Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

Media.Rich (yeast extract-peptone-dextrose [YPD]), YP-glycerol (YPG), synthetic complete (SC), synthetic dextrose (SD), 5-fluoro-orotic acid (5-FOA), and sporulation media were prepared as described previously (62). Media lacking or containing 200 μM inositol were prepared with yeast nitrogen base that contained ammonium sulfate but lacked inositol (Q-Bio Systems). NaCl, LiCl, caffeine, and hydroxyurea were added to YPD medium to final concentrations of 1.4 M, 0.3 M, 15 mM, and 100 mM, respectively. 6-Azauracil (6AU) was added to SC-Ura medium to a final concentration of 50 μg/ml. G418 medium, for selection of yeast strains expressing the kanMX4 gene, was prepared as described previously (33).

Genetic methods.With the exception of O660 and KA102 to KA106, all strains (Table 1) are isogenic to FY2, a GAL2⁺ derivative of S288C (82). RKR1, BRE1, LGE1, DOT1, SET1, and SET2 disruptions were created by a PCR-based method using the HIS3-marked plasmid pRS303 and the kanMX4-marked plasmid pRS400 (2). In each case, disruptions were made in diploid strains and confirmed by PCR or Southern analysis. Haploid mutant progeny were obtained by tetrad dissection. All disruptions remove the entire ORF and replace it with the indicated selectable marker. Genetic crosses, tetrad analyses, and yeast transformations were performed by standard methods (20, 62).

Plasmids.Standard cloning techniques were used to construct all plasmids (2). pPC65, a pRS314 (71) derivative of a library plasmid (pPC62) that was obtained through complementation of the SL505 mutation, contains a 7,447-bp insert that includes tA(AGC)M2 (alanine tRNA), RKR1, and part of FAA4. pMB11 is identical to pPC65, except for the addition of the triple hemagglutinin (HA) epitope sequence (3XHA) at the amino terminus of Rkr1. To introduce the epitope tag, pSH3 was created by subcloning the XhoI/AatII fragment of pPC65 (containing the promoter/5′ end of RKR1) into pRS406 (71). Site-directed mutagenesis (Quikchange; Stratagene) was used to introduce an NdeI site at the ATG of RKR1 in pSH3, and a PCR fragment encoding the triple HA tag flanked by NdeI sites was inserted at this site. The XhoI/AatII fragment was then subcloned back into pPC65 to create pMB11. pMB66 was created by site-directed mutagenesis using pMB11 as a template to replace cysteine 1508 of Rkr1 with alanine. pMB11 and pMB66 were sequenced over the entire RKR1 ORF to ensure that no undesired mutations were introduced. pMB26, which contains HTA1, FLAG-htb1-K123R, and HIS3, was created by replacing a 737-bp XhoI/SphI fragment from a plasmid (54) containing FLAG-HTB1 with a 737-bp fragment containing htb1-K123R (54). The presence of FLAG-htb1-K123R in pMB26 was confirmed by DNA sequencing. Derivatives of pRS314 that contain HHT2 and myc-HHF2 (pNOY436) (35), wild-type HHT2 and HHF2 (pJH18) (28), or hht2-K4R and HHF2 (6) were described previously. pMB27, which contains SPT10 and TRP1, was created by ligating a 2,503-bp SpeI/XhoI fragment from pGN1101 (50) to SpeI/XhoI-digested pRS314 (71). pMB30 was created by ligating a 1,137-bp blunt-ended BanI/StuI fragment from pPC65 to SmaI-digested pGEX-3X (74) to generate a glutathione S-transferase (GST) fusion protein containing amino acids 1251 to 1562 of Rkr1. pMB81 was created by site-directed mutagenesis of pMB30, replacing cysteine 1508 of Rkr1 with an alanine. pMB81 was sequenced throughout the RKR1 ORF to ensure that no secondary mutations were created.

Identification of RKR1. RKR1 was identified in a previously described synthetic lethal screen with rtf1Δ (10). Determination of the identity of RKR1 was performed in the same manner as that of other genes identified in this screen. Briefly, RKR1 was cloned by complementation of the SL505 mutation identified in the synthetic lethal screen. Subcloning, linkage, and DNA sequencing analyses were performed to confirm that RKR1 was allelic to SL505. To determine the nature of the SL505 mutation, pPC62 was digested with BsrGI to delete nucleotides 1086 to 3468 of the RKR1 ORF. The unligated plasmid was transformed into a strain containing the SL505 mutation for gap repair (63). DNA sequencing showed that the SL505 mutation introduced a premature stop codon at position 1133 of the Rkr1 protein.

