutant allele of SIR2 (psir2H364Y). Signal obtained from set1 sir2 double mutant cells containing the pSIR2 plasmid was subtracted as background in the analysis of these ChIP experiments (see Materials and Methods). The data revealed that the levels of K4-di- and -trimethylated H3 were higher at the rDNA in cells expressing sir2H364Y compared with cells with pSIR2 (Figure 4). Our results indicate that the deacetylase activity of Sir2 is required to maintain low levels of K4-methylated H3 at the rDNA. In addition, whereas the mutant Sir2 protein did not affect the levels of methylated H3 at RPS16A or the intergenic region, the levels of K4-di- and -trimethylated histone H3 were increased significantly at a region 0.6 kb from the right end of chr VI (Figure 4, right). Together, our results indicate that the deacetylase activity of Sir2 is required to maintain low levels of K4-di- and -trimethylated H3 at the rDNA and TEL-VIR.

View larger version (36K): [in this window] [in a new window]   Figure 4. Deacetylase activity of Sir2 is required to exclude K4-di- and -trimethylated H3 from the rDNA. Graph showing the average ratio of %IP of K4-dimethylated H3 (H3 K4 diMe, black bars) and K4-trimethylated H3 (H3 K4 triMe, hatched bars) across rDNA repeat (left) and control loci (right) in psir2H364Y/pSIR2 cells. The labels for the representation of rDNA repeat are defined in legend to Figure 1. Average ratio of %IP ± SE for three independent experiments is shown.

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View larger version (22K): [in this window] [in a new window]   Figure 5. Association of Pol II with silent loci is increased in sir2 cells. Graphs show the average ratio of %IP of Pol II at the (A) rDNA (left), the promoter of the rDNA-Ty1 element and control loci (right); (B) HMR; and (C) TEL-VIR in sir2 cells relative to wild-type cells. The representation of the rDNA in A indicates the portion of the rDNA repeat analyzed. Labels for the representations of the rDNA, HMR, and TEL-VIR are in the legends to Figures 1 and 2. The average ratio (±range) of Pol II measured in sir2:wild-type cells from two independent experiments is plotted.

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View larger version (65K): [in this window] [in a new window]   Figure 6. Endogenous transcription units in the rDNA NTS are expressed in rDNA silencing-defective mutants. (A) Schematic of rDNA NTS showing the polarity of transcripts detected by probes used in Figure 6B. Other labels, as in Figure 1. (B) Representative Northern blots of total RNA from wild-type, sir2, and set1 cells hybridized to strand-specific probes that are complementary to transcripts with the same polarity as the NTS1 top (lanes 1–3), NTS1 bottom (lanes 4–6), NTS2 top (lanes 7–9), and NTS2 bottom (lanes 10–12) strands. For each blot, PYK1 RNA levels were used to normalize for the amount of RNA analyzed. Numbers on left, transcript length in kb. Asterisks or line with asterisks, position of NTS-specific transcripts. (C) Primer extension analysis to map the 5' ends of the NTS2 bottom-strand transcripts. Schematic at top represents an NTS2 transcript and the oligonucleotide (OM454, line with arrowhead) that was used to map the 5' ends (not to scale). DNA sequence ladder and primer extension reactions revealed one major 5' end within the consensus TSS (underlined in DNA sequence representation of the bottom strand of NTS2 at base of C) and three minor start sites upstream of the TSS. Asterisks and/or bent arrows indicate 5' ends.

