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A RAP 1-interacting protein involved in transcriptional silencing and telomere length regulation

Christopher F.J. Hardy/ Lori Sussel, and David Shore^ Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032 USA

The yeast RAPl protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAPl is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(Ci_3A) sequences. Previous studies have indicated that the regulatory function of RAPl is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAPl-interacting factor (RIFl). Strains carrying gene disruptions of RIFl grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rapl" mutants. Furthermore, hybrid proteins containing rapl^ missense mutations are defective in an interaction with RIFl in the two-hybrid system. Taken together, these data support the idea that the rapT phenotypes are attributable to a failure to recruit RIFl to silencers and telomeres and suggest that RIFl is a cofactor or mediator for RAPl in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIFl that partially restores the interaction with rapl** mutant proteins. [Key Words: Transcriptional silencing; telomere structure; Saccharomyces cerevisiae-, RAPl; protein-protein interactions; two-hybrid system] Received lanuary 13, 1992; revised version accepted March 4, 1992.

Repressor/activator protein 1 (RAPl) is an essential reg­ rapV mutants have longer poly(Ci_3A) tracts, the length­ ulatory protein in yeast whose DNA-binding sites are ening effect being proportional to the strength of the si­ found at three types of chromosomal elements: promot­ lencing defect (Sussel and Shore 1991). ers, silencers, and telomeres. Analyses of rapl mutants Several observations suggest that the opposite regula­ have revealed that the protein acts at all three types of tory functions of RAPl are not intrinsic to its binding loci. Temperature-sensitive lethal mutations in RAPl sites but, instead, result from interactions with different [lapl^^] are defective in transcriptional activation, an ap­ factors at promoters and silencers. For example, the par­ parently essential function of RAPl (Giesman et al. ticular DNA sequence of a RAPl-binding site does not 1991; Kurtz and Shore 1991). Although one lapl"^ strain determine its regulatory function: Promoter-derived is also partially defective in silencing (Kurtz and Shore binding sites function in place of a normal silencer site 1991), the repression function of RAPl can be genetically and vice versa (Brand et al. 1987; Shore and Nasmyth separated from essential activation functions, as demon­ 1987; Buchman et al. 1988b). Furthermore, silencers and strated by the isolation of several viable mutants {rapl''] promoters containing RAPl-binding sites typically ap­ that are specifically defective in transcriptional silencing pear to be complex regulatory sites in which at least one (Sussel and Shore 1991). Both types of rapl mutants other regulatory element is required for either proper re­ [rapl'''' and rapT^] also display changes in the poly(C]_3A) pression or activation (Rotenberg and Woolford 1986; sequences at telomeres, which contain RAPl-binding Brand et al. 1987; Kimmerly et al. 1988; Stanway et al. sites approximately every 40 bp (Longtine et al. 1989). 1989). Several candidates exist for RAPl-interacting fac­ When grown at semipermissive temperatures, where tors at silencers (e.g., SIR1-SIR4 and ABFl) [Rine and RAPl function is limiting, rapV mutants undergo a pro­ Herskowitz 1987; Shore et al. 1987; Buchman et al. gressive loss of poly(Ci_3A) sequences (Conrad et al. 1988a; Diffley and Stillman 1988) and at promoters (e.g., 1990; Lustig et al. 1990). Conversely, silencing-defective GCRl or GAL11/SPT13) (Fassler and Winston 1989; Nishizawa 1990; Santangelo and Tomow 1990). No bio­ chemical or genetic data, however, directly indicate that ^Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, any of these proteins physically interacts with RAPl. New York 11724 USA. ^Corresponding author. Recently, we have shown that high-level expression of

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Hardy et al. the carboxyl terminus of RAPl fused to the GAL4 DNA- utilizes the separable domains of the transcriptional ac­ binding domain (GBJ^,) causes a dominant-negative effect tivator GAL4, its amino-terminal DNA-binding domain on silencing; leading to the derepression of normally si­ (GBJ3/ amino acids 1-147), and its carboxy-terminal tran­ lent genes at the HMR locus (Hardy et al. 1992). Over- scriptional activation domain (GAD, amino acids 768- expression of the carboxyl terminus of RAPl also results 881). Fields and co-workers showed that coexpression in in telomere elongation (Conrad et al. 1990; Hardy 1991), yeast of two interacting proteins (X and Y) as GBQ/X and another phenotype associated with rapl'' mutants. Dere­ GAD/Y hybrids resulted in the activation of a GALl- pression by Gj, 1^3/RAPl hybrids occurs only when they lacZ reporter gene. Activation occurs presumably by a are expressed from a strong promoter on multicopy plas- protein-protein interaction between the X and Y por­ mids and not when expressed from the RAPl promoter tions of the two hybrids that tethers the activation do­ in single copy. Furthermore, this dominant-negative ef­ main to the promoter. We have adapted the two-hybrid fect is not dependent on a DNA-bmdmg site to target the screen to isolate RAP I-interacting proteins by starting hybrids to the silencer. We reasoned, therefore, that dere­ with a GALl-lacZ reporter strain expressing the G^j^/ pression results from the titration of limiting silencer RAPl(653-827) hybrid. This strain was then transformed factors. The part of RAPl responsible for this dominant- with a library of plasmids in which yeast genomic frag­ negative effect on silencing was mapped to a region from ments (produced by partial digestion with the enzyme amino acid 667 to the carboxyl terminus (amino acid Sau3A] were fused to sequences encoding the SV40 T 827), the same region of the protein to which the silenc- antigen nuclear localization signal followed by the G^D ing-defective rapP mutants map. Our results indicated (a generous gift of P. Bartel and S. Fields). Approximately that this putative titrated factor is probably neither 120,000 transformants were replica plated onto X-gal RAPl itself nor the other silencer binding factor ABFl plates, and 11 blue colonies were identified and purified (Hardy et al. 19921, (for details, see Materials and methods). Potential acti­ On the basis of these results, we have used a novel vation domain hybrids encoded on I£ (72-containing genetic system (Fields and Song 1989; Chien et al. 19911 plasmids were isolated from these cells and transformed to identify a protein that interacts with the RAPl car­ back into two different reporter strains, one with and one boxyl terminus. We report the cloning and characteriza­ without the GBU/RAP1(653-827) hybrid plasmid. Of the tion of a gene encoding such a protein, designated RIFl 11 plasmids, 10 activated in both reporter strains and, by (for RAPl-interacting factor 1). Strains carrying disrup­ restriction mapping, appeared to contain all or portions tions of the RIFl gene are defective in silencing and have of the GAL4 gene. elongated poly(Ci_3A) tracts at telomeres, both pheno- A single G^D plasmid was found to confer GB^/ types characteristic of rapV mutants. By use of the two- RAPI(653-827)-dependent activation of the reporter gene hybrid system, we have shown that the region of RAPl (Fig. 1). Activation requires the RAPl portion of the required for the silencer derepression effect (amino acids Ggj-j/RAPl hybrid; no activation is observed when the 667-827) is also necessary for the RAPl-RIFl protein- plasmid is transformed into a strain expressing the GBD protein interaction. Furthermore, hybrids made with the alone. The hybrid is therefore not targeted to the reporter different rapP mutants display a defect m interacting gene through a direct Ggo interaction but, instead, en­ with RIFl that is proportional to the corresponding rapi- codes a polypeptide that interacts with RAPI(653-827), silencing defects. These results suggest that one function which will be referred to hereafter as RIFl. Subsequent of RAPl, defective m the rapP mutants, is to direct the analysis (see below) indicated that the plasmid contained binding of RIFl to silencers and telomeres. Interestingly, a carboxy-terminal region of the RIFl gene fused in- two rapP hybrids that appear to be completely unable to frame to G^D'Coding sequences. To demonstrate that interact with RIFl in the two-hybrid system still lead to the activation effect required the production of a GAD/ silencer derepression when overexpressed, suggesting RIFl fusion protein, the following experiments were that the RAPl carboxyl terminus may also interact with done. First, a frameshift mutation was created between other factors at silencers and telomeres not yet uniden­ GAD ^^^ RIFl sequences (at a BamHl site) to truncate tified. the fusion protein just beyond the G^D sequences. This frameshift mutation in the GAD/RIFI hybrid abolishes activation, demonstrating that the RIFl portion of the Results hybrid is required for activation of the GALl-lacZ re­ porter gene (Fig. 1), However, two downstream frame- Isolation of a RAPI-interacting piotein shift mutations, at a unique Ncol site and a distal BamHl by the two-hybrid system site, had no effect on activation by the GAD/RIFI plas­ Expression of Ggj^^/RAPl hybrids from a strong promoter mid, indicating that any coding sequences beyond these results in derepression of the silent HMR locus, presum­ two sites are not necessary for the interaction. To deter­ ably as the result of protein-protein interactions that mine the 3' end point of RIFl sequences required for either sequester or titrate another factor important for activation, two other mutations in the insert DNA were silencing (Hardy et al. 1992). In an attempt to identify constructed. Deletion of sequences 3' to an Xbal site this putative RIF, we have used the two-hybrid system, a located —500 bp beyond the fusion junction abolished genetic method to identify protein-protein interactions activation, whereas deletion of sequences beyond the dis­ (Fields and Song 1989; Chien et al. 1991). This method tal BamHl site had no effect (Fig. 1). These data indicate

