Suppression analysis of esa1 mutants in Saccharomyces cerevisiae links NAB3 to transcriptional silencing and nucleolar functions.

The acetyltransferase Esa1 is essential in the yeast Saccharomyces cerevisiae and plays a critical role in multiple cellular processes. The most well-defined targets for Esa1 are lysine residues on histones. However, an increasing number of nonhistone proteins have recently been identified as substrates of Esa1. In this study, four genes (LYS20, LEU2, VAP1, and NAB3) were identified in a genetic screen as high-copy suppressors of the conditional temperature-sensitive lethality of an esa1 mutant. When expressed from a high-copy plasmid, each of these suppressors rescued the temperature-sensitivity of an esa1 mutant. Only NAB3 overexpression also rescued the rDNA-silencing defects of an esa1 mutant. Strengthening the connections between NAB3 and ESA1, mutants of nab3 displayed several phenotypes similar to those of esa1 mutants, including increased sensitivity to the topoisomerase I inhibitor camptothecin and defects in rDNA silencing and cell-cycle progression. In addition, nuclear localization of Nab3 was altered in the esa1 mutant. Finally, posttranslational acetylation of Nab3 was detected in vivo and found to be influenced by ESA1.

Nucleosomes containing the core histones (H2A, H2B, H3, and H4) form the basic packaging unit of DNA that organizes chromatin into higher-order structures. The N-terminal tails of histones are subject to multiple covalent modifications that can influence gene expression locally at specific promoters or within large regions of chromatin. Increased histone acetylation is associated with both transcriptional activation and repression. Lysine acetyltransferases (KAT), the enzymes that catalyze the acetylation reaction on histones, have been ascribed multiple cellular functions. Recently, nonhistone targets have also been identified for many KATs, including Esa1 (Lin ) [reviewed in Yang and Seto (2008)].

The Esa1 KAT of Saccharomyces cerevisiae is a member of the deeply conserved MYST family of acetyltransferases and is essential in yeast (Smith ; Clarke ). Esa1 is the catalytic component of the NuA4 and piccolo complexes that acetylate histone H4, H2A, and its variant H2A.Z (Allard ; Babiarz ; Keogh ; Millar ). Many of the NuA4 subunits, including Esa1, are essential (Galarneau ; Loewith ; Eisen ), indicating that this complex has critical cellular roles.

Esa1 has a role in regulating expression of ribosomal protein genes (Reid ). Further, genome-wide expression analysis reveals widespread transcriptional changes in mutants (Durant and Pugh 2006), and genome-wide binding profiles show Esa1 bound to the promoters of actively transcribed genes (Robert ). Esa1 also functions in transcriptional silencing of the rDNA and at telomeres (Clarke ). The variety of genomic targets identified thus far suggests Esa1 activity regulates transcription at many loci, indicative of its function in multiple cellular processes.

Genetic analysis further defines Esa1’s role in diverse cellular functions. Temperature-sensitive mutants of display a G2/M cell-cycle arrest at the restrictive temperature that is dependent upon the DNA damage checkpoint (Clarke ) and are hypersensitive to the topoisomerase I inhibitor camptothecin (Bird ). Esa1 localizes to double-strand breaks where it functions in repair of DNA damage (Downs ). Together, these results suggest Esa1 activity is required for cell-cycle regulation and genomic integrity, although Esa1’s catalytic activity may not be its only essential role (Decker ).

Suppression analyses have linked to the deacetylase Sir2, a key silencing protein. Overexpression of Sir2 was found to suppress rDNA-silencing defects, thereby suggesting that Sir2 and Esa1 may function coordinately to silence the rDNA array (Clarke ). Several other studies have identified additional suppressors of conditional alleles of (Biswas ; Lin ; Chang and Pillus 2009; Scott and Pillus 2010).

To pursue genetic interactors of , a dosage suppression screen was performed on an mutant. Of the four high-copy suppressors identified, became a focus for two primary reasons. First, only overexpression rescued both the temperature-sensitivity and the silencing defects of mutants. Second, has known roles in RNA processing, and this functional connection to Esa1 may establish a novel link between two nuclear processes. Numerous studies have characterized roles for Nab3 and its binding partner Nrd1 in 3′-end processing of several classes of small noncoding RNAs [reviewed in Lykke-Andersen and Jensen (2007)]. These classes of RNAs include small-nuclear (sn) RNAs, small-nucleolar (sno) RNAs, and cryptic unstable transcripts (CUT). Nab3 and Nrd1 each recognize specific RNA sequences for 3′-end formation and transcription termination (Carroll ).

This study reports new mutant phenotypes of , revealing roles for Nab3 in rDNA silencing, the DNA damage response, and cell-cycle progression. Further, Nab3 was found to be posttranslationally modified by acetylation. This acetylation was reduced in an conditional mutant that displays reduced Esa1 acetyltransferase activity, providing evidence that Nab3 is a nonhistone substrate of Esa1 whose function may be influenced by acetylation.