Sequence analysis.PROSITE (http://ca.expasy.org/prosite ) sequence analysis tools were used to predict the presence of any functional motifs or domains within the primary amino acid sequence of Rkr1. A RING domain is predicted at the carboxy terminus of Rkr1, consisting of amino acids 1508 to 1554. The sequence alignment shown in Fig. 2 was obtained from BLAST (http://www.ncbi.nlm.nih.gov/BLAST ) searches, with the S. cerevisiae Rkr1 protein sequence in its entirety as the query. Sequences of the most similar proteins from Homo sapiens (accession no. NP_056380.1 ; E value, 4e−41), Mus musculus (accession no. XP_982690.1 ; E value, 7e−42), Drosophila melanogaster (accession no. NP_730427 ; E value, 1e−21), Arabidopsis thaliana (accession no. NP_200649.1 ; E value, 2e−36), and Schizosaccharomyces pombe (accession no. CAA20765.1 ; E value, 3e−51) were aligned with the S. cerevisiae Rkr1 sequence by using the ClustalW program (http://www.ebi.ac.uk/clustalw ). The alignment of the last 150 amino acids of each protein was copied into Jalview (http://www.jalview.org ), and grayscale shading was added with a threshold of 50% sequence identity.

Growth assays.Saturated cultures of each strain were grown in the appropriate media. Cells were collected by centrifugation, and cell pellets were washed two times with sterile water. Tenfold serial dilutions (1 × 10⁸ cells/ml to 1 × 10⁴ cells/ml) were made in sterile water, and 2 or 3 μl of each dilution was spotted onto the appropriate media. Spots were allowed to dry and plates were incubated at 30°C for 3 to 5 days. YPD and YPG plates were used in every assay to ensure even spotting and to assay for petite cells, respectively.

Immunoblotting analysis.Transformed cells were grown under selective conditions to a density of approximately 4 × 10⁷ cells/ml. Whole-cell extracts were prepared by glass bead lysis in lysis buffer (100 mM sodium acetate, 20 mM HEPES [pH 7.4], 2 mM magnesium acetate, 10 mM EDTA, 10% glycerol, and 1 mM dithiothreitol DTT, plus protease inhibitors), as previously described (70). Proteins (20 or 25 μg extract per lane) were separated on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels for analysis of Rkr1 levels or on 15% SDS-polyacrylamide gels for analysis of histone levels and were transferred to nitrocellulose membranes. The following antibodies were used: anti-HA (1:3,000 dilution; Roche), anti-L3 (1:5,000 dilution) (81), anti-FLAG M2 (1:1,000; Sigma), anti-myc (1:500; Covance), and anti-H3 (1:2,000; Upstate). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Biosciences) were used at a 1:5,000 dilution. Immunoreactive proteins were detected by chemiluminescence (Perkin Elmer) and visualized with a Kodak 440CF digital imaging station.

Indirect immunofluorescence.Yeast strain FY632 was transformed with pPC65 (untagged RKR1) or pMB11 (3XHA-RKR1), and transformants were grown in SC medium lacking tryptophan to a density of approximately 1 × 10⁷ cells/ml. A total of 1.4 × 10⁸ cells were processed for indirect immunofluorescence, as described previously (77). The primary antibody, anti-HA (Roche), was added at a 1:3,000 dilution, and the secondary antibody, Cy3-conjugated goat anti-mouse immunoglobulin G (Alexa Fluor 488; Molecular Probes), was added at a 1:250 dilution. DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride) was used to stain the DNA, as described previously (60). Cells were visualized with an Olympus BX60 epifluorescence microscope and photographed with QED in vivo software.

RT-PCR analysis.Total RNA was prepared from yeast strains FY14, KY1170, and FY896 grown in YPD to approximately 1 × 10⁷ cell/ml and used for reverse transcription (RT)-PCR analysis, as described previously (26). RNA was DNase treated with the Turbo DNA-free kit (Ambion), and cDNA was prepared with 1 μg of RNA, 500 ng oligo(dT₂₀), and 200 units of SuperScript II reverse transcriptase (Invitrogen) in a 20-μl reaction. Two different amounts of cDNA (0.05 and 0.15 μg) were used in separate PCRs to ensure linearity. Experiments were performed in duplicate and analyzed by ethidium bromide staining of agarose gels.