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RAPl mediator at silencers and telomeres

Bam(P) Xba Ncol Bam(D) Isolation and DNA sequence of the RIFl gene 3 II Using the proximal BamHl-Xbal RIFl fragment to probe GAD RIF1... a yeast genomic library, a large overlapping clone of ~8.0 500 bp kb was obtained. The DNA sequence of 6.5 kb of this fragment was determined and is shown in Figure 2. The sequence contains a single large open reading frame that DNA Binding Activation ^gal Units Domain Hybrid Domain Hybrid could encode a protein of 1916 amino acids with a mo­ lecular mass of 219 kD. Although this open reading frame contains the splicing signal 5'-TACTAAC-3' (at GBD <1 nucleotide 4947), we found no nearby match to the con­ GBD/RAP! <1 served 5' splice site sequence (5'-GTATGT-3') (Guthrie 1991 and references therein). Northern analysis of both GBD/RAPI GAD/RIF! 616 total and poly(A)-selected RNA shows that this 8.0-kb GAD/R'FI <1 fragment hybridizes to an ~6.3-kb RNA, consistent with GBD GAD/RIFI <1 the 5.8-kb open reading frame present in the fragment (data not shown). The RIFl sequence present in the GAD^ GBD/RAPI GAD/R'f1-Bam(P)fs <1 RIFl hybrid begins at amino acid 1614 of the predicted protein, indicating that a carboxy-terminal portion of GBD/RAPI GAD/RIF1-NCOI fs 681 RIFl is sufficient to interact with RAPL We failed to GBD/RAPI GA[yRtF1-Bam{D)fs 750 find any strong similarities between the predicted RIFl protein and sequences present in available protein data GBD/RAP1 GA[yRIF1{Bam(P)-Xbal) <1 bases (for details, see Material and methods), and the GBD^RAPI G AiyRIFI (Bam-Bam) 770 protein sequence does not appear to indicate any distinc­ tive structural features. Interestingly, there is a single GBD/Rlf^l (Bam-Bam) <1 Mlul site (ACGCGT) in the 6.5-kb sequence (at nucle­ otide 262), -200 bp upstream of the putative initiator GBD/RAPI RIF1 2nm <1 ATG of RIFl. Mlul sites are often found within the pro­ moters of genes whose mRNA levels increase at the Gj/ Figure 1. Isolation of a plasmid encoding a RAPl-interacting S-phase boundary (Andrews and Herskowitz 1990). Sev­ protein using the two-hybrid system. A plasmid (GAU/RIFI) was isolated from a library of fusions in the expression vector eral genes encoding DNA replication proteins contain pGAD2 on the basis of its ability to activate a GALl-lacZ re­ one or two Mlul sites in their upstream regions, and porter gene in the presence of the GB]3/RAP(653-827) hybrid (see these sites appear to be at least in part responsible for Material and methods). A partial restriction map of GAD/RIFI is their cell cycle-regulated transcription (Pizzagalli et al. shown above. Quantitative p-galactosidase assays were con­ 1988; Bauer and Burgers 1990; Brill and Stillman 1991; ducted on a variety of strains transformed with the designated Lowndes et al. 1991). plasmids, and the average of four independent transformants is given. All the strains contain 2-|JLm-based high copy number plasmids. The GBD hybrids are expressed from the strong ADHl promoter, whereas the G^^ hybrids are expressed from a cryptic Disruption of the RIFl gene affects silencing promoter, perhaps lying within the ADHl terminator fragment. To examine directly the in vivo function of RIFl, the Gj^j^/Rl¥[Bam{?]-Bam{'D]\ and Gj^T:)/Kl¥[Bam{?]-Xba\] contain the corresponding restriction fragments from the original G^o/ cloned gene was used to construct a disruption of the RIFl isolate cloned into the pGAD2 vector (see Materials and chromosomal copy. An Mlul-Xbal fragment of RIFl (Fig. methods). RIFl 2 ^JLm contains the full-length RIFl gene, includ­ 2, base pairs 262-5685) was deleted and replaced by a ing putative upstream promoter sequences, on a 2-fjLm-based fragment containing the URA3 gene. This results in de­ L£(72-containing plasmid. letion of >90% of the predicted RIFl open reading frame and removes all of the amino-terminal coding sequences. Initially, a ura3 ~ homozygous diploid was transformed that the RIFl sequences necessary and sufficient for an with this construct. Ura"^ transformants were selected interaction with the RAPl carboxyl terminus are located and screened by Southern blotting for those in which a upstream of the Ncol site and that the sequences con­ single copy of the gene had been replaced by the tained on the small BamHl-Xbal fragment are not suf­ nfl::URA3 deletion/substitution. Four different isolates ficient for this interaction. Activation by GA^/RIFI is with the appropriate structure were sporulated, and most also dependent on the G^D portion of the hybrid because tetrads yielded four viable spores with URA3 segregating a GBD/RIFI hybrid; which targets the carboxy-terminal 2 : 2, indicating that RIFl is not an essential gene (data part of RIFl from the original G^D/RIFI hybrid to the not shown). The growth of hfl::URA3 cells on YEPD reporter gene, does not result in activation (Fig. 1). Fur­ plates is indistinguishable from isogenic RIFl cells, in­ thermore, overexpression of full-length RIFl (from a dicating that RIFl is not required for any essential tran­ 2-jjLm plasmid containing the complete gene) in the pres­ scriptional activation functions mediated by RAPl. ence of the GBD/RAP1(653-827) hybrid does not result in To determine whether hfl::URA3 mutants are defec­ activation of the reporter gene (Fig. 1; RIFl 2 |xm). tive in silencing, the effects of the RIFl disruption at the

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AAAAGTATGCAAAATTAACOCT*TCAGCCAAGTATAACGAACTGCTGTAATCCTTTACAAATCGTTAAAAAAGGGCCGCAGC*AATATTTATATAGTAAOGACAGCTATCTTAGATTTAC 10 30 50 70 90 110

ATCGTOAATATACTCTACTTTTGATAAGAAACAAGAAGTCAACAGAAGGCAGGTGATTAGTGCCATTTTGATTTTCAGTTCTTTGTGTTTTTCCTCTTATGTCATATTTACTACAATAAT 130 1B0 170 190 210 230