Materials and Methods

Dosage suppressor screen

A -marked 2μ genomic library (generously provided by P. Hieter) was transformed into two isolates of the strain LPY3291 in six independent experiments, yielding a total of 130,000 transformants with an approximate 70-fold coverage of the genome. Transformants were grown under permissive conditions on SC-Trp-Ura plates, and then replica-plated and incubated at 28°, 35°, and 37°. Two hundred colonies were able to grow at 35° but not 37° (this was a secondary screen used to avoid recovering wild-type ). These candidates were tested for plasmid dependence by growing original transformants on 5-fluoroorotic acid (5-FOA) plates. The resulting resistant strains, which had lost the -marked plasmid, were tested for temperature sensitivity at 35°. This resulted in 34 suppressor strains being classified as plasmid-dependent. Suppressing plasmids were rescued from yeast, and inserts were sequenced using T3 and T7 primers. Of the 34 plasmids, 22 were WT , 3 were unidentified, and the remaining 9 comprised six independent clones containing one of the four following genes: , , , or . Library fragments that contained multiple ORFs were dissected by subcloning to identify the gene responsible for suppression. Strategy for identification of the four suppressors is described in detail (Clarke 2001). The plasmid subclones were retransformed into LPY3291 to confirm the suppressing phenotype.

Yeast methods and strain and plasmid construction

All yeast strains and plasmids used in this study are listed in Tables 1 and 2. The silencing markers rDNA:: (Fritze ) and TELVR:: (Renauld ) were introduced through standard genetic crosses. All strains originate from YPN100 (provided by M. Swanson) (Conrad ). Nab3 Flag-tagging was carried out by amplification of pFA6a-2FLAG-kanMX6 and transformation into LPY5 (W303-1a) using the method described (Longtine ) to make LPY15000. All library plasmids are in the pRS202 (pLP1402) backbone. pLP1238 ( in pRS202) and pLP2018 ( in pRS426) were subcloned from pLP1419 ( library construct) using EcoRI and XhoI. pLP1310 ( in pLP271) was subcloned from pLP1419 using EcoRI. Dilution assays for growth, silencing, and drug sensitivity were performed as described (Chang and Pillus 2009) and represent 5-fold serial dilutions starting from an A600 of 1.0. Images were captured after 2–4 days of growth at the indicated temperatures.

Table 1

Yeast strains used in this study

Strain	Genotype	Reference
LPY5 (W303-1a)	MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1	Thomas and Rothstein 1989
LPY3291	MATa his3Δ200 leu2-3,112 trp1Δ1 ura3-52 esa1Δ::HIS3 + pLP863 (esa1-414)	Clarke et al. 1999
LPY4774	W303 MATa esa1-414
LPY4909	W303 MATα rDNA::ADE2-CAN1	Clarke et al. 2006
LPY4911	W303 MATα esa1-414 rDNA::ADE2-CAN1	Clarke et al. 2006
LPY4917	W303 MATα TELVR::URA3	Clarke et al. 2006
LPY4919	W303 MATα esa1-414 TELVR::URA3	Clarke et al. 2006
LPY4979	W303 MATα sir2Δ::HIS3 TELVR::URA3
LPY5406	W303 MATa nab3-10 rDNA::ADE2-CAN1
LPY5407	W303 MATa nab3-10 TELVR::URA3
LPY10622	W303 MATa nab3-10
LPY11286	W303 MATa nab3-10 adh4::ADE2 TELVIIL
LPY11300	W303 MATa adh4::ADE2 TELVIIL
LPY12154	W303 MATa rpd3::kanMX	Chang and Pillus 2009
LPY15000	W303 MATa NAB3-2Flag::kanMX
LPY15004	W303 MATa esa1-414 NAB3-2Flag::kanMX

Except where noted, strains were constructed during the course of this study or are part of the standard lab collection.

Table 2

Plasmids used in this study

Plasmid (Alias)	Description	Source/Reference
pLP362 (pRS426)	Vector URA3 2µ	Sikorski and Hieter 1989
pLP1402 (pRS202)	Library vector URA3 2μ	P. Hieter
pLP37	SIR2 URA3 2μ
pLP271	Vector TRP1 2µ
pLP796	ESA1 URA3 2μ	Clarke et al. 2006
pLP798	ESA1 TRP1 2µ
pLP863	esa1-414 TRP1 CEN	Clarke et al. 1999
pLP1238	NAB3 URA3 2μ
pLP1259	VAP1 URA3 2μ
pLP1310	NAB3 TRP1 2µ
pLP1412	LYS20 URA3 2μ
pLP1405	LYS20-library clone URA3 2μ
pLP1406	VAP1-library clone URA3 2μ
pLP1417	LEU2-library clone URA3 2μ
pLP1419	NAB3-library clone URA3 2μ
pLP2018	NAB3 URA3 2µ
pLP2054	NRD1 URA3 2µ

Except where noted, plasmids were constructed during the course of this study or are part of the standard lab collection. “Library clone” represents a clone obtained directly in the suppressor screen, whereas others are subclones as detailed in Clarke (2001).

