Copyright 0 1995 by the Genetics Society of America

Extra Telomeres, but Not Internal Tracts of Telomeric DNA, Reduce Transcriptional Repression at Saccharomyces Telomeres

Emily A. Wdey and Virginia A. Zakian

Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, and Department of Pathology, University of Washington, Seattle, Washington 981 95

Manuscript received June 16, 1994 Accepted for publication September 13, 1994

ABSTRACT Yeast telomeric DNA is assembled into a nonnucleosomal chromatin structure known as the telosome,

which is thought to influence the transcriptional repression of genes placed in its vicinity, a phenomenon called telomere position effect (TPE). The product of the RAP1 gene, Raplp, is a component of the telosome. We show that the fraction of cells exhibiting TPE can be substantially reduced by expressing large amounts of a deletion derivative of Raplp that is unable to bind DNA, called RaplABBp, or by introducing extra telomeres on a linear plasmid, presumably because both compete in trans with telo- meric chromatin for factor(s) important for TPE. This reduction in TPE, observed in three different strains, was demonstrated for two different genes, each assayed at a different telomere. In contrast, the addition of internal tracts of telomeric DNA on a circular plasmid had very little effect on TPE. The product of the SIR3 gene, SirSp, appears to be limiting for TPE. Overexpression of Sir3p completely suppressed the reduction in TPE observed with expression of Rap1 ABBp, but did not restore high levels of TPE to cells with extra telomeres. These results suggest that extra telomeres must titrate a factor other than Sir3p that is important for TPE. These results also provide evidence for a terminus-specific binding factor that is a factor with a higher affinity for DNA termini than for nonterminal tracts of telomeric DNA and indicate that this factor is important for TPE.

T HE term heterochromatin refers to regions of chro- mosomes that remain highly condensed through-

out the cell cycle. Heterochromatic regions, such as the inactive X chromosome in mammalian females, typi- cally replicate late in S phase and are transcriptionally repressed (reviewed in CATTANACH 1975). In Drosoph- ila, this transcriptional repression can spread to nearby genes as observed when euchromatic genes are juxta- posed near or within heterochromatin. In these cases, the translocated gene has an on-off pattern of expres- sion, commonly called position effect variegation (PEV) (reviewed in EISSENBERG 1989; SPRADLING and WEN 1990). Many euchromatic loci that suppress or enhance PEV in a dosagedependent manner have been identi- fied (reviewed in REUTER and SPIERER 1992). Some of these loci, for example HP-1, encode proteins found in heterochromatin. In addition, PEV can be modified by the dosage of heterochromatin (SPOFFORD 1976). For example, PEV is suppressed in trans by the presence of extra Y chromosomes, which are entirely heterochro- matic in somatic cells (GOWEN and GAY 1934). This derepression is thought to be due to the ability of the Y chromosome to titrate proteins like HP-1 away from heterochromatin (SPOFFORD 1976).

Telomeres, the ends of eukaryotic chromosomes, are

Cmesponding author Emily A. Wiley, Fred Hutchinson Cancer Re-

WA 98104. search Center, Basic Sciences, A2-168, 1124 Columbia St., Seattle,

Genetics 139 67-79 (January, 1995)

composed of simple repetitive DNA sequences. For ex- ample, Saccharomyces telomeres have -350 bp of C1-&TG1-3 DNA. In ciliated protozoa like Oxytricha, the Grich strand of the repeat sequence is extended to form a single-strand tail on the ends of macronuclear DNA molecules (reviewed in ZAKIAN 1989). In Oxytri- cha, two proteins form a heterodimer that binds tena- ciously to this single-strand tail, staying bound even in the presence of 2 M salt (GOTTSCHLING and ZAKIAN 1986). These proteins are terminus-specific binding proteins in that they require both the specific single- strand tail and adjacent duplex DNA for their most efficient binding. In vitro, the Oxytricha proteins pro- tect DNA termini from exonucleolytic degradation (GOTTSCHLING and ZAKIAN 1986), mediate telomere- telomere interactions (LIPPS et aZ. 1982), and promote the formation of G-quartet DNA (FANG and CECH 1993). In vivo, the presence of these proteins might explain how cells distinguish a telomere from a broken end and how telomeres protect chromosome ends from degradation or end-to-end fusion. Recently, a salt-stable terminus-specific binding factor was identified from Xenopus eggs although there is, as yet, no evidence that this protein interacts with telomeres in vivo (WE-

Telomeres have properties reminiscent of hetero- chromatin. In some higher eukaryotes, the ends of chromosomes are heterochromatic by cytological crite-

NAS f?t al. 1993).