AGAAGTTACTATAATTGACCGACGCGTCGATTTTTTATTTATTGGCGACTCATCGTTAGCAAGAAGATCGAAAGTAATAACGCAGTCTTAGAGCGATTTGAGGCAATTCGTCGGCATCAQ 250 270 290 310 330 3B0 MSKDFSDKK TACCTAAOGAGATAAAAGTGAATATATATATATATATTCGTCAACGTTATTACAGGATTGCCATTGCAAAATCGTTTTTGTGGTCAATTTGCAATGTCGAAAGATTTTTCAGATAAAAAQ 370 390 410 430 450 470 KHTIDRIDQHILRRSQHDNYSNGSSPWMKTNLPPPSPQAH AAACATACGATAGATCGAATTGATCAGCATATCCTCCGTAGGTCTCAACACGATAATTATTCCAATGGAAGCTCTCCGTGGATGAAAACTAACTTGCCACCTCCATCTCCACAAGCTCAT 490 B10 530 550 570 590 MHiqSDLSPTPKRRKLASSSDCENKQFDLSAINKNLYPED ATGCATATACAAAGCGATCTATCGCCAACGCCAAAGAGGCGAAAGCTAGCGTCTTCAAGTGATTGCGAGAATAAACAATTTGATTTATCAGCTATTAACAAGAATTTATATCCAGAAGAT 610 630 650 670 690 710 TGSRLMQSLPELSASNSDNVSPVTKSVAFSDRIESSPIYR ACAGGAAGCAGGCTAATGCAAAGTCTTCCAGAGTTGTCTGCCTCTAATAGTGATAATGTATCACCGGTTACAAAAAGTGTAGCTTTTTCTGATAGGATAGAATCATCACCGATTTACCGC 730 750 770 790 810 830 IPGSSPKPSPSSKPGKSILRNRLPSVRTVSDLSYNKLQYT ATACCAGGTTCCTCACCTAAACCCTCTCCATCAAGCAAACCAGGAAAATCAATTTTGAGGAATAGGTTGCCTTCAGTGAGGACTGTAAGCGACTTGAGTTACAATAAACTCCAATATACC 860 870 890 910 930 950 QHKLHNGNIFTSPYKETRVNPRALEYWVSGEIHGLVDNES CAACACAAATTACACAACGGTAATATTTTCACATCACCTTATAAGGAAACAAGGGTAAATCCTCGCGCCTTAGAGTATTGGGTGAGTGGTGAGATCCATGGTTTAGTTGATAATGAAAGT 970 990 1010 1030 1050 1070 VSEFKEIIEGGLGILRQESEDYVARRFEVYATFNNIIPIL GTTTCGGAGTTTAAAGAAATTATTGAGGGCGGATTGGGCATCTTGCGACAAGAATCTGAAGATTACGTCGCGAGGAGGTTTGAGGTATACGCAACCTTTAATAATATTATACCCATTCTA 1090 1110 1130 1150 1170 1190 TTKNVNEVOqKFNILIVNIESirEICIPHLQIAQDTLLSS ACCACTAAAAATGTTAACGAGGTTGATCAGAAATTCAATATTCTAATTGTGAATATAGAAAGTATCATTGAAATATGCATACCGCATCTACAAATAGCACAGGACACATTATTAAGTAGT 1210 1230 12B0 1270 1290 1310 SEKKNPFVIRLYVgiVRFFSAIMSNFKIVKWLTKRPDLVN AGTGAAAAAAAAAATCCATTTGTAATCAGACTATATGTTCAAATAGTTCGATTCTTCAGTGCTATTATGTCTAATTTCAAAATCGTTAAGTGGCTAACGAAAAGGCCAGATTTGGTTAAT 1330 1350 1370 1390 1410 1430 KLKVIYRWTTGALRNENSNKIIITAQVSFLRDEKFGTFFL AAGTTAAAAGTTATTTACAGGTGGACCACCGGTGCGCTAAGAAATGAAAATTCAAACAAGATAATCATAACAGCACAAGTCTCTTTCTTGAGAGACGAAAAATTTGGTACTTTTTTCTTQ 1460 1470 1490 1610 1630 1650 SNEEIKPIISTFTEIMEINSHNLIYEKLLLIROFLSKYPK AGCAACGAAGAAATTAAACCTATCATTAGCACTTTCACTGAAATAATGGAAATCAATAGTCACAACCTAATATATGAAAAATTCCTACTTATAAGGGGATTTTTGAGCAAGTATCCAAAA 1570 1690 1610 1630 1650 1670 LMIETVTSWLPGEVLPRIIIGDEIYSMKILITSIVVLLEL TTAATGATTGAGACGGTAACTTCTTGGTTACCAGGTGAGGTACTACCTAGGATTATTATCGGAGATGAAATTTACTCCATGAAAATTCTCATAACTTCAATAGTTGTTTTACTGGAACTA 1690 1710 1730 1750 1770 1790 LKKCLDFVDEHERIYQCIMLSPVCETIPEKFLSKLPLNSY CTAAAAAAATGCTTAGATTTTGTTGATGAACATGAAAGGATTTACCAGTGTATCATGCTATCTCCAGTATGCGAAACAATCCCGGAAAAATTTTTATCTAAACTACCGTTAAATTCATAT 1810 1830 1850 1870 1890 1910 DSANLDKVTIGHLLTqqiKNYIVVKNDNKIAMDLWLSMTG GACAGCGCTAATTTAGATAAAGTGACAATTGGTCATCTTCTTACACAGCAGATAAAAAACTATATTGTGGTCAAAAATGACAATAAAATAGCAATGGATTTGTGGCTTTCAATGACCGGA 1930 1950 1970 1990 2010 2030 LLYOSGKRVYDLTSESNKVWFDLNNLCFINNHPKTRLMSI TTACTATACGATAGCGGAAAGAGAGTTTATGATTTGACATCAGAATCTAACAAAGTTTGGTTTGATTTAAATAACCTGTGCTTTATCAATAATCACCCCAAGACTCGCTTAATGTCCATC 2060 2070 2090 2110 2130 2160 KVWRIITYCISTKISqKNQEGNKSLLSLLRTPFOMTLPYV AAAGTTTGGAGAATTATTACCTACTGCATATCCACGAAAATATCACAGAAAAATCAGGAAGGAAACAAAAGTTTACTGTCACTTTTACGAACCCCTTTTCAAATGACGCTTCCTTATGTC 2170 2190 2210 2230 2260 2270 NOPSAREGIIYHLLGVVYTAFTSNKNLSTDMFELFWOHLI AATGATCCAAGCGCTAGAGAAGGGATCATCTACCACCTTTTAGGTGTTGTTTACACTGCATTTACAAGTAATAAGAATTTGTCAACAGATATGTTTGAACTATTTTGGGACCACCTAATA 2290 2310 2330 2350 2370 2390 TPIYEDYVFKYDSIHLQNVLFTVLHLLIGGKNADVALERK ACCCCGATTTATGAAGATTATGTTTTCAAATATGATTCGATTCACTTGCAAAATGTACTCTTCACAGTTTTGCATTTGTTAATTGGGGGAAAGAATGCAGATGTTGCATTGGAAAGAAAA 2410 2430 2450 2470 2490 2510 YKKHIHPMSVIASEGVKLKDISSLPPQIIKREYDKIMKVV TATAAAAAGCATATTCACCCAATGAGTGTTATAGCCTCAGAAGGTGTAAAGTTAAAAGACATTTCAAGCCTGCCCCCGCAGATCATCAAGCGTGAATACGATAAAATCATGAAAGTTGTO 2630 2550 2570 2590 2610 2630 FqAVEVAISNVNLAHDLILTSLKHLPEDRKDQTHLESFSS TTCCAGGCTGTTGAAGTAGCCATTTCTAACGTTAATTTGGCTCATGATCTGATCCTTACATCTTTGAAGCATCTTCCAGAGGATAGAAAGGACCAAACTCATTTAGAATCCTTTAGCAQT 2650 2670 2690 2710 2730 2750 LILKVTqNNKDTPIFRDFFGAVTSSFVYTFLDLFLRKNDS CTTATATTAAAAGTCACGCAAAACAACAAAGATACACCCATTTTTCGTGACTTTTTTGGTGCTGTTACTTCATCGTTTGTTTATACATTTTTGGACCTGTTTTTAAGAAAGAATGATTCA 2770 2790 2810 2830 2860 2870 SLVNFNiqiSKVGISQGNMTLOLLKDVIRKARNETSEFLI TCATTAGTAAATTTCAATATCCAAATTTCCAAAGTTGGTATTTCGCAAGGAAACATGACCCTTGATCTTTTAAAAGATGTTATCAGGAAAGCTCGAAACGAAACATCTGAATTTCTGATA 2890 2910 2930 2950 2970 2990 lEKFLELODKKTEVYAQNWVGSTLLPPNISFREFQSLANI ATAGAAAAGTTTCTAGAATTGGATGATAAGAAAACAGAAGTATATGCCCAAAATTGGGTTGGATCAACGTTATTACCACCAAATATATCTTTCAGAGAATTTCAGAGTTTGGCGAATATT 3010 3030 3050 3070 3090 3110 VNKVPNENSIENFLDLCLKLSFPVNLFTLLHVSMWSNNNF GTGAACAAAGTTCCGAATGAAAATTCCATTGAAAATTTCTTGGACCTATGTTTGAAGCTCAGCTTTCCAGTTAATTTGTTTACACTACTTCATGTGTCTATGTCGTCGAACAATAATTTT 3130 3150 3170 3190 3210 3230 lYFiqSYVSKNENKLNVDLITLLKTSLPGNPELFSOLLPF ATTTATTTCATTCAAAGCTATGTTTCAAAAAATGAAAACAAATTAAACGTGGATCTGATCACGCTGTTAAAAACTTCTTTACCAGGGAATCCTGAACTGTTTTCCGGTCTGCTCCCATTT 3250 3270 3290 3310 3330 3350 LRRNKFMDILEYCIHSNPNLLNSIPDLNSOLLLKLLPRSR TTAAGGCGTAACAAATTTATGGATATTTTGGAGTACTGTATTCATTCCAATCCAAATTTACTAAATTCCATCCCTGATCTAAATTCAGATCTCCTATTGAAGCTQTTACCACGTAGCAQA 3370 3390 3410 3430 3460 3470 ASYFAANIKLFKCSEqLTLVRWLLKGqqLEQLNQNFSEIE GCTTCATATTTTGCAGCAAACATAAAACTATTCAAGTGCTCCGAACAGCTTACATTAGTTCGTTGGCTGTTGAAGGGTCAACAACTTGAACAGCTAAATCAAAATTTTTCAGAAATCGAG 3490 3510 3530 3550 3570 3590 Figure 2. {See facing page for legend.