Northern analysis, protein immunoblots, and immunoprecipitations

RNA was isolated using the hot acid phenol protocol as described (Collart and Oliviero 2001). Northern blotting was performed as described (Cox and Walter 1996), and results were obtained by phosphorimager (Storm, GE Healthcare). Yeast extracts were prepared by bead beating as described previously (Clarke ), separated on SDS-polyacrylamide gels (18% for detection of histones, 8% for Sir2 and Rpd3), and transferred to nitrocellulose (0.2 μm). Primary antisera used were anti-H4K5Ac (Serotec), anti-H4K8Ac (Serotec), anti-H4K12Ac (Serotec), anti-H4K16Ac (Upstate), anti-Sir2 (Garcia and Pillus 2002), anti-Rpd3 (Rundlett ), anti-PGK (Baum ), anti-FLAG (Sigma-Aldrich, F3165), and anti-acetyl-lysine (Cell Signaling, #9681). Secondary antibodies conjugated to horseradish peroxidase in combination with chemiluminescence reagents were used for detection on film. FLAG-Nab3 was immunoprecipitated with anti-FLAG M2 Affinity Gel (Sigma-Aldrich, A2220), eluted in SDS sample buffer, separated on a SDS-polyacrylamide gel, and immunoblotted with either anti-FLAG or anti-acetyl lysine. All experiments were performed in triplicate or more and a representative blot was chosen for quantification. Quantification of all immunoblots was performed with ImageQuant software.

Nab3 and Sir2 immunofluorescence

Immunofluorescence was performed as described (Gotta ; Stone ). WT and strains were grown in YPD for four hours at either 28° or 37°. Cells were fixed by adding paraformaldehyde to the cultures at a final concentration of 3.3% at 30° for 10 min. Samples were washed twice in YPD, resuspended at 1 ml per 0.1 g of cells in 0.1 M EDTA, KOH pH 8.0, and 19 mM DTT, and then incubated at 30° for 10 min with gentle agitation. The primary antibodies used were anti-Nab3 (mouse monoclonal 2F12) (Wilson ) and anti-Sir2 (Garcia and Pillus 2002). Texas Red-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit were used as secondary antibodies. Staining was visualized with an Applied Precision Deltavision optical sectioning deconvolution microscope.

Flow cytometry

Cell-cycle profiles were obtained by flow cytometry of propidium iodide stained cells on a FACSCalibur machine (Becton Dickinson) and analyzed with CellQuest software (Becton Dickinson). Cells were grown to an A600 of between 0.6 and 1.0, fixed in ethanol overnight, and stained with propidium iodide. Stained cells were sonicated and then analyzed by flow cytometer. For each sample, 100,000 cells were counted and analyzed.

Results

Four suppressors of the esa1 temperature-sensitive phenotype

To identify genes that interact functionally with , a dosage-suppressor screen was performed utilizing a genomic 2μ plasmid library. The temperature-sensitive strain was transformed with the library, and transformants were tested for growth at both permissive and restrictive temperatures. Plasmids were rescued from transformants that grew at the restrictive temperature to determine the identity of suppressing genomic fragments. The results of this analysis revealed four dosage suppressors: , , , and (Figure 1). None of these suppressors bypassed the inviable . When tested with other previously characterized alleles (Clarke ), some allele-specificity was observed (supporting information, Table S1). The series of alleles was also tested for suppression of other phenotypes (see below).

Figure 1 

The esa1 temperature-sensitive growth defect is partially suppressed by four genes expressed from 2μ plasmids. Increased gene dosage of LYS20 (pLP1405), VAP1 (pLP1406), LEU2 (pLP1417), or NAB3 (pLP1419) from a 2μ plasmid moderately suppresses the esa1-414 (LPY3291) growth defect at 35°. The esa1 growth defect at 35°, demonstrated by vector (pLP1402) transformants, is completely restored in cells transformed with an ESA1 plasmid (pLP796). All strains were plated on SC-Ura-Trp media. Suppression was not observed at higher temperatures. Multiple independent transformants were tested to examine any variability between transformants.

and are nonessential genes required for the biosynthesis of leucine and lysine [reviewed in Kohlhaw (2003) and Xu , respectively]. is also involved in amino acid metabolism, encoding a transporter of several amino acids, including tyrosine, tryptophan, valine, and leucine (Schmidt ). Characterization of as a suppressor of revealed additional roles for this metabolic gene in DNA damage repair (Scott and Pillus 2010). , as noted, is an essential gene critical for 3′-end processing of nonpolyadenylated transcripts [reviewed in Lykke-Andersen and Jensen (2007)].