68 E. A. Wiley and V. A. Zakian

ria (ZAKIAN 1989). In addition, yeast telomeres replicate late in S phase (MCCARROLL and FANGMAN 1988; WEL LINGER et al. 1993). In Drosophila (LEVIS et al. 1985) and in yeast (GOTTSCHLING et al. 1990), genes placed near telomeres are transcriptionally repressed. Yeast telomeres repress the constitutive expression of genes in a reversible manner, similar to the on-off pattern of expression in PEV. A gene under telomeric position effect (TPE) can switch between transcriptionally active and inactive states, with each state being stable for many generations (GOTTSCHLING et al. 1990).

The ability of telomeres to repress transcription of nearby genes may be the consequence of a specialized chromatin structure of the telomeric repeats and adja- cent DNA. The yeast C1-JA/TG1-3 repeats are assem- bled into a nonnucleosomal chromatin structure called the telosome, while adjacent DNA is in nucleosomes (WRIGHT et al. 1992). The chromatin of the adjacent DNA must be unusual, however, since DNA in this re- gion is not accessible to dam methylase when this gene is expressed in yeast (GOITSCHLING 1992), and the his- tones in subtelomeric chromatin are hypoacetylated compared with histones in active regions of the genome (BRAUNSTEIN et al. 1993). Certain histone modifications such as single amino acid substitutions in histone H4 (APARACIO et al. 1991) relieve TPE, further supporting the idea that chromatin structure is important for TPE. Moreover, genes required for TPE, including SIm, SIR3 and SIR4, are required for silencing at HML and HMR, two loci at which transcriptional repression is thought to result from a specialized chromatin structure (APARA- CIO et al. 1991).

Biochemical (CONRAD et al. 1990; WRIGHT et al. 1992), cytological (Klein et al. 1992) and genetic experiments (CONRAD et al. 1990; LUSTIG et al. 1990; KYRION et al. 1992) indicate that Raplp, an essential and multifunc- tional protein, associates with yeast telomeres in vivo. Moreover, expression of large amounts of a deletion derivative of Raplp, called RaplABBp, which lacks most of the Raplp DNA binding domain, causes telo- meres to lengthen (CONRAD et al. 1990). To explain this result, RaplABBp was proposed to compete with telomere-bound Raplp for a factor that prevents telo- mere lengthening.

Yeast chromosomes acquire and lose TGI-3 single- strand tails in a cell cycle-dependent manner (WEL LINGER et al. 1993). However, there is no biochemical evidence in yeast for salt-stable terminus binding pro- teins such as those in Oxytricha (J. WRIGHT and V. A. ZAKIAN, unpublished results). Using a genetic ap- proach, the addition of extra CI-&TGI-.S DNA was shown to cause telomeres to lengthen, presumably by competing for factors that determine telomere length (RUNGE and ZAKIAN 1989). Since C1-+4Z/TG1-3 tracts on a circle were as effective as extra telomeres in causing

telomere lengthening, the titrated factor that affects telomere length did not appear to be terminus specific.

We reasoned that conditions that make telomeres more accessible to lengthening might also make telo- mere adjacent genes more accessible to transcription. This paper shows that, indeed, addition of extra telo- meres or expression of high levels of RaplABBp re- lieved TPE, but that effects on telomere length were separable from effects on TPE. These data provide the first evidence for a terminus-specific binding protein in Saccharomyces and indicate that the presence of this protein is important for TPE.

MATERIALS AND METHODS

Yeast strains, transformations, plasmid construction: Yeast strains used were WS105 (MATa ade2 a d d lys2-801 leu2- 3,112 trplA ura3A aro2 canl) , 452 (MATa leu2 ura3 t rp l his3 reg1 cyh2 c u d ) (SCHUIX and ZAKIAN 1993) and 47925-111 (MATa his7 leu2 t rp l ura3 ade2 ade3 canl) (WELLINGER and ZAKIAN 1989). Yeast cells were transformed as described (SCHIESTL and GIETZ 1989) except that cells were not soni- cated. URA3 was placed at the chromosome VZJL telomere by fragment-mediated transformation using a 2.5-kb fragment from pVII-L URASTEL (GOTTSCHLING et al. 1990). The struc- ture of chromosome VZZ-L and all other chromosome and plasmid modifications were confirmed by Southern analysis.

To make the RIFl disruption plasmid, a pUC19-based plas- mid containing RlFl called pCH450 (obtained from DAVID SHORE) was digested with HpaI, thereby deleting 3.6 kb of the lilFl open reading frame. A 1.7-kb BglII fragment containing TRPl was blunt-ended by treatment with T4 DNA polymerase and then ligated with T4 DNA ligase to HpaI-digested pCH450 to make pCH450RT. pCH450RT was digested with AuuI and NdeI to liberate a 2.8-kb fragment used for integration at the ml locus.