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To determine whether the NTS2 transcripts were made by Pol II, we used wild-type and sir2 strains lacking the genomic RPB1 gene that encodes the largest subunit of Pol II with either the wt RPB1 gene or a ts mutant allele, rpb1-1 provided from a plasmid (see Materials and Methods). Strains with rpb1-1 make functional Pol II at 24°C; however, upon shift of these cells to 36°C, Pol II becomes nonfunctional. RNA from cultures grown continuously at 24°C and from cultures shifted from 24 to 36°C for 30 min was hybridized with probes specific for the NTS2 transcripts, Pol II-transcribed ACT1 RNA, and Pol I-transcribed rRNA (Figure 7A). In wild-type and sir2 cells with RPB1 (wt RNA Pol II), ACT1 and rRNA transcript levels were maintained in cells shifted to 36°C (Figure 7A, lanes 1–4, middle and bottom), as were the NTS2 transcripts in total RNA from sir2 cells (Figure 7A, lanes 3 and 4, top). However, in cells with rpb1-1 (ts RNA Pol II, Figure 7A, lanes 5–8), ACT1 RNA was present in cultures grown at 24°C but disappeared after 30 min at 36°C (compares lanes 5 and 6 or 7 and 8). Likewise, the NTS2 transcripts present in the sir2 cells were gone after 30 min at 36°C (compare lanes 7 and 8). The level of transcript from the Pol I-transcribed 35S rRNA gene was stable at 36°C. These data suggest that the NTS2 transcripts were made by Pol II and not by Pol I. In addition, it is unlikely that the NTS2 transcripts were made by Pol III, as numerous polyT_>5 stretches, which act as terminators for Pol III (Allison and Hall, 1985; Braglia et al., 2005), are present in the rDNA NTS.

View larger version (76K): [in this window] [in a new window]   Figure 7. NTS2 transcripts are made by RNA polymerase II. (A) Cultures of SIR2+ and sir2 cells containing a wild-type allele of RPB1 (wt RNA Pol II) or a ts allele rpb1-1 (ts RNA Pol II) were grown at 24°C and either maintained at 24°C or shifted to 36°C for 30 min (see Materials and Methods). Northern analysis of total RNA shows that NTS2 and ACT1 transcript levels are stable at 24 and 36°C in cells with wt RNA Pol II (lanes 1–4, top and middle) but are depleted after 30 min at 36°C in cells with ts RNA Pol II (lanes 5–8). The levels of Pol I transcribed rRNA are stable at 24 and 36°C (lanes 1–8, bottom). (B) Time-course analysis reveals that NTS2 transcripts are depleted 15 min after inactivation of Pol II. Cultures of cells grown at 24C were shifted to 36°C and aliquots were removed at 0, 15, 30, and 60 min. Total RNA was analyzed as described in A. Between 15 and 30 min after the shift to 36°C, the NTS2 transcripts were depleted significantly in cells containing the ts RNA Pol II but continue to be made in cells with the wt RNA Pol II (compare lanes 13–16 to 5–8, top). By 60 min, the level of ACT1 RNA was reduced significantly in cells with ts RNA Pol II, whereas rRNA levels remain stable (compare lanes 1–8 to 9–16, middle and bottom). Asterisks indicate positions of NTS2 transcripts.

sir2

Nucleosome Positioning at NTS2 Is Altered in Cells with rDNA-silencing Defects
Previous studies revealed that cells lacking Sir2 have altered chromatin structure in the rDNA (Fritze et al., 1997; Cioci et al., 2002); however, these studies did not focus on NTS2 where we predict changes in nucleosome positioning might occur because of increased transcription of the NTS2 by Pol II. To determine whether nucleosome positioning in the NTS2 region of the rDNA was altered in cells lacking Sir2 or Set1, we performed indirect end-labeling analysis of MNase-digested chromatin. The rDNA repeats in S. cerevisiae exist in two configurations that are correlated with their accessibility to a cross-linking reagent psoralen and Pol I transcription activity (Dammann et al., 1993). Open rDNA repeats are accessible to psoralen and transcribed by Pol I, whereas closed repeats are inaccessible to psoralen and not transcribed. It is important to consider that similar to the ChIP analyses, MNase experiments at the rDNA evaluate populations of cells, with each cell containing 150–200 rDNA repeats that are in different conformations with variable MNase accessibilities. It is possible that there are stronger effects on individual repeats, however, the alterations we observed represent the average accessibility of all the rDNA repeats. Thus, the ability to see even modest alterations in MNase cleavage over 150–200 repeats is significant.