Increased dosage of NAB3 suppresses multiple esa1 mutant phenotypes

To understand the connection between the suppressors and Esa1 function, overexpression of the four genes was tested for suppression of mutant defects other than temperature sensitivity. One phenotype of mutants is a strong rDNA-silencing defect and a slight increase in mitotic rDNA recombination (Clarke ). Previously, it was shown that increased gene dosage of suppresses the rDNA-silencing defect (Clarke ). Using an strain with the dual reporter integrated at a single 25S rDNA repeat (Fritze ) (Figure 2A), the suppressors were tested for their effect on silencing of the rDNA locus. Only increased dosage of robustly suppressed the rDNA-silencing defect, restoring silencing to near wild-type levels (Figure 2B). By contrast, slightly exacerbated ’s silencing defect, whereas and had little to no effect (Figure 2B). Unlike increased gene dosage of in a wild-type strain (Smith ), did not enhance wild-type rDNA silencing (Figure 2C). None of the suppressors had significant effects on rDNA recombination. As there appeared to be a link between and for both silencing and growth, we chose to characterize in greater detail.

Figure 2 

The esa1 rDNA silencing defect is suppressed by increased gene dosage of NAB3. (A) Diagram and location of rDNA::ADE2-CAN1 silencing marker within the rDNA array on chromosome XII. (B) The esa1 rDNA silencing defect (vector) is restored in cells transformed with an ESA1 plasmid, and by NAB3. Increased dosage of LYS20, LEU2, or VAP1 does not rescue esa1’s rDNA silencing defect. An esa1 strain with the 25S rDNA::ADE2-CAN1 reporter (LPY4911) was transformed with vector (pLP1402), ESA1 (pLP796), LYS20 (pLP1412), NAB3 (pLP1238), VAP1 (pLP1259), SIR2 (pLP37), or LEU2 (pLP1417). To test for rDNA silencing defects, strains were plated on SC-Ade-Arg-Ura (growth) with and without 32 µg/ml canavanine (rDNA silencing) at 33°. (C) NAB3 overexpression (pLP1238) has no effect on rDNA silencing of a WT strain (LPY4909).

In addition to their rDNA-silencing defects, mutants are defective in telomeric silencing (Clarke ) (Figure 3A), as shown by diminished growth on 5-FOA when using a reporter gene on the right arm of chromosome V (TELVR) (Renauld ). Increased dosage of in mutants allowed for increased growth on 5-FOA, thereby rescuing the sensitivity shown in the mutant (Figure 3A). Recent studies have shown that readout of this reporter-based assay for some genes may reflect changes in nucleotide metabolism instead of telomeric-silencing defects (Rossmann ; Takahashi ). Thus, based on these new studies, rescue of ’s 5-FOA sensitivity by in strains carrying the telomeric reporter gene can be interpreted as the ability of overexpression to suppress telomeric-silencing defects or nucleotide metabolism changes in an mutant. Because has no known defects in HM silencing or mating efficiency (Clarke ), dosage was not tested for effects on mating efficiency.

Figure 3 

Overexpression of NAB3 affects multiple esa1 mutant phenotypes. (A) Top: Diagram of TELVR::URA3 telomeric silencing marker on the right arm of chromosome V. Bottom: Increased gene dosage of NAB3 suppresses the esa1 5-FOA sensitivity in this assay. An esa1 strain with the TELVR::URA3 reporter (LPY4919) was transformed with vector (pLP271), ESA1 (pLP798), or NAB3 (pLP1310), and plated on SC-Trp (growth) with and without 5-FOA (telomeric silencing) at 33°. (B) Increased gene dosage of NAB3 exacerbates esa1’s sensitivity to the DNA damaging agent camptothecin. An esa1 strain (LPY4774) was transformed with vector (pLP326), ESA1 (pLP796), or NAB3 (pLP2018), and plated on SC-Ura with DMSO (growth) and 20 µg/ml camptothecin (DNA damage). (C) Overexpression of NAB3 does not increase global acetylation levels of H4K5, H4K8, H4K12, or H4K16 in esa1 mutants. Whole-cell extracts were made from wild-type (LPY5) and esa1 (LPY4774) strains with vector (pLP362) or 2μ NAB3 (pLP2018) grown in SC-Ura media at both permissive (28°) and elevated (34°) temperatures. These were immunoblotted for amounts of isoform-specific H4 acetylation and total H3. An H3 reprobe was performed for each individual H4 acetylation blot. Quantification data shown are normalized for H3 loading. (D) Overexpression of NAB3 does not influence esa1’s G2/M cell-cycle block. The same strains as in (C) were grown at 28° and shifted to 37° for 4 hr before fixing and staining with propidium iodide. Cell-cycle profiles were analyzed by flow cytometry.

Another phenotype of mutants is sensitivity to DNA damage induced by camptothecin, a topoisomerase I inhibitor that triggers double-strand breaks (Bird ). overexpression was tested for its ability to suppress this mutant defect in the DNA damage response and was found to exacerbate ’s camptothecin sensitivity (Figure 3B). This result is in contrast to -mediated suppression of ’s silencing defects, highlighting a difference between Nab3 and Esa1’s functions in transcriptional silencing and DNA damage repair.