To make a galactose-inducible SZR3 gene for integration at LYS2, the YCp50-based plasmid YCp125H (a gift fromJksPER RINE) was digested with SalI and PuuI to liberate a fragment containing SZR3 fused to the GAL1,IO promoter. This frag- ment was blunt-ended by treatment with T4 DNA polymerase and then ligated with T4 DNA ligase to SmuIdigested pDP6, which contains LYS2 (FLEIG et al. 1986). The resulting plas- mid, called pDPS3, was digested with XhoI for integration into the LYS2 locus. To induce Sir3p overexpression, cells carrying the integrated GAL-SZm construct were grown in medium containing 3% galactose as the sole carbon source.

To make the ADE2 disruption plasmid, a 3.6-kb BamHI fragment containing ADE2 from pL909 (a gift from R. KEII,), was ligated to BamHI-digested pVZl (HENIKOFF and EGHTE- DARZADEH 1987) to make pVADE2. pVADE2 was digested with BglII and StuI, thereby deleting 224 bp of the ADE2 OW. The resulting 6.5-kb fragment was isolated, blunt-ended by treatment with T4 DNA polymerase and ligated with T4 DNA ligase to a 2.3-kb fragment containing HZS3, obtained from a BamHI digestion of FATRS303’b’ (see below), which was blunt-ended by treatment with T4 DNA polymerase. The re- sulting plasmid, pVA2H3, was digested with BamHI to liberate a 5.6-kb fragment containing ade2::HZS3, which was used for integration at the ADE2 locus.

TO place ADE2 at the chromosome VR telomere, pVADADE2(+) (GOTTSCHLING et ul. 1990) was digested with NcoI and SalI to remove the entire adh4 sequence and a por- tion of the URA3 sequence. The resulting vector fragment

A Yeast Telomere-Specific Factor 69

was treated wi th T4 DNA polymerase. Plasmid pRG-IOH (a gift from C. NIWI,ON) was digested with Hind111 to liherate a 2.8-kh fragment containing sequences adjacent t o the srlh telomeric Y’ element on chromosome VR. This fragment was treated with T4 DNA polymerase and ligated with T4 DNA ligase t o the digested pVADADE2(+) to create p5RADE2, which was then digested with %/I and Sj,/ll to liberate a 7.2- kh fragmrnt IISCCI lor integration. Integration ofthis fragment replaces DNA distal to the Y’-adjacent sequence on chromo- some VRwith AIX’2and an 81-hp tract of C-:A/TGl-:,, which is elongated in v i r m t o form a new full-length telomere. For colony color detection, colonies were grown for 3 days at 30” and then were incubated for 1 week at 4’.

All episomal yeast plasmids used in this study with the ex- ception of YEpFAT.8 were derivatives of the circular plasmid YEpFATIO (Rrsc;~:. and ZAKIAN 1989). YEpFAT10 was linear- ized as descrihed (RCIN(;I:. and ZAKIAN 1989) to produce YLp FATIO. YEpFATl0.3 was made by inserting a 280-bp EcoRI fragment containing 276 hp of Cl-:A/TGl-:, isolated from pM,PV (WEI.I.ISGEK r/ n/. 1993) into the RnmHI site of YEp FATIO. YEpFAT10-based plasmids containing portions of the fitl’l gene have hecn described previously (CONMI) r / nl. 1990, and Figure 1R). To make FATlOSS and FATARRSJ, a 4.4-kh Sol1 fi-agment containing S I R ? from pKL3 (pUCl9- based plasmid from R. STERNGIANZ) was ligated to 1W”II- digested YEPFATIO and FATRAPARB. The plasmid YEP FAT.8 was constructed previously (FATRSS03’h“TT; STAVESr I W E N and ZAKIAN 1994). Briefly, three 2 7 M p tracts of <;I-J/TGI-:I were ligated together to form an 828-hp tract, which was then ligated to Spl-digested pRS303’h’ (obtained from D. G(YITSCIII.ING). pRS303‘b’ was constructed hv ligat- ing the 2.Gkh Xhnl fragment containing the 2 pm ARS and the hc2-d allele from YEpFATI 0 to the An/ll-digested pRSSO3 (SIKORSKI and HIFTEK 1989).