Five positioned nucleosomes have been mapped in the NTS2 region of the rDNA (Figure 8, open circles labeled 1–5; Vogelauer et al., 1998). We detected alterations in MNase accessibility at several positions in the NTS2 region in sir2 cells and set1 cells (Figure 8, A–C). Between the ARS and the transcription start site of the 35S rRNA gene, in the region containing the TSS for the NTS2 transcripts, an MNase cleavage site was clearly missing in chromatin from sir2 cells and set1 cells that was present in wild-type chromatin (Figure 8, B and C, arrow marked e). This change may reflect protection by Pol II or its associated factors in sir2 and set1 cells. Other changes in MNase accessibility were detected in sir2 cells only, including a new cleavage site in NTS2 upstream of the Pol III-transcribed 5S gene (Figure 8, A–C, arrow marked a) and a missing cleavage site near the ARS (Figure 8, A and C, arrow marked d). In addition, we detected subtle changes in two MNase cleavage sites between nucleosomes 2 and 3 (Figure 8, A–C, arrows marked b and c). Our results indicate that rDNA chromatin structure was altered near the TSS sequence in the NTS2 region of the rDNA repeat in sir2 cells and set1 cells. These changes in chromatin structure are consistent with the observation that sir2 and set1 cells, which have profoundly different types of modified histones at the rDNA, both have defects in silencing of Pol II-transcribed genes at the rDNA.

View larger version (58K): [in this window] [in a new window]   Figure 8. MNase accessibility of chromatin in the NTS2 region of the rDNA is altered in set1 and sir2 cells. DNA purified after MNase treatment of spheroplasts from wild-type, set1, or sir2 cells was digested with EcoRI (A) or PvuII (B). Schematic of NTS2 with five positioned nucleosomes (numbered ovals) identified here and in a previous study (Vogelauer et al., 1998) is shown on the left of each panel and in C. Triangles, increasing amounts of MNase; N, naked DNA; arrows with lowercase letters, altered MNase accessibility (see text); other labels as in Figure 1. (C) The NTS2 region of the rDNA. Numbered circles, positioned nucleosomes; + and –, extent of MNase digestion in sir2 cells relative to wild-type cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

K4-Methylated Histone H3 and Pol II Are Excluded from Silent Chromatin by Sir2
We have analyzed K4-methylated H3 at silent loci in wild-type and sir2 cells by ChIP. Our results show that Sir2 excludes K4 mono-, di- and -trimethylated histone H3 from silent chromatin domains, including HMR, TEL-VIR, and the rDNA (Figures 1–3). Using a catalytically inactive form of Sir2, we have determined that the deacetylase activity of Sir2 is required to exclude K4-di- and -trimethylated H3 from the rDNA (Figure 4). Consistent with the primary role of Sir2 at silent chromatin, no significant difference in the levels of K4-methylated H3 was detected at the euchromatic and non-rDNA intergenic regions in sir2 cells.

In S. cerevisiae, high levels of K4-trimethylated histone H3 are associated with actively transcribed genes in euchromatic domains of the genome (Bernstein et al., 2002; Santos-Rosa et al., 2002). These observations are supported by work showing that the Pol II elongation complex Paf1C recruits the histone H3 K4-methylation complex COMPASS to genes being transcribed by Pol II (Krogan et al., 2003; Ng et al., 2003b, c). The results of experiments in Figures 5–7 show that the level and activity of Pol II are increased at the rDNA NTS in sir2 cells. Interestingly, in cells lacking Paf1, the levels of K4-methylated H3 at the rDNA NTS are reduced, suggesting that even the low levels of K4-methylated H3 observed at the rDNA are dependent on transcription (Mueller et al., 2006). Finally, our MNase data show that the structure of chromatin in NTS2 is altered in sir2 cells. Based on these results, we propose that the deacetylase activity of Sir2 prevents the conversion of silent chromatin at the rDNA, normally inaccessible to Pol II, to active chromatin with the potential to be transcribed. This conclusion is supported by results showing that the level of K4-dimethylated H3, a mark of chromatin with the potential to be transcribed by Pol II, was increased over the rDNA repeat in cells lacking Sir2 and that the highest level of K4-trimethylated H3 was in the NTS, where Pol II transcription was occurring.