At a molecular level, global H4 acetylation is dramatically reduced in mutants when grown at restrictive temperatures (Clarke ). To determine whether increased dosage of restores wild-type levels of histone acetylation to mutants, a series of protein immunoblots with isoform-specific antibodies was performed to define the global acetylation state in strains overexpressing . All the histone H4 lysine residues that Esa1 is known to acetylate (K5, K8, K12, and K16) (Clarke ) were tested in these experiments (Figure 3C). Total histone levels were determined by probing with a control antibody specific to the C-terminus of histone H3. This series of immunoblots shows that increased dosage of in strains did not restore H4 acetylation. Therefore, overexpression does not rescue mutants by restoring global acetylation defects at substrate residues in the H4 N-terminal tail.

A distinct potential mechanism for suppression is through Esa1’s role in the cell cycle. Since Esa1 is required for cell-cycle progression through G2/M, cell-cycle profiles of mutant strains with increased dosage of were examined by flow cytometry to distinguish cellular DNA content before (1C) and after (2C) replication. The mutants at restrictive temperature have a well-defined G2/M cell-cycle block, visualized as a decrease in the 1C peak and an accumulation of the 2C peak (Clarke ). With overexpression, no change in the cell-cycle profile was observed (Figure 3D), indicating that overexpression does not bypass the G2/M cell-cycle block of mutants. Thus, increased dosage of suppresses a defined subset of mutant phenotypes, which includes silencing defects and temperature sensitivity.

Nab3 does not affect protein or transcript levels of histone-modifying enzymes

In addition to their function for termination of noncoding RNAs, there is evidence that Nab3 and its partner Nrd1 participate in 3′-end formation of protein-coding transcripts (Sugimoto ; Arigo ; Darby ). We considered the possibility that Nab3 might bind to mRNA to direct its 3′-end formation. Nab3 binding sites have the simple UCUU consensus sequence (Carroll ) that is found at several positions within the transcript. Northern blotting was performed to determine whether there were any -dependent changes in the transcript. is an essential gene (Wilson ) and, thus, the temperature-sensitive mutant was used in this study. The allele was described previously and specifies a single F371L amino acid substitution in its RNA-recognition motif (RRM) domain (Conrad ). When transcripts were examined in the mutant (Figure 4A), there were no detectable changes in either transcript levels or migration. Transcript levels of were also found to be constant with or without increased dosage of (Figure 4A). Increased dosage of , which encodes a binding partner of Nab3, also failed to influence mRNA. In conclusion, does not affect the transcriptional regulation of itself.

Figure 4 

NAB3 overexpression does not change transcript levels of ESA1 or protein levels of Rpd3 and Sir2. (A) NAB3 overexpression does not alter ESA1 mRNA levels. Total RNA was isolated from both WT (LPY5) and esa1 (LPY4774) mutant strains grown at an elevated temperature (35°) with vector control (pLP362), NAB3 overexpression (pLP2018), or NRD1 overexpression (pLP2054). Northern analysis was performed with an ESA1-specific probe, and results were obtained by phosphorimager scan. (B) Overexpression of NAB3 does not influence Rpd3 protein levels. Whole-cell lysates from WT and esa1 strains grown at an elevated temperature (35°) with vector control or NAB3 overexpression [same strains as in (A)] were examined by immunoblot with anti-Rpd3. An rpd3Δ strain (LPY12154) transformed with vector (pLP362) was used as a negative control, and anti-PGK1 (phosphoglycerate kinase) was used to determine equal loading between samples. (C) Overexpression of NAB3 does not influence Sir2 protein levels. Whole-cell lysates were made from WT (LPY5) and esa1 (LPY4774) strains grown at an elevated temperature (37°) with vector control (pLP1402) or NAB3 overexpression (pLP1238) and immunoblotted with anti-Sir2. Extract from a sir2Δ strain was used as a negative control. A nonspecific band (*) detected by anti-Sir2 was used to determine equal loading between samples.

We considered the possibility that affects transcription of a histone deacetylase (HDAC) that acts in opposition to Esa1. Transcriptional downregulation of an HDAC could compensate for the lack of functional Esa1 and restore the imbalance of acetylation in the cell. For example, deletion of the histone deacetylase gene suppresses the temperature-sensitivity and silencing defects of an mutant (Chang and Pillus 2009). To test whether suppression of is mediated through changes in Rpd3 levels, its protein levels were examined by immunoblot. Comparing Rpd3 levels between wild type and strains with and without increased dosage of revealed no -dependent changes (Figure 4B). Another HDAC candidate of interest is Sir2, an HDAC critical for establishment and maintenance of silent chromatin [reviewed in Rusche ]. Similar to overexpression, overexpression has been shown to rescue rDNA silencing in an mutant (Clarke ). Sir2 levels were determined in wild-type and mutant strains overexpressing by immunoblot, and no -dependent differences were observed (Figure 4C). Therefore, overexpression does not alter expression of either Rpd3 or Sir2, demonstrating that suppression is not mediated through transcriptional regulation of either HDAC.