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RAPl mediator at silencers and telomeres

NVLQNASDSELEKSEIIRELLHLAMANPIEPLFSGLLNFC AACGTTTTACAAAATGCGTCTGATAGCGAGTTAGAAAAATCAGAAATAATCAGAGAACTCCTTCATCTGGCGATGGCAAATCCCATTGAACCATTATTCAGTGGCCTATTGAATTTTTGC 3610 3630 36B0 3670 3690 3710 IKNNMADHLDEFCGNMTSEVLFKISPELLLKLLTYKEKPN ATAAAGAATAATATGGCGGATCATTTGGATGAATTTTGCGGAAATATGACCAGCGAAGTTCTTTTCAAAATAAGTCCAGAGCTACTGTTAAAATTACTGACGTATAAGGAGAAGCCTAAT 3730 3750 3770 3790 3810 3830 CKLLAAVIEKIENGDDDYILELLEKIIIQKEIQILEKLKE GGGAAGTTACTGGCAGCAGTTATTGAAAAAATTGAAAATGGAGATGATGATTATATATTGGAGTTGCTTGAAAAGATTATTATACAAAAAGAGATCCAAATTTTGGAAAAATTAAAGGAG 3850 3870 3890 3910 3930 3950 PLLVFFLNPVSSNMQKHKKSTNMLRELVLLYLTKPLSRSA CCACTTTTAGTTTTCTTCTTAAATCCAGTTAGCTCTAATATGCAAAAACACAAAAAGAGTACAAATATGCTCCGTGAATTAGTCTTATTATATCTCACTAAACCGCTTTCTAGAAGTGCC 3970 3990 4010 4030 4050 4070 AKKFFSMLISILPPNPNYQTIDMVNLLIDLIKSHNRKFKO GCCAAAAAATTTTTTTCCATGTTAATTAGTATATTACCGCCAAACCCTAACTATCAAACTATTGATATGGTTAATCTGTTGATTGACCTCATCAAATCACATAATCGTAAGTTTAAAGAT 4090 4110 4130 4150 4170 4190 KRTYNATLKTIGKWIQESGVVHQGDSSKEIEAIPDTKSMY AAGAGAACCTATAATGCAACATTAAAGACAATAGGAAAGTGGATACAGGAATCGGGCGTGGTTCATCAAGGGGACTCAAGTAAGGAAATTGAAGCTATACCTGACACTAAGTCGATGTAT 4210 4230 4250 4270 4290 4310 IPCEGSENKLSNLQRKVDSQDIQVPATQGMKEPPSSiqiS ATTCCATGCGAAGGGAGTGAAAATAAACTTTCAAACTTACAGAGAAAAGTGGATTCTCAAGATATCCAGGTTCCGGCTACGCAGGGAATGAAAGAGCCTCCCTCCTCAATTCAAATATCT 4330 4350 4370 4390 4410 4430 SqiSAKDSDSISLKNTAIMNSSQQESHANRSRSIDDETLE TCCCAGATTAGTGCGAAAGATTCAGATTCAATATCGCTTAAAAATACTGCAATAATGAATTCTTCACAGCAAGAATCACATGCTAACCGAAGCAGATCAATTGATGATGAAACACTAGAQ 4450 4470 4490 4510 4530 4550 EVDNESIREIDQQMKSTQLDKNVANHSNICSTKSDEVDVT GAAGTGGATAATGAAAGCATTAGAGAAATAGATCAGCAGATGAAGAGTACGCAGTTAGATAAAAACGTGGCCAATCATAGCAACATTTGTTCTACTAAAAGCGATGAGGTGGATGTTACT 4570 4590 4610 4630 4650 4670 ELHESIOTQSSEVNAYqPIEVLTSELKAVTNRSIKTNPDH GAGCTGCATGAAAGTATTGATACACAATCCTCGGAAGTGAACGCATACCAACCGATAGAAGTTCTCACTAGCGAACTGAAGGCGGTAACAAATAGATCTATCAAAACGAATCCTGATCAT 4690 4710 4730 4750 4770 4790 NVVNSDNPLKRPSKETPTSENKRSKGHETMVDVLVSEEQA AACGTTGTTAACAGTGATAATCCTCTAAAACGACCTTCCAAAGAGACGCCTACCTCTGAAAATAAAAGATCGAAAGGTCATGAAACGATGGTGGACGTTTTAGTTTCTGAGGAACAAGCQ 4810 4830 4860 4870 4890 4910 VSPSSDVICTNIKSIANEESSLALRNSIKVETNCNENSLN GTGTCGCCTAGCAGTGACGTTATATCTACTAACATCAAGAGTATAGCCAACGAAGAATCTTCGTTAGCTTTAAGGAATAGCATAAAAGTGGAAACAAACTGTAATGAAAATTCCTTGAAT 4930 4950 4970 4990 5010 5030 VTLDLDqQTITKEDGKGQVEHVQRqENQESMNKINSKSFT GTCACTTTAGATCTCGACCAGCAAACCATAACAAAGGAAGATGGAAAAGGGCAAGTTGAACATGTTCAAAGACAGGAGAACCAGGAAAGTATGAATAAAATCAACAGTAAAAGTTTTACQ 5050 6070 5090 6110 6130 B160 QDNIAqYKSVKKARPNNEGENNDYACNVEqASPVRNEVPQ CAAGACAATATTGCACAGTACAAGAGCGTGAAGAAAGCCCGTCCTAATAATGAGGGTGAAAACAACGATTATGCTTGTAATGTTGAACAAGCCTCTCCAGTAAGGAATGAGGTGCCAGGA 5170 5190 6210 5230 B250 6270 DGIQIPSGTILLNSSKqTEKSKVDOLRSOEOEHGTVAQEK GACGGCATTCAGATCCCGAGTGGGACTATACTTCTCAATAGTTCAAAGCAGACAGAAAAATCAAAAGTTGATGACTTGCGTAGTGATGAAGATGAACATGGAACGGTTGCGCAAGAGAAA 5290 5310 B330 5350 5370 5390 HqVGAINSRNKNNDRMDSTPiqCTEEESREVVMTEEGlNV CATCAAGTGGGTGCAATAAACAGTAGAAACAAAAATAATGACAGGATGGACAGCACACCCATTCAAGGCACTGAAGAAGAATCTAGGGAAGTTGTTATGACCGAAGAAGGTATCAATGTA 5410 5430 5450 5470 5490 5610 RLEDSGTCELNKNLKGPLKGOKDANINDDFVPVEENVRDE CGCTTAGAAGATAGTGGAACATGCGAACTTAATAAAAATTTGAAGGGACCGCTTAAAGGAGATAAGGATGCCAATATTAATGACGATTTTGTACCTGTTGAAGAAAATGTCAGAGATGAQ BE30 E550 5B70 5590 5610 6630 GFLKSMEHAVSKETGLEEqPEVADISVLPEIRIPIFNSLK GGCTTTTTAAAATCAATGGAACATGCGGTATCTAAAGAGACAGGTCTAGAAGAACAACCCGAAGTTGCTGATATTTCAGTGTTACCAGAAATTCGTATACCCATTTTCAATTCTTTGAAA 5650 5670 5690 5710 5730 5760 MqCSKSqiKEKLKKRLqRNELMPPDSPPRMTENTNINAqN ATGCAAGGGTCAAAGAGCCAAATAAAAGAGAAATTGAAAAAGAGATTGCAAAGGAATGAGTTAATGCCACCAGATTCACCTCCTCGAATGACAGAAAATACAAATATCAACGCGCAGAAT 5770 5790 5810 5830 5850 6870 GLDTVPKTIGGKEKHHEiqLGqAHTEADGEPLLGGDONED GGCTTAGACACCGTACCAAAAACAATTGGTGGAAAAGAAAAACACCATGAAATTCAATTAGGCCAAGCCCACACAGAAGCAGATGGCGAACCTTTGTTGGGGGGAGATGGTAACGAAGAT 5890 5910 5930 5950 5970 5990 ATSREATPSLKVHFFSKKSRRLVARLROFTPGDLNGISVE GCTACATCTAGGGAAGCCACACCTTCATTAAAAGTGCACTTTTTTTCAAAAAAGTCGAGAAGGTTAGTGGCCAGATTGAGAGGGTTCACACCGGGCGACTTGAACGGAATATCTGTGGAA 6010 6030 6050 6070 6090 6110 ERRNLRIELLDFMMRLEYYSNRDNDMN* GAAAGAAGAAACTTGAGAATTGAATTATTAGATTTTATGATGAGGCTCGAATATTACTCAAACAGGGATAATGATATGAATTGATTGTTAGTATGTAGAATAGATCAAAATGGCAATAAA 6130 6150 6170 6190 6210 6230

TTAATTACAATAATAAAATCTCTCGAAACAATATATAGTTACGATGACACACTAGAAATCGAGAGAGATATATAATAAGTAGAAAGAATTTTTTTTTTTTTTTGATCAGTGCATCTTAAC 6250 6270 6290 6310 6330 8350

CCTTCTTTTGTCTCCATTTTTTTCAGTTGGGAATTAGGATAATATCCCTACAGATAGTTGAAATTTTCTTGAACAATCTTCTCCATTCTAAAGGATCACCAGTTTTTCTTTCAAATCCOA 6370 6390 6410 6430 6450 6470

CACTTTTCAACTCTTCTTCAATCTTTATGATGGAAATGCTACCGCATAATTGATAACCCCC 6490 6510 6530 Figure 2. Nucleotide and deduced amino acid sequence of the RIFl gene. The DNA sequence shown begins 454 bp upstream of the putative translational start site (first methionine) of RIFl and extends 337 bp beyond the TGA termination codon. The predicted amino acid sequence of RIFl is shown in the one-letter code.