The observation of increased K4-methylated H3 and increased transcription at the rDNA in S. cerevisiae cells lacking Sir2 is reminiscent of recent reports regarding the role of modified histones in regulating Pol I transcription and rDNA chromatin structure in higher eukaryotes (reviewed in Grummt and Pikaard, 2003; McStay, 2006). In several Arabidopsis species, disruption of the histone deacetylase (HDAC) activities of HDT1, a plant-specific HDAC, or HDA6, a member of the RPD3 family of HDACs, caused normally silenced rDNA repeats to acquire characteristics of active rDNA repeats, including high levels of Pol I and K4-methylated H3, and low levels of K9-methylated H3 and DNA methylation (Lawrence et al., 2004; Probst et al., 2004; Earley et al., 2006). Unlike the Arabidopsis system, in S. cerevisiae, transcription by Pol I is not increased in cells that lack the histone deacetylase RPD3 (Oakes et al., 1999, 2006). Likewise, no changes in Pol I transcription have been observed in cells lacking Sir2 (Oakes et al., 1999, 2006). Despite the differences, a common thread between the yeast and plant systems is that loss of specific HDACs results in rDNA that is more like euchromatin, and, subsequently, has altered function.

It is important to note that each rDNA repeat in S. cerevisiae contains an ARS; however, only a small fraction (20%) of these replication origins fire during S phase to replicate the rDNA locus (Linskens and Huberman, 1988). Molecular imaging experiments have shown that a large proportion of rDNA origins (50%) are activated in cells lacking Sir2 (Pasero et al., 2002). The high level of K4-trimethylated H3 at the rDNA may be associated with increased firing of the rDNA ARS elements in sir2 cells. Experiments to address the role of K4-methylated H3 and NTS transcription in origin firing at the rDNA are currently underway.

Additional Factors May Limit K4-Methylated H3 at the rDNA
In cells lacking Sir2, increased levels of K4-trimethylated histone H3 were observed across an 4-kb region of HMR and up to 2.8 kb from TEL-VIR, whereas the increase at the rDNA was limited primarily to NTS2 (Figures 1 and 2). The region of HMR where we detect changes in the association of K4-methylated histone H3 falls within the boundaries of the silent domain mapped in previous studies (Donze et al., 1999; Donze and Kamakaka, 2001). From these observations, we conclude that COMPASS has greater access to HMR and TEL-VIR than to the rDNA in sir2 cells. Given that Pol II transcription units have been identified in the 35S rRNA gene and NTS1 (see below), it is possible that Sir2-independent mechanisms exist that limit the association of Pol II and K4-methylated H3 with these other regions. One possibility is that transcription by Pol I excludes factors required for methylation of H3. This can be tested by measuring the levels of K4-methylated H3 at the rDNA in cells that lack Pol I and make rRNA from a plasmid-borne 35S rRNA gene under the control of a Pol II promoter, similar to the cells used to demonstrate the requirement for Pol I transcription in rDNA silencing (Buck et al., 2002; Cioci et al., 2003).

In addition, a trans-histone regulatory pathway has been identified where the ubiquitylation of histone H2B by Rad6 is required for the di- and trimethylation of histone H3 on K4 and K79 (Briggs et al., 2002; Sun and Allis, 2002; Shahbazian et al., 2005). In cells lacking Ubp10, an enzyme required for the removal of ubiquitin from H2B at silent domains in S. cerevisiae, the level of K4-trimethylated H3 was increased at the 5S and 35S rRNA gene (Emre et al., 2005). We measured low levels of K4-trimethylated H3 at these regions in sir2 cells, suggesting that Ubp10 limits K4-methylated H3 even in the absence of Sir2.

Silencing at the rDNA, HM loci, and telomeres requires the deacetylase activity of Sir2. The known protein targets of Sir2 are lysine residues in histones H3 and H4, including K9 and K14 of H3 and K16 of H4 (reviewed in Rusche et al., 2003). Recent work has demonstrated that O-acetyl-ADP-ribose (AAR), a product of the Sir2 deacetylation reaction, promotes a conformational change in the Sir2-3-4 complex that may contribute to the formation of silent chromatin (Liou et al., 2005). Although we have determined that Sir2 restricts the access of Pol II and the K4-methylation machinery to silent chromatin, our in vivo studies do not separate the contributions of hypoacetylated histone tails and AAR. In vitro studies using purified factors and chromatin templates should distinguish between the functions of these products of Sir2.