nab3 mutants share phenotypes with esa1 mutants

To characterize further the role of in relation to , mutants were examined for established phenotypes of mutants. Since overexpression rescued the telomeric- and rDNA-silencing defects (Figures 2B and 3A), it was possible that mutants might be defective in silencing. Telomeric silencing was tested using the same reporter assay as before (Figure 3A), and it revealed that mutants display growth on 5-FOA comparable to wild-type strains (Figure 5A). Use of an independent color-based telomeric-silencing assay also showed no defects for mutants (Figure S1). Combined with the lack of defects observed for mutants in both our assays (Figure 5A), the earlier observation that overexpression rescued ’s 5-FOA sensitivity (Figure 3A) likely results through an indirect mechanism. In contrast, when assayed for rDNA-silencing defects, mutants displayed a strong defect, similar to that observed in (Figure 5B). Together, these data suggest that Nab3 functions directly in rDNA silencing but not telomeric silencing.

Figure 5 

nab3 mutants display defects similar to esa1 mutants. (A) nab3 mutants display no defect in the telomeric 5-FOAS assay. WT (LPY4917), nab3-10 (LPY5407), esa1 (LPY4919), and sir2 (LPY4979) strains with a TELVR::URA3 reporter were plated on SC and 5-FOA. (B) nab3 mutants have an rDNA silencing defect. WT (LPY4909), esa1 (LPY4911), and nab3 (LPY5406) strains with the 25S rDNA::ADE2-CAN1 reporter were assayed for rDNA silencing defects on SC-Ade-Arg with and without 16 μg/ml canavanine. (C) The nab3 mutant is sensitive to the DNA-damaging agent camptothecin. WT (LPY5), esa1 (LPY4774), and nab3 (LPY10622) were plated on DMSO (control) and camptothecin (40 μg/ml) to test for drug sensitivity. (D) nab3 mutants display a G2/M block when grown at an elevated temperature. The same strains as in (C) were fixed and stained with propidium iodide to analyze cell-cycle profiles after being grown at 28° and shifted to 37° for 4 hr. (E) nab3 mutants have wild-type levels of global H4K5 acetylation. The same strains as in (C) and (D) were grown in YPD at 28° and shifted to 37° for 2 hr before whole-cell extract preparation. Samples were immunoblotted to detect global H4K5 acetylation levels. Compared with the H4K5 acetylation levels in esa1 mutants shown in Figure 3C, a more severe temperature challenge is shown here, accounting for the greater magnitude in decreased acetylation.

overexpression did not suppress the DNA damage and cell-cycle phenotypes of mutants (Figure 3, B and D). However, when mutants were examined for defects in DNA damage repair and cell-cycle progression, the results revealed a role for in these processes. As seen in Figure 5C, mutants are sensitive to the topoisomerase I inhibitor camptothecin, although less so than . Cell-cycle profiles of mutants also showed a G2/M block resembling that of mutants (Figure 5D). In addition to the defective rDNA silencing of , the identification of these phenotypes for mutants reveals a more extensive functional overlap with mutants.

We earlier considered the possibility that a molecular link for Nab3 and Esa1 functions would be that Nab3 influences histone acetylation (Figure 3C). When tested for changes in acetylation of H4K5, the primary in vivo target of Esa1 (Clarke ), global acetylation in mutants was maintained at wild-type levels (Figure 5E). Therefore, does not directly influence the global histone acetylation activity of Esa1’s primary target.

Localization and posttranslational acetylation of Nab3 are altered in esa1 mutants

The nucleolus is a key compartment for RNA processing in the nucleus. Ultrastructural analysis has shown mutants to have aberrant nucleoli (Clarke ), and mutants display strong rDNA-silencing defects and rDNA chromatin structure defects (Clarke ). Because of these connections of Esa1 to nucleolar function and Nab3′s influence on rDNA silencing (Figure 5B), Nab3 localization was visualized in mutants. Immunofluorescence was performed using an antibody directed against Nab3 in wild-type and strains. In addition, Sir2 staining was used to demarcate the nucleolus.

Nab3 localization has been previously described as dispersed throughout the nucleus but distinct from nucleolar structure proteins (Wilson ) (Figure 6A, top). At permissive temperatures, Nab3 localization appeared normal in both wild-type and cells. However, at restrictive temperature, Nab3 localization in became diffuse and no longer confined to the nucleus as defined by DAPI staining (Figure 6A, middle), indicating that Nab3 localization is altered in the mutant. Sir2 staining was also affected in the mutant and no longer found in discrete nucleolar and telomeric foci, although Sir2 protein expression appeared essentially normal at elevated temperature (Figure 4C). Nab3 protein levels were also found to be equal by immunoblot between wild-type and cells at both permissive and restrictive temperatures (Figure 6A, bottom).