HMR locus were assayed. Because the HMRE silencer is silencers that retain complete function. In addition to a redundant regulatory element, it was necessary to ex- the RAPl-binding site, the HMRE silencer contains two amine a wild-type silencer and two different mutated other regulatory sites (called A and B), either one of

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Hardy et al.

SC -Trp SC

RIF1 HMR wt rif1::URA3

RIF1 Figure 3. hfl:: URA3 mutants are defective m hmrAA silencing at hmrAA::TRPl. The effect of a rif1::URA3 rifl::URA3 gene disruption on transcriptional silencing in strains containing i3mr;.Ti?Pi loci RIF1 hmrAB with either wild-type or mutated silencer ele­ rif1::URA3 ments is shown. Tenfold serial dilutions of overnight liquid cultures grown m rich me­ dium (YEPDl were spotted onto plates contain­ rap1-11 ing synthetic medium lacking tryptophan rap1-12 (SC - Trp, left) and synthetic complete me­ hmrAA dium (SC, right). The plates were incubated at rap1-13 30°C for 2-3 days before being photographed. For purposes of comparison, the growth of the rap1-14 four different rapT strains jm a hmrAAr.TRPl background) and a strain carrying the defective hmrAEB RAP1 RIF1 hmrAEB silencer are shown below.

which, together with the RAPl site (E), is sufficient for the riflrURAS mutant strains. Total yeast genomic complete repression (Brand et al. 1987; Kimmerly et al. DNA was digested with Xhol and probed with radiola­ 1988). The A element is an autonomously replicating beled poly[d(G-T)] • poly[d(A-C)], which hybridizes with sequence (ARS) consensus clement, whereas the B ele­ the terminal poly(Ci„3A) sequences (Shampay et al. ment is a binding site for the ABFl protein (Shore et al. 1984; Walmsley et al. 1984). A prominent heterogeneous 1987; Buchman et al. 1988a). We noted previously that band of —1.2 kb results from the large number of yeast rapT mutants display a defect m silencing only when telomeres that contain a subtelomeric Y' element. As the A element at HMR is deleted [hmrAA] (Sussel and shown in Figure 4, riflrURAS mutants display a signif­ Shore 1991). Furthermore, the hmiAA silencer is affected icant increase in the average length of this terminal frag­ most severely by overexpression of GBD/RAPI hybrids ment (—200-300 bp), as compared to the wild-type par­ (Hardy et al. 1992). ent strain. We presume that this results from an increase To assay for silencer function, strains in which the m the length of the poly(Ci_3A) sequences within these TRPl gene has been placed at HMR (Miller et al. 1984; fragments. In addition, the heterogeneity in length of the Brand et al. 1985) were used. The ability of such strains chromosome ends appears to increase in riflrURAS to grow in the absence of tryptophan is a sensitive and strains relative to their wild-type parent. This effect is accurate assay for the loss of silencer function (Susscl very similar to the telomere lengthening displayed by and Shore 1991). Haploid strains containing i^m/.-.Ti^Pi, the silencing-deiective rapl -12 and rapl -13 mutants (Fig. hmrAA::TRPl, or hmrABr.TRPl silencers were trans­ 4). formed with lifl:: URA3 DNA and screened by Southern blotting for the correct chromosomal disruption. Analy­ sis of these strains showed that only the hmrAAr.TRPl A carboxy-terminal domain of RAPl silencer strain was affected by the chromosomal disrup­ required for RIFl binding tion of RlPl and was thus able to grow m the absence of A previously characterized series of GBQ/RAPI hybrids tryptophan (Fig. 3). Furthermore, the derepression of (Flardy et al. 1992) was used to determine which RAPl hmrAAr.TRPl caused by the riflrURAS mutation was sequences in Ggj^/RAPl are required for G^D complete and indistinguishable from that of the strong /RIFl-de­ rapr alleles, rapl-12 and rapl-13. Strains containing pendent activation of the GALl-IacZ reporter gene. Fig­ a wild-type silencer [hmir.TRPl) or a AB silencer ure 5 shows that there is a very close correlation between [hmrABr.TRPl) showed normal repression of the TRPl the sequences required for derepression by GBQ/RAPI reporter gene in the presence of the riflrURA3 disrup­ and those required for an interaction with GA.D/RIF1- For tion. example, GBD/RAP1(653-827) and GBD/RAP1(655-827) both completely derepress the hmrAAr.TRPl locus and cooperate with G^Q/RIFI to give comparable levels of RIFl affects telomere length regulation p-galactosidase expression. In addition, the GBD/ To determine whether RIFl plays a role at telomeres, as RAP1(679-827) hybrid displays a reduced ability to both does RAPl, Southern blot analysis was used to measure derepress [-10- to 20-fold lower than GBD/RAP1(653- the average length and heterogeneity of telomeres from 827) and GBD/RAP1(655-827)] and to participate in G^D/ RIFl-dependent activation (36 units, compared with 616

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RAPl mediator at silencers and telomeres

Xho1 and 747 (Sussel and Shore 1991). The rapl" mutants Y"(0-4 ) cause either strong [rapl-12 and rapl-13] or weak {rapl- 11 and iapl-14] derepression of the iimr^A::TRPl locus (Ci-3A)n (Ci-3A)n and variable elongation of poly(Ci„3A) tracts at telo­ meres. A series of four GBLVrapP(653-827) hybrids were constructed that incorporated the mutant changes of the rapr alleles into the GBO/RAP1(653-827) hybrid. These Gg^/rapl^ hybrids were then coexpressed with the G^^/ RIFl hybrid in the GALl-lacZ reporter strain. As shown in Table 1, there is a striking inverse corre­ lation between the extent of derepression caused by the different rapV alleles and the ability of the correspond­ ing Ge^/rapF hybrids to activate the GALl-lacZ re­ porter when expressed together with GAD/RIFI • The two «.-.,. strongrapi"* mutants, iapl-12 andrapl-13, are both com­ pletely defective in silencing the hmrAA::TRPl locus 4.82 and also fail to activate in conjunction with G^^^ /RIFl. 4.32 Perhaps more significant is the observation that the 3.68 weak zapl" alleles [rapl-ll and iapl-14, with only a par­ tial defect in silencing of hmrAAr.TRPl] display a mea­ surable but reduced ability as Ggi^/rapP hybrids to in­ teract with GAD/RIFl- These results suggest that the 2.32 strong lapV mutants have a totally defective RIFl inter­ 1.93 action site, whereas the weak lapl^ mutants retain a partially functional RIFl interaction site. Furthermore, they signify that the defect in silencing by the lapV mu­ 1.37 1.26 tants may be explained by a failure to interact with RIFl. Interestingly, though, the GB^/rapP hybrids derepress the hmiAA::TRPl locus to the same extent as the GB^/ RAP(653-827) hybrid from which they were derived (data not shown; see Discussion). Figure 4. Telomeric poly(Ci_3A) tracts are lengthened in nfl::URA3 mutants. (A) Schematic representation of yeast telomeres. Note that many, but not all, telomeres contain the Isolation of a Gj^^/hfl mutant that partially restores Y' element, which has a Xhol site near the poly(Ci__3A) tract. [B] an interaction vdth Giny/rapV hybrids Southern analysis. Xhol-digested genomic DNA from the indi­ We reasoned that if rapP proteins were defective in an cated strains was probed with -^^P-labeled poly[d(G-T) • d(C-A)]. interaction with RIFl, then mutations in RIFl may sup­ A heterogeneous band of —1.2 kb in the wild-type strain (indi­ cated by the arrow) arises from the many yeast telomeres that press this defect. The two-hybrid system was utilized contain at least one Y' element. further to screen for a mutated GAo/rifl hybrid that could suppress the activation defect of the GBo/rapF hybrids. The GAD/RIFI plasmid was mutagenized by passage through an Escherichia coli mutDS strain (see and 895 units). Finally, all of the additional hybrids that Materials and methods) and transformed into a GALl- we examined arc completely defective in both activities lacZ reporter strain expressing GBD/rapl-12, which is (Fig. 5, rows 5-11). Taken together, these results indicate completely defective in GAo/RIFl'^icdi^ted activation that both derepression and GAD/RIFI "dependent activa­ (see Table 1). From > 15,000 transformants, one plasmid tion by GBD/RAPI hybrids require the same carboxy- was obtained that conferred a blue colony color on X-gal terminal domain of RAPl. plates and thus appeared to suppress the GBL^/rapl-12 activation defect. The plasmid, encoding what will be referred to as Gj^Y:,/nil-l, was retransformed into the re­ Silencing-defective lapV mutants are defective porter strain coexpressing the original GBD/RAPI(653- in G^i^/RIFl-dependent activation 827) hybrid and into strains coexpressing each one of the Having shown that a carboxy-terminal part of RAPl, four different GBo/rapP mutant derivatives of this hy­ whose overexpression causes derepression, is necessary brid. Interestingly, the p-galactosidase levels for each for a RAPl-RIFl interaction, we then asked whether si- strain converged to an intermediate value between 100 lencing-defective point mutations in this region of RAPl and 200 units, less than one-third of the value obtained disrupt the RAPl-RIFl interaction. To answer this ques­ with the two wild-type hybrids, GBD/RAPI and G^D/ tion we made use of four different lapl''' alleles that con­ RIFl (Table 2). The mutation was mapped to the RIFl tain either single or double missense mutations in the part of the GAT,/RIFl hybrid by exchanging restriction RAPl carboxyl terminus, affecting amino acids 726, 727, fragments with the wild-type GAD/RIFl plasmid and