Purification of recombinant proteins and in vitro ubiquitylation assays.BL21(DE3) cells were transformed separately with plasmids expressing wild-type (pMB30) or mutant (pMB81) GST-Rkr1 fusion proteins or GST alone (pGEX-3X). Cells were grown at 37°C and induced for 1.5 h at an optical density at 600 nm of 0.6 to 0.7 with 0.3 mM IPTG (isopropyl-β-d-thiogalactopyranoside). Cell lysates were made by sonication in a buffer containing 50 mM Tris-Cl, pH 8.0, and 1 mM EDTA, as described previously (11). Lysate (10 ml) was incubated for 1 h at 4°C with 1 ml prewashed GST-Sepharose beads in the presence of 1% Triton X-100 and protease inhibitors. Sepharose beads were pelleted and washed three times with phosphate-buffered saline (PBS) (pH 7.3) (2) plus 10% glycerol, 1% Triton X-100, and protease inhibitors. Fusion proteins were eluted in PBS plus 1% Triton X-100 and 50 mM glutathione for 10 min at room temperature.

Ubiquitylation assays were performed as previously described (19), with some modifications. Ubiquitin-activating enzyme (yeast recombinant Uba1), ubiquitin-conjugating enzyme (human recombinant UbcH5a), and ubiquitin (human recombinant) were purchased from Boston Biochem. One hundred nanograms of Uba1, 200 ng of UbcH5a, and ∼500 ng of GST fusion proteins were combined in reaction buffer (50 mM Tris-Cl [pH 7.5], 2 mM ATP, 2.5 mM MgCl₂, and 0.5 mM dithiothreitol) with 2.5 μg of ubiquitin in 30-μl reaction volumes. Reaction mixtures were incubated for 1.5 h at 30°C. Proteins were separated on a 7 to 20% gradient SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoblotting analysis was performed with antiubiquitin antibody (34) at a 1:50 dilution to detect ubiquitin-conjugated substrates and anti-GST antibody (Invitrogen) at a 1:500 dilution to detect and normalize the levels of GST fusion proteins. HRP-conjugated secondary antibodies (Amersham Biosciences) were used at 1:5,000 dilutions. Chemiluminescent signals (Perkin Elmer) were detected with a Kodak 440CF digital imaging station.

Identification of Rkr1.The Paf1 complex functions in transcription elongation, RNA 3′ end formation, and histone modification. Original support for a role of the Paf1 complex in transcription elongation came from a genetic screen for mutations that cause lethality in combination with an rtf1Δ mutation (10). This screen identified synthetic lethal mutations in CTK1, FCP1, and POB3, which encode an RNA Pol II CTD kinase, an RNA Pol II CTD phosphatase, and a component of the FACT transcription elongation complex, respectively. In addition to these well-characterized RNA Pol II transcription factors, this screen also identified a synthetic lethal mutation, the SL505 mutation, in a previously uncharacterized gene, YMR247c. YMR247c is a 4,686-bp ORF that is predicted to encode a protein of 1,562 amino acids. Linkage analysis confirmed that YMR247c contained the SL505 mutation, and DNA sequence analysis of a gap-repaired plasmid containing the YMR247c SL505 mutation showed that it led to a premature stop codon at amino acid 1133. Further analysis, which is described below, showed that truncation of this nonessential protein results in the loss of a functionally important RING domain at the carboxy terminus. We therefore decided to rename the gene RKR1, for “RING domain mutant killed by rtf1Δ.”

The results of the plasmid-based synthetic lethal screen were confirmed by tetrad analysis of spores derived from diploid strains that were doubly heterozygous for complete deletions of RTF1 and RKR1. No viable haploid rtf1Δ rkr1Δ strains were obtained after tetrad dissection (Fig. 1). This synthetic lethal interaction indicates that Rkr1 and Rtf1 regulate similar processes in yeast.

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FIG. 1.