Figure 6 

Nab3 localization and acetylation is altered in esa1 cells. (A) Nab3 localization is aberrant in the esa1 mutant. Top: At a permissive temperature (28°), Nab3 staining in wild type (LPY4909) and esa1 (LPY4911) cells appears as punctate nuclear foci interspersed with diffuse nuclear staining. Sir2 localization demarcates the nucleolus in a crescent shape (inset, green) and is normal. At a restrictive temperature (37°), Nab3 staining is diffuse in the esa1 mutant but appears normal in the wild-type strain. No Sir2 foci are observed in the esa1 mutant. Bottom: WT and esa1 strains used above were grown at permissive and elevated temperatures and used for immunoblots to detect total Nab3 levels using anti-Nab3. Anti-PGK1 (phosphoglycerate kinase) was used as a loading control. (B) Nab3 is acetylated in vivo in an ESA1-dependent manner. To detect posttranslational acetylation of Nab3, a WT (LPY15000) and esa1 (LPY15004) strain containing a chromosomal FLAG-tagged version of Nab3 were grown at an elevated temperature (37°) and used in an anti-FLAG immunoprecipitation followed by an immunoblot with anti-acetyl lysine. Decreased levels of Nab3 acetylation are observed in the esa1 mutant. Quantification of films from independent experiments shows a 48% decrease in Nab3 acetylation in esa1 compared with wild-type. An untagged WT strain (LPY5) is used as a negative control. Nab3-FLAG levels are not themselves altered in the esa1 mutant, as demonstrated by control immunoblotting of immunoprecipitations and inputs with anti-FLAG.

Because WT levels of Nab3 were observed in mutants, whereas simple overexpression suppressed phenotypes, we considered the possibility that in the mutants, Nab3 protein differs not quantitatively but qualitatively. One such qualitative difference could be at the level of its posttranslational modification. We tested the idea that Nab3 might itself be an in vivo substrate for Esa1, a possibility first raised by a proteomics survey suggesting that Esa1 could acetylate Nab3 in vitro (Lin ). To examine whether this modification occurs in vivo, an antibody that recognizes proteins with acetylated lysines was utilized. Immunoprecipitation of Nab3 followed by immunoblot detection with anti-acetyl-lysine revealed Nab3 to be acetylated in vivo (Figure 6B). To test whether Nab3 is a substrate for Esa1 acetylation, Nab3 acetylation levels were evaluated in an mutant. Since is essential, the temperature-sensitive mutant was grown at nonpermissive temperatures and samples were prepared. As expected if Esa1 acetylates Nab3 in vivo, a decrease in acetylated-Nab3 was observed in the mutant. Quantification of the anti-acetyl-lysine immunoblot shows that Nab3 acetylation was reduced in the mutant to 48% of the level observed in the wild-type strain. Thus, it appears that a fraction of the acetylation of Nab3 was -dependent, although our data did not distinguish whether this acetylation was by Esa1 on Nab3 directly as a target or indirectly through another acetyltransferase influenced by Esa1.

Discussion

Genetic suppression has provided a valuable tool for expanding the understanding of Esa1’s nuclear functions (Biswas ; Lin ; Chang and Pillus 2009; Scott and Pillus 2010) and, in this case, its interactions with the RNA binding protein Nab3. Increased dosage of was found to suppress a subset of mutant phenotypes, including temperature sensitivity and silencing defects. In addition, mutants shared overlapping phenotypes with mutants, displaying defects in rDNA silencing, cell-cycle progression, and the DNA damage response. Further strengthening these genetic interactions, nuclear localization and posttranslational acetylation of Nab3 were both altered in the mutant.

Nab3 is found in a complex with the RNA binding protein Nrd1 and the Sen1 helicase. This Nab3 complex ensures proper termination and 3′-end formation of many nonpolyadenylated transcripts, including snRNAs, snoRNAs, and CUTs (Steinmetz ; Arigo ; Thiebaut ). In addition, Nab3 physically associates with the nuclear exosome for processing and degradation of these transcripts (Vasiljeva and Buratowski 2006). Nab3 and Nrd1 form a heterodimer (Carroll ), and each protein has a different consensus RNA recognition sequence (Carroll ). Domain analysis suggests that both proteins bind RNA transcripts, whereas Nrd1 also physically associates with the C-terminal domain of Pol II (Conrad ). In accordance with these tightly linked functions of Nab3 and Nrd1, we found that overexpression of also suppresses some mutant phenotypes (Figure S2). Because genetic suppression by was less dramatic than that by , our focus in this study was on ’s genetic interaction with , but our observations with support the idea that suppression is mediated by Nab3 in the context of the Nab3-Nrd1-Sen1 complex, and not via an independent role of Nab3 alone.