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(3 gal Units SC-Trp-His SC-His

616 GAL4(1-147) RAP1 (653-827) :3::iii 895 • -^ GAU(n47) RAP1 (655-827) 145 •^Bi I^^B 1^^^ ^ RAP1 (667-827) ^tgF ^^ ^^ 36 ^ RAP1 (679-827)

GAL4{1-147) RAPl(691-827) <1

GAL4(1-147) RAPl (702-827) <1 ^ ^ ^ >' .4{1-147) RAPl (653-751) <1

L4(1-147) RAPl (653-799) <1

GAL4(1-147) RAP1(653-727'754-827) <1 <1 GAL4(1-147) RAPl (653-743,764-827) <1 GAL4<1-147) RAPl (653-716/721-827) Figure 5. RIFl interacts with a carboxy-termmal domain of RAPl. A series of GJJD/RAPI hybrids was assayed for the ability to interact with the G^^/RIFl hybrid and to activate the GALl-lacZ reporter gene. Quantitative p-galactosidase assays were performed for strains containing both the indicated GBD/RAP plasmids and the G^^p/RIFI plasmid (see Fig. 1 and Materials and methods). The GBo/RAP-expressmg plasmids were also assayed for their effect on silencing at the hmiAA::TRPl locus (see Fig. 3; Fiardy et al. 1992).

was identified by DNA sequencing as a G ^^ A change at G^o/RIFl hybrid to distinguish between wild-type Ggj^/ nucleotide 6169 (Fig. 2). This mutation would be pre­ RAPl and the two classes of rap*" hybrids. The failure of dicted to result m a change from glutamic acid to lysine the Gj:^^/hil-l hybrid to distinguish between GB^^/RAPI at amino acid 1906 of RlFl, only 10 codons from the and the different G^i^/rap'' hybrids suggests that the carboxyl terminus of the protein. rifl-1 mutation may abolish a specific interaction with Two features of the GAo/rifl-l mutant hybrid are RAPl, defined at least in part by codons 726, 727, and worth emphasizing. First, the mutant hybrid appears to 747 in RAPl, rather than compensating for the alteration interact less well with the wild-type Gp^n/RAPl hybrid in the iapl-12 mutant. than does the wild-type G^i^/RIFl hybrid, as shown by the significantly lower (3-galactosidase value. This result indicates that the G^-^^^/nil-l mutant does not work by Discussion simply raising the affinity for wild-type RAPl and thus We have isolated a novel gene, RIFl, on the basis of the compensating for a defect m rap 1-12. Second, although ability of a portion of its protein product to associate the rifl-1 mutant is not allele specific, that is, it im­ with the RAPl carboxyl terminus in vivo. Loss of RIFl proves the interaction with both the rap 1-13 and rap 1-14 function results in derepression of an HMR silencer, hybrids, it has the striking property of appearing to in­ whose ARS consensus element has been deleted, and in teract approximately equally well with all four mutant the elongation of telomeres, two properties characteris- rapP hybrids and with the wild-type Gu^/RAPl hybrid. This property is m marked contrast to the ability of the Table 2. The G^^^/iifl-l mutant suppresses the activation defect of Ggi^/iapV mutants

Table 1. Gjuj/rapr mutant hybrids are defective m Activation domain hybrid GAij/RlFl-dependent activation DNA-binding G^r^/RIF1 G^r^/rifl-l Silencing defect domain hybrid (P-gal units ) DNA-binding p-gal m context of domain hybrid units full-length RAPl GBO/RAPI 616 165 GBn/rapl-12 <1 110 GBD/RAPI 616 none Gnu/rap 1-13 <1 177 GBo/rapl-ll 172 weak GBD/rapl-14 35 188 GgD/rapl-ll <1 strong GBo/rapl-ll 172 126 GgD/rapl-lS <1 strong GBD <1 ND'' GBi^/rapl-M 35 weak None <1 <1 <1 ''Not determined.

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RAPl mediator at silencers and telomeres tic of silencing-defcctive rapl^ alleles (Sussel and Shore 1991); confirming an involvement of RIFl in both tran­ scriptional silencing and telomere length regulation. The genetic experiments presented here provide com­ pelling evidence that the RAPl and RIFl proteins can physically associate and that this association is impor­ tant for repression at hmiAA silencers and length regu­ lation at telomeres. Activation by G^u/RIFl would ap­ pear to depend on a physical association with the RAI^l carboxyl terminus. Furthermore, using the two-hybrid system, we have shown that Gyo/rapl* mutant hybrids RIFl interact poorly or not at all with GAD/RIFI- What is particularly striking is the strict correlation between the severity of the silencing defects of individual lapl'' mu­ tant alleles and the ability of the corresponding G^^/ rapP hybrids to interact with the GAO/RIFI hybrid. The two weakest rapV alleles lead to an intermediate level of activation together with GAD/RIFI/ whereas the two strongest rapl'' alleles fail to produce a detectable signal in the two-hybrid system. It seems unlikely that the Ggo/rapF defects reflect a general instability of the rapP carboxy-terminal domains, as the lapl^ mutants have both normal protein levels and do not display any temperature sensitivity (Sussel and Shore 1991). In addi­ tion, the Gpn/rap'' hybrids are all capable of silencer derepression, implying that they are also stable and prop­ erly folded (data not shown). The properties of the compensating GAo/rifl-l mu­ tant argue strongly that there is a specific protein-pro­ Figure 6. A model for the RAPl-RIFl interaction based on tein interaction between RIFl and the small carboxy- properties of mutant proteins in the two-hybrid system. (A) The terminal region of RAPl affected by the rapT'' alleles. complementary triangular surfaces are intended to represent a The G^Yi^/rifl-I hybrid appears to interact less well with proposed interaction site between RAPl and RIFl, defined in wild-type GB^/RAPI than does G^n/RIFl/ implying part by amino acids 726, 726, and 747 in RAPl and amino acid that the rifl-1 mutation does not work by increasing the 1906 in RIFl. The complementary rectangular surfaces repre­ affinity of RIFl for both wild-type RAPl and rapF pro­ sent the sum of all other RAPl-RIFl interactions. The rapl-12 teins. Instead, we suggest that the rifl-1 mutation alters mutation creates an unfavorable interaction that significantly a specific interaction with RAPl defined by the rapV reduces or abolishes binding (J5). The rifl-1 mutation removes this unfavorable contact with rapl-12, allowing rifl-I to inter­ alleles, such that the rifl-1 protein can no longer distin­ act equally well with rapl-12 and wild-type RAPl protein {C,D). guish between wild-type RAPl and the four different In the weaker alleles, rapI-14 and rapl-11, we imagine that the rapl"" mutants and thus interacts equally well with all of repulsive rapI-RIFI interaction seen for the rapl-I2-RIFI com­ them. The properties of the hf 1-1 mutation are similar to bination [B] is either minimal (rapl-I4) or essentially nonexis­ those described for loss-of-contact mutations in se­ tent (rapl-II). quence-specific DNA-binding proteins (Ebright et al. 1987). A simplified model for the RAPl-RIFl interac­ tion, based on the mutant data, is shown schematically in Figure 6. The model predicts that the rapl'' alleles (at amino acids 726, 727, and 747) define a specific interac­ genes (Fiaber and George 1979; Klar et al. 1979; Rine and tion site on the RAPl carboxyl terminus and that strong Ficrskowitz 1987), HHF2 (encoding histone Fi4) (Kayne rapV alleles {rapl-12 and rapl-13) create a repulsive in­ et al. 1988; Megee et al. 1990), NATl and ARDl (encod­ teraction with RIFl at this site that is abolished by the ing an amino-terminal protein acetyltransferase) (White- hfl-1 mutation. Further experiments, involving site-di­ way et al. 1987; Mullen et al. 1989), CDC7 (Axelrod and rected mutagenesis of both proteins, will test the valid­ Rine 1991), SUMl (Klar et al. 1985; Livi et al. 1990; ity of this proposal. Laurenson and Rine 1991), RAPl (Shore and Nasmyth 1987; Kurtz and Shore 1991; Sussel and Shore 1991), and presumably the gene encoding the other silencer binding factor ABFl (Diffley and Stillman 1989; Halfter et al. RIFl: a co-factor or mediator for RAPl 1989; Rhode et al. 1989; Francesconi and Eisenberg at silencers and telomeres 1991). It is worth noting that in searching directly for a Previous studies have identified a large number of trans­ RAPl-interacting factor involved in silencing, we have acting regulators of silencing, including the four SIR identified a novel gene. Our results suggest that the con-