Loss of RKR1 and RTF1 leads to synthetic lethality. Yeast strains of opposite mating types containing deletions of RKR1 (KY1172) or RTF1 (KY957) were mated and sporulated. Tetrads were dissected and incubated for 3 days at 30°C.

Rkr1 contains a conserved, functionally important RING domain.In an attempt to identify a cellular process that requires Rkr1, rkr1Δ strains were exposed to a wide range of phenotypic tests. We tested rkr1Δ mutants for the ability to grow on media lacking inositol or on media containing 6AU, caffeine, formamide, hydroxyurea, benomyl, sucrose, raffinose, glycerol, or high concentrations of salt (sodium chloride or lithium chloride). We also assayed for growth defects at 37° and 15°C on YPD. For most of these phenotypes, the rkr1Δ strains appeared to be similar to the wild type. However, we observed that rkr1Δ strains grow poorly on media lacking inositol, a phenotype associated with general defects in transcription (Fig. 2A) (22). This phenotype correlates well with the genetic interaction with RTF1, as defects in the Paf1 complex also cause inositol auxotrophy (4).

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FIG. 2.

Rkr1 contains a conserved RING domain that is essential for in vivo function. (A) An rkr1Δ strain (KY1168) was transformed separately with the TRP1-marked CEN/ARS plasmids pPC65 (untagged Rkr1), pMB11 (HA-Rkr1), pMB66 (HA-Rkr1-C1508A), and pRS314 (empty vector). Cultures were spotted as 10-fold serial dilutions onto media lacking tryptophan in the presence or absence of inositol (Ino) and grown at 30°C for 3 days. (B) Schematic representation of the Rkr1 protein sequence. The conserved RING domain is highlighted in gray. The position of the amino acid change encoded by the SL505 mutation is indicated by an asterisk. (C) Alignment of primary amino acid sequences of the carboxy-terminal 150 amino acids of Rkr1 homologs as determined by BLAST searches. The degree of shading correlates with amino acid identity among the six sequences listed. The residues that make up the RING domain are underlined. The critical cysteine and histidine residues within the RING domain are marked with asterisks. (D) Immunoblotting analysis of strains used in panel A shows that the wild-type and C1508A forms of Rkr1 are expressed equally. L3 levels served as a loading control.

In parallel with the phenotypic analyses, we performed several database searches in an effort to identify a cellular role for Rkr1. Blast analysis showed that the protein is conserved with other uncharacterized proteins of similar size in many eukaryotes, including humans. There is significant homology throughout the protein, but a region at the carboxy terminus is the most highly conserved. Sequence analysis indicated that a RING domain exists in this region between amino acids 1508 and 1554 (Fig. 2B and C). RING domains consist of eight critical amino acids, often seven cysteine residues and one histidine residue, which bind two zinc ions to form a cross-brace structure (reviewed in reference 32). The RING domain of Rkr1 is of the C4HC3 type and is the only recognizable domain or motif within the protein. To determine whether the RING domain is important for the in vivo function of Rkr1, we mutated the first cysteine of the RING domain, changing it to an alanine (Rkr1-C1508A). Similar substitutions have been shown to disrupt the functions of other RING domain-containing proteins (11, 80). Strains lacking the genomic copy of RKR1 were transformed with plasmids expressing wild-type and C1508A forms of Rkr1. Growth assays showed that strains expressing wild-type Rkr1 grow on media lacking inositol, while strains expressing Rkr1-C1508A grow as poorly as rkr1Δ strains (Fig. 2A). Furthermore, the Rkr1-C1508A derivative failed to complement the synthetic lethality between rtf1Δ and rkr1Δ (Table 2). Immunoblotting analysis demonstrated that the inability of Rkr1-C1508A to complement the rkr1Δ allele is not due to instability of the Rkr1 mutant protein (Fig. 2D). Together, these data demonstrate that the conserved RING domain of Rkr1 is required for the function of the protein in vivo.