Cells lacking Set1 exhibit defects in silencing at the rDNA and telomeres (Nislow et al., 1997; Briggs et al., 2001; Bryk et al., 2002; Nagy et al., 2002; Mueller et al., 2006). Unlike what has been observed at telomeres (Ng et al., 2002, 2003a; Meneghini et al., 2003; Martin et al., 2004; Katan-Khaykovich and Struhl, 2005), silencing factors remain at the rDNA in set1 cells (Bryk et al., 2002), and thus the mechanism behind loss of silencing at the rDNA in set1 cells is not consistent with the model proposed to explain loss of silencing at telomeres. One model that we are currently testing is that release of Sir proteins from telomeres in set1 cells interferes with rDNA silencing. However, at this time, we are unable to determine whether the changes in chromatin accessibility or the loss of rDNA silencing seen in set1 cells are direct or indirect.

Other Pol II Transcription Units in the rDNA
In addition to the NTS2 transcripts that we have identified, the rDNA contains other Pol II transcription units. A naturally occurring Pol II-transcribed gene TAR1 has been identified on the antisense strand of the 35S rRNA gene 3–4 kb away from NTS2 (Coelho et al., 2002). Although its expression was shown to be responsive to Sir2, we did not observe high levels of K4-trimethylated H3 over the TAR1 ORF. Previous studies have characterized mutants that lack subunits of Pol I and survive by transcribing the 35S rRNA genes with Pol II (Conrad-Webb and Butow, 1995; Vu et al., 1999). However, in RNA from sir2 cells, Pol II-derived 35S rRNA transcripts have not been detected (Oakes et al., 1999, 2006). Moreover, we did not detect high levels of K4-trimethylated H3 or Pol II over the 35S rRNA gene, which would be expected if the 35S rRNA gene was being transcribed by Pol II.

Recent work has shown that in sir2 cells, bidirectional transcripts from NTS1 displace cohesin complexes from the rDNA leading to high levels of unequal sister chromatid exchange and rDNA repeat amplification (Kobayashi and Ganley, 2005). In addition to the transcripts from NTS1, we have identified transcripts from NTS2. Although transcription and the level of Pol II is increased clearly over both NTS1 and NTS2 in sir2 cells, we were surprised that the level of K4-trimethylated H3 over NTS1 was increased only 1.5- to 2.0-fold. Despite our efforts to correct for total histone H3, we suspect reduced levels of total H3 at NTS1 (Supplemental Figure S1) may have contributed to the lower level of K4-trimethylated H3 measured at NTS1 in ChIP experiments (Figure 1 and Supplemental Figure S2). Nonetheless, the NTS transcription units display histone marks found on euchromatic genes, suggesting that not only Pol II- but also Pol II-associated factors required for the trimethylation of H3 on K4 have access to the rDNA in sir2 cells.

Three possible ORFs of 71, 64, and 55 amino acids were identified in the sequence of the 1.7-kb NTS2 transcript, but no significant matches to known eukaryotic proteins from the nonredundant protein database were identified using BLASTp searches. Likewise, significant conservation of S. cerevisiae NTS DNA sequences was noted in several yeast species related to S. cerevisiae by BLASTn, but not in nonyeast organisms. It is possible that the NTS RNAs are noncoding RNAs. Intergenic transcription has been associated with regulatory pathways involving chromatin and gene expression in several organisms (reviewed in Bernstein and Allis, 2005). Interestingly, intergenic transcripts from the rDNA spacer that are made by Pol I have recently been shown to regulate the heterochromatin structure of rDNA repeats in mouse cells (Mayer et al., 2006). Experiments are currently underway to analyze the Pol II-transcribed NTS transcripts from S. cerevisiae in detail and to determine whether transcription through NTS2 alters rDNA recombination, silencing of Pol II marker genes, and/or DNA replication from the origin present in each rDNA repeat.

	ACKNOWLEDGMENTS

 
We thank Julie Huang for rDNA primer and Kevin Struhl for control primer sequences, Danesh Moazed for Sir2 antisera, Adam Rudner for plasmids, Catalina Alfonso for initiating micrococcal nuclease studies, and Neva Barker for analyzing rDNA sequences. We are grateful to Craig Kaplan, Mike Kladde, and Kelly Williamson for comments on the manuscript. This work was supported by grants from the American Heart Association, Texas Affiliate (0365004Y), the American Cancer Society (RSG-04-049-01-GMC), and the National Institutes of Health (GM-070930) to M.B.