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Hardy et al. nection between the silencer DNA-binding factors and hmiAB silencers to work. With this in mind, one can other proteins responsible for silencing (e.g., histone H4 imagine that RIFl is required to stabilize interactions and SIR proteins) may be complex, involving RIFl and with replication factors (e.g., an ARS consensus se­ possibly other cofactors or mediators. Recent studies of quence binding factor) at an hmiAA silencer, where a transcriptional activation also point to intermediary fac­ perfect match to the ARS consensus sequence is not tors in the interaction of DNA-binding activators and present. In such a scheme, RIFl function may be needed the basic transcriptional machinery (Dynlacht et al. only during the S phase oi the cell cycle, when the es­ 1991; Flanagan ct al. 1991). tablishment of silencing is thought to occur (Miller and Data presented here are consistent with a model m Nasmyth 1984). It will be interesting to determine which RAPl targets the binding of RIFl to silencers and whether RIFl transcription is elevated during S phase, as telomeres. We propose that this interaction leads to the suggested by the presence of the Mlul site upstream of recruitment of essential silencer factors (e.g., SIR pro­ the RIFl gene. teins), which themselves function as modifiers of chro­ Genes placed near telomeres are subject to a position- matin structure at both silent matmg-type loci and effect repression (Gottschling ct al. 1990) that requires telomeres (Nasmyth 1982; Gottschlmg et al. 1990; Apa- many of the same trans-actmg regulators necessary for ricio et al. 1991). It should be noted, however, that we silencing at HML and FIMR (Aparicio et al. 1991). Fiow- cannot rule out a model in which RIFl modifies RAPl to ever, telomeric silencing is metastable, a phenomenon allow it to function at silencers and telomeres but is seen at silent mating-type loci only in strains with cer­ itself not stably associated with these loci. It is possible tain cis- or tians-silencing mutations (Pillus and Rine that the proposed RAPI-RIFI interaction is restricted to 1989; Mahoney et al. I99I; Sussel and Shore 1991; L. silencer and telomere loci, because deletion of RIFl has Sussel and D. Shore, unpubl.) and is independent of SIRl no effect on essential RAPl transcriptional activation function (Aparicio et al. 1991). These observations have functions and RIFl interacts with a part of RAPl that led to the suggestion that the silencers at HML and HMR may be important only at silencers and telomeres. One have additional redundant pathways for repression not explanation for such a restriction is that RIFl interacts present at telomeres (Aparicio et al. I99I). The silencing with specific DNA sequences and/or other proteins pathway defined by the rapl'' and hfl mutations may not found exclusively at silencers and telomeres. Modula­ function at telomeres or it may also be redundant there. tion of the function of a DNA-bmdmg regulatory protein This may explain why rapl'^ mutants appear to have no by additional protcm-protcm and protem-DNA interac­ effect on telomeric silencing (B. Billington and D. tions occurs in other systems. For example, the MATa2 Gottschling, pers. comm.), despite the occurrence of repressor protein in yeast uses the MCMl protein, itself many RAPl-bindmg sites within the telomeric poly(Ci_ an activator in many contexts, as a corepressor at some 3A) tracts. It remains to be seen whether telomeric si­ loci through a combination of protem-protein interac­ lencing is also independent of RIFl. The reason for tions and sequence-specific DNA binding (Keleher et al. telomere elongation in both rapl^ and hfl mutants re­ 1988, 1989). One interpretation of our results is that mains unclear and is puzzling in light of the observation RAPl does not interact directly with any of the silencer that mutations in SIR2-SIR4, which abolish repression factors identified previously (e.g., SIRI-SIR41 and that its at silent mating-type loci and telomeres, have no effect essential function in silencing at hmrAA is to target the on telomere length (D. Gottschling, pers. comm.). How­ binding of RIFl to this locus, which then leads to the ever, the correlation between the strength of rapl" si­ recruitment of SIR proteins. This idea can be tested m lencing defects and the extent of telomere elongation part by providing RIFl with a heterologous DNA-bmdmg (Sussel and Shore 1991) suggests that these two phenom­ domain and targeting the resultant hybrid protein to a ena are related at some level. Perhaps mechanisms con- silencer containing the corresponding DNA-bindmg site troling telomere length are more sensitive to the effects and lacking a binding site for RAPl. Whether RIFl is a of rapT and hfl mutations than is the telomeric silenc­ mediator or a cofactor for RAPl at silencers and telo­ ing machinery. meres, it seems likely that it interacts with other factors at these loci (e.g., SIRs) and that such interactions will have important functional consequences. Other RIFs A particularly notable feature of the HMRE silencer is its functional redundancy (Brand et al. 1987; Kimmerly Given both the multiple functions of RAPl in silencing etal. 1988; Stone etal. 1991). We noted recently that this and the requirement for RIFl in only one RAPl-medi­ redundancy extends to the RAPl protein itself (Sussel ated silencing pathway, it seems reasonable to assume and Shore 1991) because lapl'' mutants do not affect the that other RIFs contribute to silencing at wild-type and ability of the protein to contribute to repression of wild- hmrAB silencers. We showed recently that overexpres- type or hmiAB silencers. Because the hflr.URAS mu­ sion of a GRD/RAPI hybrid partially derepresses HMR tants also display a silencing defect only when the A wild-type and hmrAB silencers (Fiardy et al. 1992). A element (an ARS consensus sequence) is deleted at disruption of the RIFl gene, however, does not derepress HMRE, RIFl could function only for this one redundant either the wild-type or hmrAB silencers nor does it mit­ RAPl silencing function. This RIFl-associated silencing igate the partial derepression effect of a Ggj^/RAPl hy­ pathway is not required for either the wild-type or brid on these silencers (data not shown). Because the

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RAPl mediator at silencers and telomeres