Rkr1 is a nuclear protein.A large-scale study has been performed to determine the subcellular localization of all yeast proteins (29). This study involved the construction of carboxy-terminal green fluorescent protein (GFP) fusions for the majority of ORFs in yeast and microscopy to detect localization of the GFP signal. Rkr1 was not localized to any cellular compartment in this study, possibly because incorporation of the GFP tag at the carboxy terminus disrupted the structure and/or function of the RING domain. Database analyses did not reveal any cellular localization signals within the primary amino acid sequence of Rkr1. Therefore, we used indirect immunofluorescence to determine the cellular localization of Rkr1. To detect Rkr1, we constructed an amino-terminal HA epitope-tagged version of the protein. This tag does not appear to disrupt activity, as determined by complementation of the Ino⁻ phenotype of an rkr1Δ strain (Fig. 2A), and immunoblotting analysis showed that HA-Rkr1 migrates in denaturing gels at its predicted molecular mass of approximately 180 kDa (Fig. 2D). Indirect immunofluorescence experiments showed that HA-Rkr1 is localized to the nucleus (Fig. 3).

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FIG. 3.

HA-Rkr1 localizes to the nucleus. Indirect immunofluorescence experiments were performed with transformants of a wild-type strain (FY632) expressing HA-Rkr1 (A and B) or untagged Rkr1 (C and D). Localization of the triple HA epitope was detected with Cy3-coupled secondary antibody (A and C), and nuclear and mitochondrial DNAs were detected with DAPI stain (B and D). HA-Rkr1 and untagged Rkr1 were expressed from the CEN/ARS plasmids pMB11 and pPC65, respectively.

Genetic interactions suggest that Rkr1 is functionally connected to chromatin modification.The synthetic lethal relationship between rkr1Δ and rtf1Δ suggests that Rkr1 and Rtf1 regulate a common process within the cell. We undertook a genetic approach to begin to identify this process. We first wanted to learn whether the genetic interaction between RKR1 and RTF1 extended to other members of the Paf1 complex, Paf1, Ctr9, Cdc73, and Leo1. Interestingly, although rkr1Δ paf1Δ and rkr1Δ ctr9Δ double mutants grow very slowly, only rkr1Δ rtf1Δ double mutants are unviable (Table 2). This result is particularly striking because deletion of PAF1 or CTR9 generally causes stronger mutant phenotypes than deletion of any other member of the Paf1 complex, including RTF1 (4, 76). Therefore, the synthetic lethality between RKR1 and RTF1 most likely relates to a function of the Paf1 complex that is primarily carried out by Rtf1, potentially histone H2B ubiquitylation or histone H3 K4 or K79 methylation. The Paf1 complex physically and genetically interacts with several transcription elongation factors, including Spt4-Spt5 and Spt16-Pob3 (yFACT) (76). To determine whether Rkr1 is involved in transcription elongation, we performed genetic crosses with rkr1Δ strains and strains containing mutations in genes that encode transcription elongation factors, including SPT4, SPT5, SPT6, SPT16, and PPR2, which encodes TFIIS. While we observed several enhanced mutant phenotypes in the double mutant strains, we found no severe genetic interactions to indicate that Rkr1 is solely or primarily involved in transcription elongation (Table 2).

To better define why Rtf1 is required for viability when Rkr1 is absent, we took advantage of a set of rtf1 internal deletion mutations that disrupt specific functions of the protein (M. H. Warner, K. L. Roinick, and K. M. Arndt, submitted for publication). Discrete regions of Rtf1 are important for its association with actively transcribed genes, interactions with other members of the Paf1 complex, and posttranslational histone modifications. We crossed strains lacking RKR1 to the different rtf1 deletion mutants and found that the rtf1Δ3 and rtf1Δ4 mutations, which together eliminate amino acids 62 to 152 from the Rtf1 protein, show a strong genetic interaction with rkr1Δ (Table 3 and Fig. 4A). Specifically, the rkr1Δ rtf1Δ3 and rkr1Δ rtf1Δ4 double mutants grow very poorly on minimal synthetic dextrose medium. Notably, deletion of amino acids 62 to 152 in Rtf1 eliminates histone H2B ubiquitylation and histone H3 K4 and K79 methylation (Warner et al., submitted).

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FIG. 4.

rkr1Δ exhibits synthetic growth defects with mutations that eliminate histone H2B K123 ubiquitylation and histone H3 K4 methylation. (A) rkr1Δ rtf1Δ3 strains exhibit the SD⁻ phenotype seen with many double mutants. Cultures were spotted as 10-fold serial dilutions onto SC or SD medium and grown at 30°C for 3 days. (B) Plasmids expressing FLAG-HTB1 or FLAG-htb1-K123R were introduced by plasmid shuffle into wild-type (FY406), rtf1Δ (KY982), and rkr1Δ (KY981) strains lacking both genomic copies of HTB. Tenfold serial dilutions of cultures were spotted onto medium lacking or containing 5-FOA and incubated at 30°C for 3 days. (C) Plasmids expressing HHT2 or hht2-K4R were introduced by plasmid shuffle into wild-type (FY1990) or rkr1Δ (KY1064) strains. 5-FOA^R colonies were obtained and analyzed for growth on SD or SC complete medium as described for panel A.