Because the transcript was unchanged in the mutant (Figure 4A), this implies that the Nab3-Nrd1-Sen1 complex does not direct 3′-end termination of the transcript. It should be noted that our study was restricted to this loss-of-function mutation. Thus, considering the genetic limitations of studying essential genes such as , we cannot fully eliminate the possibility that the Nab3 complex processes the transcript, as we have not studied multiple mutant alleles of . However, we consider our in vivo data showing that Nab3 acetylation is influenced by Esa1 either directly or indirectly (Figure 6B) to provide a more likely explanation for the dosage suppression observed between and . Consistent with these data, one potential model for the suppression is that Esa1 acetylation of Nab3 influences its function such that the reduced Nab3 acetylation in mutants results in its reduced cell viability and defects in rDNA silencing. Thus, suppression of these defects is obtained in the mutant by overexpressing to compensate for the decreased pools of acetylated Nab3.

In S. cerevisiae, the rDNA is a repetitive array in the genome that is mainly transcribed by Pol I and Pol III. Reporter genes that are transcribed by Pol II and inserted in the array are known to undergo Sir2-mediated transcriptional silencing. An endogenous Pol II transcript has been detected in the “nontranscribed” spacer region (NTS1) of the rDNA. This transcript is a CUT that is processed by the Nab3 complex and degraded by the exosome (Houseley ; Vasiljeva ). In addition to uncovering an rDNA-silencing defect for mutants (Figure 5B), we observed that overexpression of rescued the rDNA-silencing defects of mutants (Figure 2). Esa1 binding is enriched at the rDNA, and histone acetylation at the rDNA is reduced in the mutant (Clarke ). Although Nab3 does not appear nucleolar by immunofluorescence (Wilson ) (Figure 6A), a recent study found that Nab3 localizes to the rDNA via chromatin immunoprecipitation (Lepore and Lafontaine 2011). Thus, one possibility is that Nab3 recruitment to the CUTs within the rDNA is regulated by its acetylation status through Esa1 activity. Future studies will establish how Esa1 functions with the Nab3-Nrd1 complex in contributing to transcriptional silencing at the rDNA.

The number of nonhistone proteins known to be acetylated by Esa1 and the MYST family of KATs has expanded in recent years. Several schools of thought exist about the function of this posttranslational modification. In parallel with the models for histone acetylation, acetylation of nonhistone proteins may change the activity of these proteins or may serve as a recruitment platform for physical binding of other proteins [reviewed in Sapountzi and Côté (2011)]. Our finding that Nab3 is acetylated in vivo raises several possibilities regarding the function of this posttranslational modification. Whereas Nab3 acetylation is reduced in an mutant, overall levels of Nab3 remain constant (Figure 6). Therefore, it is unlikely that acetylation affects Nab3 stability but, rather, that it influences its activity or function. Knowing that Nab3 is aberrantly localized in the mutant, one possible scenario is that acetylation of Nab3 by Esa1 promotes proper Nab3 nuclear localization.

In contrast to , the other three suppressors identified in our dosage-suppression screen (, , and ) are all involved in amino acid metabolism. A separate study defined the connections between and through DNA repair that could be distinguished from Lys20’s role in amino acid biosynthesis, potentially through a noncanonical role in acetylation (Scott and Pillus 2010). Recent findings report the prevalence of lysine acetylation as a posttranslational modification in the regulation of metabolic proteins in mammals (Zhao ). In light of these studies and ours, it is possible that Esa1 acetylates the protein products of the genes we identified as dosage suppressors. Only Nab3, and not the other suppressors, was identified as a substrate in the in vitro proteomics study (Lin ). However, a number of other metabolic enzymes were found, including the gluconeogenic enzyme Pck1 that is reciprocally deacetylated by Sir2, providing a link to our earlier suppression studies between and (Clarke ). One potential explanation for our current findings of dosage suppression of by and is that Leu2 and Vap1 are acetylated by Esa1 in vivo. Future studies to determine in vivo Esa1 targets of nonhistone proteins will shed light on additional substrates and their functions.

Although it has been assumed that Esa1’s catalytic activity is its essential activity, it is unclear exactly why strains are inviable. One recent study found that an strain bearing a mutation in a residue important for catalysis retained viability, proposing that there may be more to the essential nature of Esa1 than its histone acetyltransferase activity (Decker ). Given that our screen highlights a strong genetic interaction between and the essential gene , along with several genes encoding metabolic proteins (, , ), one of Esa1’s essential functions may be the recognition and acetylation of important nonhistone substrates.

Suppressor analysis is a widely used strategy that facilitates the identification of functional relationships between different proteins. A recent investigation of hundreds of dosage suppressors in yeast revealed that dosage suppression provides functional links between two genes (Magtanong ). In addition, dosage suppression can identify unique interactions that are not discovered through other types of genome-wide studies, such as protein-protein and synthetic sickness interactions. In our study, genetic suppression has provided an effective platform for identifying and characterizing potential new substrates for an enzyme primarily studied as an acetyltransferase targeting histones.