GB^/RAPI hybrid can dcrepress in the absence of RIFl in plasmid pGAD2 (Chien et al. 1991). The 3' end points of the activity, we propose that it may work on these silencers inserts are at the unique Xbal site in pGAD2. by titrating a different RIF. The existence of other RAPl- interacting proteins involved in silencing is highlighted further by the observation that the Ge^^/rapP hybrids are Isolation of RIFl using the two-hybrid system all effective derepressors, including the GR^/rapl-ll and The yeast GALl-lacZ reporter strain GGYT71, containing a GBi:,/rapl-13 hybrids, which fail to interact with GAC,/ plasmid expressing the GBO/RAP1|653-827) hybrid, was trans­ RIFl in the two-hybrid system. Although the simplest formed with a library of genomic DNA fragments in the pGAD2 model would propose that RIFl is the factor titrated by expression vector (a generous gift of P. Bartell and S. Fields) the overexpression of GKO/RAPI hybrids, these results using the high-efficiency method of Schiestl and Geitz (1989). raise the possibility that a protein-protein interaction (as Transformants ( — 1000 per plate) were selected on SC-His-Leu yet unidentified) may, at least in part, underlie this phe­ medium at 30°C. After 3-5 days of growth, colonies (1- to 1.5- nomenon. Consequently, we are continuing to screen mm diam.) were replica-plated onto SC-His-Leu plates contain­ ing X-gal. After 1-5 days, blue colonies were identified, purified the library from which RIFl was isolated and indepen­ by restreaking on SC-His-Leu plates, and retested by replica dent libraries in which yeast sequences are joined to G^^ plating onto X-gal plates. Positive colonies were picked from the through the two other possible reading frames in search SC-His-Leu plates and grown overnight in 10 ml of SC-Leu liq­ of new RIFs. It will be interesting to compare the results uid medium. DNA prepared from these cultures was trans­ of these screens with pseudoreversion (extragenic sup­ formed into the Leu" E. coli strain BAl, and Amp^ Leu^ trans­ pressor) studies of rapl'' mutants, which are currently formants were selected. Plasmid DNA was prepared from at under way (L. Sussel and D. Shore, unpubl.). least six independent BAl transformants and tested by transfor­ In conclusion, using the two-hybrid system, we have mation into the yeast reporter strain GGYT71 with or without the GBD/RAPl(653-827)-expressing plasmid. Yeast transfor­ identified a protein that interacts with RAP I and func­ mants were also assayed for lacZ expression by replica-plating tions in both transcriptional silencing and telomere onto nitrocellulose (Breeden and Nasmyth 1985). length regulation. These studies provide the first direct evidence that a silencing function of RAPl is mediated by selective interactions with another protein. RIFl is not the product of any previously identified tians-acting Isolation of a GA^/rifl mutant regulator of silencing. Further study of RIFl, identifica­ The original GAD/RIFI plasmid was transformed into a mutD5 tion of additional RIFs, and characterization of their mo­ E. coli strain (Echols et al. 1983), selecting for ampicillin resis­ lecular targets should provide new insights into the reg­ tance on minimal M65 medium. Eight individual transformants ulation of silencers and telomeres. were picked and grown to saturation in 10 ml of LB media containing 20 |JLg/ml of ampicillin. DNA preparations from these eight cultures were pooled and used to transform yeast strain GGY:171 containing a plasmid expressing the GBD/rapl- 12 hybrid. Transformants were selected on SC-His-Leu plates. After 2-3 days of growth, replicas were made onto nitrocellu­ Materials and methods lose filters and tested for p-galactosidase activity. One blue col­ Strains and DNAs ony was detected from —15,000 transformants. Plasmid DNA was rescued from the single blue transformant and tested as Growth and manipulation of yeast strains was done according described above. The rescued plasmid was shown to confer the to standard procedures (Sherman et al. 1983). All experiments blue colony phenotype only upon retransformation of GGY: 171 involving the two-hybrid system were performed m strain strains containing GBD/rapl'* or GBD/RAPI hybrids. The muta­ GGY:171 [leu2-2,112 his3 A200 Aga.\4 AgalSO GALl-lacZ). A tion was localized to a small restriction fragment of the GAR/ library of partial SauSA-digested yeast genomic sequences in rifl-1 plasmid by exchanging corresponding fragments with the the vector pGAD2 (Chien et al. 1991) was generously provided original plasmid encoding the GAD/RIFI hybrid. An Xbal-Ncol by P. Bartel and S. Fields. Plasmid DNAs were rescued from fragment from the mutagenized plasmid conferred the mutant GGY:171 by transformation into CaClj-treated £. coli strain phenotype when placed in the G^D/RIFI plasmid background. BAl (thr}euB6 thi thyA tipClll? hisB str^), selecting simulta­ The sequence of the complete RIFl-coding sequence on this neously for ampicillin resistance and leucine prototrophy. RIFl fragment was determined using synthetic oligonucleotide prim­ gene disruptions were done in strain W303-1B [HMLa MATa ers, and a single-base change was found at nucleotide 6169 HMRa ade2-l canl-100 his3-ll,-lS leu2-3,-112 tipl-1 ma3-l), (G^A). derivatives of W303-1B containing hmT::TRPl loci (Sussel and Shore 1991), and an isogenic MATa/MATa diploid (W303). GBQ/ RAPl carboxy-terminal hybrids have been described previously (Hardy et al. 1992). GBo/rapP hybrids were constructed by re­ Other methods placing a Bglll-Xbal fragment from the original GBD/RAP1(653- Liquid assays for p-galactosidase were performed as described 827) clone with the corresponding fragment from the mutant previously (Hardy et al. 1992). The average value from at least alleles. The resulting GBD/rapl*" hybrids differ from the original four transformants of each plasmid construct is reported. Values GBD/RAP1(653-827) hybrid only in those amino acids affected for individual colonies differed by <30% from the average. in the lapl"" mutants. Deletion derivatives of the original GAQ/ Transcriptional silencing was assayed in strains containing RIFl plasmid were constructed by placing either the BamjP)- hmrr.TRPl loci (with wild-type or mutated silencer elements) Bam{D] or the Bam[P]-Xbal fragment encoding RIFl sequences by measuring colony-forming ability on media lacking tryp­ (see Fig. 1) downstream of the BamHl site at the fusion junction tophan (Sussel and Shore 1991). Telomere tract lengths were

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Hardy et al. measured by Southern blot analysis of Xhol-digested genomic gene. Cold Spring Harbor Symp. Quant. Biol. 50: 643-650. DNA using poly[d(G-T)] • poly(d(C-A)] probes (Shampay and Brill, S.J. and B. Stillman. 1991. Replication factor-A from Sac- Blackburn 1988). DNA sequencing was done primarily on an charomyces cerevisiae is encoded by three essential genes Applied Biosystems 3 73A, using the universal primer and a se­ coordinately expressed at S phase. Genes &. Dev. 5: 1589- ries of nested deletion clones generated by exonuclease III di­ 1600. gestion or using synthetic oligonucleotide primers correspond­ Buchman, A.R., W.J. Kimmerly, J. Rine, and R.D. Kornberg. ing to sequence generated from the deletions. The sequence 1988a. Two DNA-binding factors recognize specific se­ reported has been determined on both strands. Details of prim­ quences at silencers, upstream activating sequences, auton­ ers and deletions used in the sequencing are available on re­ omously replicating sequences, and telomeres in Saccharo- quest. We compared the RIFl sequence to sequences m data myces cerevisiae. Mol. Cell. Biol. 8: 210-225. bases (SWISS-PROT 19.0, and Gcnl^ept 69.0) using the FASTA Buchman, A.R., N.F. Lue, and R.D. Kornberg. 1988b. Connec­ program (Pearson and Lipman 1988). In addition, Dr. M. Goebl tions between transcriptional activators, silencers, and compared the RIFl sequence to an independent data base and telomeres as revealed by functional analysis of a yeast DNA- failed to find any significant similarities to other proteins (M. bindmg protein. Mol. Cell. Biol. 8: 508^5099. Goebl, pers. comm.'. Chien, C.-T., P.L. Battel, R. Sternglanz, and S. Fields. 1991. The two-hybrid system; A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. 88: 9578-9582. Acknowledgments Conrad, M.N., J.H. Wright, A.J. Wolf, and V.A. Zakian. 1990. We thank Stan Fields and Paul Barrel for the very generous gift RAPl protein interacts with yeast telomeres in vivo: Over­ of the yeast genomic DNA-pGAD2 library and advice through­ production alters telomere structure and decreases chromo­ out the course of this work. We also thank Rolf Sternglanz and some stability. Cell 63: 739-750. Chien-Ting Chien for considerable advice and encouragement; Diffley, J.F. and B. Stillman. 1988. Purification of a yeast protein members of the Shore laboratory for helpful discussions; Ula that binds to origins of DNA replication and a transcrip­ Beauchamp for DNA sequencing; Aaron Mitchell, Stan Fields, tional silencer. Proc. Natl. Acad. Sci. 85: 2120-2124. and Susan Wente for comments on the manuscript; and Mark . 1989. Similarity between the transcriptional silencer Goebl for sequence comparisons. This work was supported by binding proteins ABFl and RAPl. Science 246: 1034-1038. grants from the National Institutes of Health (NIFFl |GM40094I, Dynlacht, B.D., T. Fioey, and R. Tjian. 1991. Isolation of coac- the American Cancer Society (ACS! (IFRA-231 and MV-5341, tivators associated with the TATA-binding protein that me­ the Searle Scholars Fund/Chicago Community Trust, and the diate transcriptional activation. Cell 66: 563-576. Irma T. Hirschl Charitable Trust to D.S., and by an ACS Insti­ Ebright, R.H., A. Kolb, H. Buc, T.A. Kunkel, J.S. Krakow, and J. tutional Research Grant iIRG-177A) to the Comprehensive Beckwith. 1987. Role of glutamic acid-181 in DNA-sequence Cancer Center at Columbia University. 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A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation.

C F Hardy, L Sussel and D Shore

Genes Dev. 1992, 6: Access the most recent version at doi:10.1101/gad.6.5.801

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