To investigate further a potential connection between RKR1 and posttranslational histone modifications, we crossed rkr1Δ strains with strains lacking specific histone-modifying enzymes. Rad6 is the ubiquitin-conjugating enzyme and Bre1, in association with Lge1, is the ubiquitin-protein ligase required for histone H2B K123 ubiquitylation (31, 61, 83). Set1, Set2, and Dot1 are the methyltransferases responsible for methylating K4, K36, and K79 of histone H3, respectively (18, 66, 78). The Paf1 complex is required for each of these modifications (36, 38, 52, 53, 84). Interestingly, rkr1Δ rad6Δ double mutants exhibit a strong synthetic growth defect (Table 3). Further genetic analysis using strains lacking both RKR1 and the H2B ubiquitylation site (htb1-K123R) indicates that Rkr1 is important for cell growth in the absence of H2B ubiquitylation (Fig. 4B). Moreover, rkr1Δ strains lacking BRE1, LGE1, or SET1 grow very poorly on SD medium, similar to the rtf1Δ3 or rtf1Δ4 genetic interactions (Table 3). No strong genetic interactions were observed with dot1Δ and set2Δ strains. Defective growth on SD medium was also observed for an rkr1-C1508A set1Δ double mutant strain, indicating a requirement for the Rkr1 RING domain when Set1 is absent (Table 3). Because Set1 has been shown to methylate substrates other than histone H3 K4 (87), we examined the phenotype of an rkr1Δ strain in which histone H3 K4 could not be methylated (hht2-K4R). Like an rkr1Δ set1Δ strain, the rkr1Δ hht2-K4R strain grows poorly on SD medium (Fig. 4C), suggesting that the synthetic phenotype between rkr1Δ and set1Δ is due to a lack of histone H3 K4 methylation. While deletion of RKR1 causes significant growth defects in strains defective for histone H2B K123 ubiquitylation or H3 K4 methylation, Rkr1 itself does not appear to affect these modifications. Histone H3 K4 trimethylation and K79 dimethylation as well as histone H2B K123 ubiquitylation occur at wild-type levels in strains lacking RKR1 (data not shown). Taken together, our findings suggest that Rkr1 acts in parallel with Rtf1-dependent histone modifications.

Strains lacking RKR1 have defects in telomeric silencing.Transcriptional silencing of genes that are positioned near telomeres requires the proper modification of histones. Telomeric chromatin is enriched in hypoacetylated and hypomethylated histones, which provide interaction sites for the Sir proteins (reviewed in reference 64). Strains lacking Paf1 complex members exhibit defects in telomeric silencing (36, 52), most likely because the genome-wide loss of histone H3 methylation leads to the redistribution of Sir proteins away from their normal sites of action (65). To determine whether Rkr1 is important for telomeric silencing, wild-type and rkr1Δ strains, both containing a telomeric URA3 reporter gene, were plated to medium containing 5-FOA. Wild-type strains grow robustly on this medium, indicating silencing of the telomeric URA3 gene (Fig. 5). However, strains lacking RKR1 grow poorly on the 5-FOA medium, suggesting that telomeric silencing is disrupted in the rkr1Δ strains (Fig. 5). In contrast, transcriptional silencing at the ribosomal DNA and silent mating-type loci occur normally in rkr1Δ strains (data not shown). A role in telomeric silencing is consistent with the idea that Rkr1 modulates chromatin structure or function.

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FIG. 5.

Strains lacking RKR1 have defects in telomeric silencing. RKR1 (O660) and rkr1Δ (KA102 and KA103) strains were spotted in 10-fold serial dilutions onto medium containing or lacking 5-FOA. KA102 and KA103 are two independent rkr1Δ strains that contain the TELVR::URA3 telomeric silencing marker.

RKR1 genetically interacts with SPT10, a gene encoding a potential histone acetyltransferase. SPT10 is one of many SPT genes that were originally identified in a screen for suppressors of transposable element insertion mutations in yeast (17, 50). Strains lacking Spt10 exhibit global changes in chromatin structure and gene expression (14, 86). Spt10 and its interacting partner Spt21 bind to the promoters of histone genes and activate their transcription, providing a potential explanation for the broad transcriptional effects of spt10 mutations (13, 14, 25, 86). Interestingly, Spt10 contains a predicted acetyltransferase domain (51) that is required for its transcription activation activity (25), and spt10 mutants have reduced histone H3 K56 acetylation at histone gene promoters (86). However, direct acetylation of histones by Spt10 has yet to be demonstrated (25, 86), and a recent study indicates that global levels of histone H3 K56 acetylation are greatly reduced in strains lacking Rtt109 or Asf1, but not Spt10 (67). A synthetic lethal screen involving spt10Δ identified mutations in RKR1 (D. Hess and F. Winston, personal communication). We confirmed this result by tetrad analysis using complete deletions of RKR1 and SPT10 (Fig. 6A).

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FIG. 6.

RKR1 genetically interacts with SPT10. (A) Loss of RKR1 and SPT10 results in synthetic lethality. Strains of opposite mating types containing deletions of RKR1 (KY1169) or SPT10 (FY896) were mated and sporulated. Tetrads were dissected and incubated at 30°C for 5 days. Growth differences among spt10Δ single mutants are most likely due to spontaneous secondary mutations that suppress the growth defects of the spt10Δ strain (J. Chang and F. Winston, personal communication). (B) RT-PCR analysis of histone gene transcription. Total RNA was isolated from wild-type (FY14), rkr1Δ (KY1170), or spt10Δ (FY896) strains and used for RT-PCR analysis. Two different amounts of cDNA (1× and 3×) were used in the reactions to demonstrate linearity of the PCR. Reactions programmed with RNA confirmed that the RNA preparations used for cDNA synthesis were free of genomic DNA. Results shown are representative of those obtained in two independent experiments. (C) Immunoblot analysis of histone protein levels. For detection of histones H2B and H3, wild-type (FY406) and rkr1Δ (KY981) strains were transformed with plasmids that express untagged or FLAG-tagged H2B (54). For detection of histone H4, wild-type (FY1990) and rkr1Δ (KY1064) strains were transformed with plasmids that express untagged (pJH18) or myc-tagged H4 (pNOY436). L3 served as a loading control for the FLAG-H2B immunoblot, and cross-reacting bands, marked by asterisks, served as loading controls for the myc-H4 and total H3 immunoblots. (D) rkr1Δ is not synthetically lethal with a mutation that alters K56 of histone H3. TRP1-marked CEN/ARS plasmids expressing HHT1 or hht1-K56R were introduced by plasmid shuffle into RKR1 (FY1990) or rkr1Δ (KY1064) strains that lacked both genomic copies of HHT. Cultures were serially diluted and spotted onto YPD and synthetic medium containing 5-FOA. Plates were incubated at 30°C for 3 days. (E) Strains lacking SPT10 have defects in telomeric silencing. Tenfold serial dilutions of SPT10 (KA104) or spt10Δ (KA106 and KA105) cultures were spotted onto medium containing or lacking 5-FOA. Plates were incubated at 30°C for 5 days. KA106 and KA105 are independent strains that contain the TELVR::URA3 telomeric silencing marker and spt10Δ.

Because Spt10 has a well-studied role in histone gene transcription, we measured histone mRNA levels in an rkr1Δ strain using RT-PCR analysis and oligonucleotide primers that distinguish among the highly related histone genes (25). We found that all of the histone genes are expressed at nearly wild-type levels in the rkr1Δ strain (Fig. 6B), while HTA2, HTB2, and HHF2 mRNA levels are significantly decreased in the spt10Δ strain, as expected from earlier studies (14, 25). We confirmed by immunoblotting analysis that histone H2B, H3, and H4 levels are unaffected by the rkr1Δ mutation (Fig. 6C) (H2A levels were not tested). Therefore, the inviability of rkr1Δ spt10Δ double mutant strains is most likely not due to insufficient histone gene expression. Consistent with this idea, rkr1Δ spt21Δ double mutants are viable (Table 3), even though spt21Δ and spt10Δ mutations both reduce histone mRNA levels (13, 25).

To determine whether Rkr1 is essential in the absence of Spt10 because histone H3 K56 acetylation is defective, we performed a plasmid shuffle experiment with RKR1⁺ and rkr1Δ strains that lacked both chromosomal copies of the genes for histones H3 and H4 and carried a URA3-marked HHT2-HHF2 CEN/ARS plasmid. Cells were transformed with a TRP1-marked plasmid that expressed a histone H3 derivative in which K56 was replaced with arginine (43). Following growth on synthetic medium containing 5-FOA, the histone H3 K56R derivative was the only version of histone H3 available in the cell. The results of the plasmid shuffle revealed that rkr1Δ strains grow as well as RKR1⁺ strains in the presence of the histone H3 K56R derivative (Fig. 6D). In addition, histone H3 K56 acetylation occurs at wild-type levels in rkr1Δ cells (data not shown). Therefore, the basis for the synthetic lethality between rkr1Δ and spt10Δ remains unclear but appears not to be related to the proposed role of Spt10 in histone H3 K56 acetylation (86).

Since the loss of Rkr1 or Rtf1 alleviates telomeric silencing, we wanted to determine whether the loss of SPT10 also causes this phenotype. Strains lacking SPT10 and containing the telomeric URA3 reporter gene were constructed and plated on 5-FOA medium to monitor URA3 expression. Consistent with a role for Spt10 in chromatin structure or function, the spt10Δ strains grow extremely poorly in the presence of 5-FOA, indicating a strong defect in telomeric silencing (Fig. 6E).

The RING domain of Rkr1 possesses ubiquitin-protein ligase activity.Proteins that contain a RING domain often possess ubiquitin-protein ligase activity (16). Within the ubiquitylation pathway, RING domain ubiquitin-protein ligases are thought to bring the ubiquitin-conjugating enzyme and substrate together to facilitate the transfer of ubiquitin to the substrate (16). Since the only identified domain or motif in Rkr1 is the carboxy-terminal RING domain, we determined whether Rkr1 possesses ubiquitin-protein ligase activity. Because attempts to express full-length, recombinant Rkr1 were unsuccessful, GST fusions to the carboxy terminus of Rkr1 (amino acids 1251 to 1562) were constructed. Wild-type Rkr1 and Rkr1-C1508A GST fusions, as well as GST alone, were purified from bacteria. In vitro ubiquitin-protein ligase assays were performed with recombinant ubiquitin, ubiquitin-activating enzyme (yeast Uba1), ubiquitin-conjugating enzyme (human UbcH5a), and GST-RING proteins. These conditions have been used previously to show that other RING domain-containing proteins can facilitate polyubiquitylation (19). Reactions lacking individual components of the ubiquitylation pathway were performed to show that all components of the pathway are required for efficient ubiquitylation. The results show that the RING domain of Rkr1 has ubiquitin-protein ligase activity that is dependent on the presence of the first cysteine residue in the RING motif (Fig. 7). Neither the Rkr1-C1508A fusion protein nor GST yielded detectable levels of protein ubiquitylation. These data suggest that Rkr1 may also act as a ubiquitin-protein ligase in vivo. Combined with previous data, we propose a model in which the ubiquitin-protein ligase activity of Rkr1 targets proteins important for transcription and/or chromatin function.

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FIG. 7.

The RING domain of Rkr1 possesses ubiquitin-protein ligase activity in vitro. Purified recombinant ubiquitin-activating enzyme (Uba1), ubiquitin-conjugating enzyme (UbcH5a), or GST-RING fusion proteins were combined with recombinant ubiquitin. Reactions contained GST fusions to the wild-type Rkr1 RING domain (lanes 2 to 4), the Rkr1-C1508A RING domain (lanes 5 to 7), or GST alone (lane 8). Proteins were separated by SDS-polyacrylamide gel electrophoresis analysis, and immunoblotting was performed with antiubiquitin antibody to detect ubiquitylated substrates or anti-GST antibody to detect GST-RING fusions. Numbers on the left indicate protein molecular mass standards, in kilodaltons. All GST proteins migrated at predicted molecular masses. The GST-only reaction lane originated from the same gel but was aligned with reactions containing GST fusion products for presentation purposes.