Nucleic acid and protein analysis: Total yeast genomic DNA was prepared by a glass head lysis procedure (RCNGE and ZAKIAS 1989). All enzymes were obtained from New England Biolahs and used as directed except that restriction enzymes were used in a different buffer (MIKKOWTC:H rt nl. 1984) with the addition of 10 mM dithiothreitol. Unless noted otherwise, yeast DNAs were analyzed by electrophoresis in 1% agarose gels and by Southern hyhridization as described previously ( W A I I I . r / nl. 1987). Probes for hyhridization were radiolabeled hy a random primer procedure (FEINREKG and VOGEISTEIN 1983). Plasmid copy numbers were determined as descrihed (R~YGE r / a/. 1991), except that genomic DNA was digested with 1:‘coRV. This digestion yields a 3.0-kb fragment from the chromosomal copy o f IXU2 and a 2.6kb fragment from the plasmid when probed with the 113.J2 I:’coRI-Kjlnl fragment. The amount of hyhridization to the plasmid and chromo- somal hands was quantitated by storage phosphor imaging (JOHNSTON r/ nl. 1990). To determine the structure of M,p FATl0, DNA from cells containing linear plasmids was also analpcd hy 2-D gel electrophoresis (RKF.WEK and FANGMAN 1987) and Southern hyhridization using a 3.5kb I k X l frag- ment of pVZl as a prohe for plasmid (HENIKOFF and EGMI‘E- I)AKA.AI~EH 1987). To monitor telomere lengths, total genomic DNA was either digested with Xhol, which releases an -1.3- kb fragment from the ends of Y’ hearing telomeres, or with pS/l, which cuts once within URA3 to liberate an -1.3-kh fragment containing the telomere of chromosome VfI-L. A 121-hp fragment containing 81 hp of Cl-:A/TGl-:, DNA from plasmid pYTcA-1 (Rlw;F. and ZAKIAN 1989) was used as a probe for X/toI-digestetl DNA while a 1.1-kh Hind111 fragment from URA? was used to detect the VfI-L telomere from h / I - digested DNA.

To determine the amount of Raplp and RaplARRp in

A M

B

DVZ

FI(;L:RE 1 .-Strain and plasmid constructions. (A) Insertion of URA3 adjacent to the telomere of chromosome Vlf-L. A portion of the left arm of chromosome V I 1 with AIIH4 is shown. Strains A, R and C were transformed with a fragment containing URA3 flanked by 81 bp of C-:A/TGl-:, and a portion of AIlH4. This step deletes 20 kh of DNA distal to ADH4. The short CI-:A/TGI-:, tract is elongated in u i 7 m to form a full-length telomere (GO~S(:F-II ,IN<; r / ( I / . 1990). As a result, URA? was placed immediately adjacent to the newly formed chromosomal end. The arrowhead represents the telomere. The region designated as “probe” was r~sed to probe Southern blots for the length of the chromosome VII- L telomere. (B) To make YEpFAT-hased plasmids containing a deletion derivative of Raplp (FATRAPABB), RAP1 was in- serted into the 6.8-kh plasmid YEpFATl0 (RL‘s(;E and ZAKIAS 1989) to make FATRAP1 then digested with HnmHI and reli- gated (CONRAD PI nl. 1990). FATRAPARRF was made by di- gesting FATRAPARB with HnmHI, tilling in the ends, and religating to generate a frameshift within the RAP1 ORF. Por- tions of Raplp: striped box, activation domain; checked hox. DNA binding domain; box with waved lines, nonessential re- gion; B, RnmHI. The 7.1-kh plasmid YEpFAT10.3 was created by inserting -270 bp of Cl-:A/TGl-:, sequence into YEP FATIO. (C) To make YEpFAT.8, 827 hp of Cl-:A/TG1-:, was inserted into FATRS303‘h‘ (S~AVENH.I\(;ES and ZAKIAN 1994; FATRS303’b‘ was constructed by DAN G o ~ s ( ; I I I . I N ( ; ) . (D) YLpFAT10 was created by linearizing YF,:pFATIO by digesting with Sal1 and ligating a fragment containing 81 hp ofCl-:A/ TGI-:, onto each end. The 81-bp tracts are elongated in vivo to form hdl-length telomeres (RoNc;~.: and ZAKIAS 1989).

ceIIs, total yeast protein was isolated from -.i X 10‘’ cells from the strains containing YEpFATl 0, FATRAPABR, FATARRSS, FATIOSS or no plasmid as previously dcscrihed (CONMI) P/

nl. 1990). Protein samples (80 pg) were electrophoresed on a 10% SDSpolyacrylamide gel and electrohlottetl to nitrocel- lulose. The hlot was stained with 0.01% amido hlack to verify

70 E. A. Wiley and V. A. Zakian

that equal amounts of protein were in each lane and was prepared as directed, for probing with antibody and detection with horseradish peroxidase-labeled secondary antibody (ECL Detection kit; Amersham). The anti-Raplp serum, which was diluted 1:1000, was a generous gift from SUSAN GASSER.

To verify Sir3p overexpression, total yeast protein was iso- lated as described above from strain A containing YLpFATlO and from strain A with the GAL-SIR3 construct integrated at LYS2 (strain A-GS3) containing YLpFATlO. Both strains were grown in YC-trp with galactose as the sole carbon source. From each strain, equal amounts of protein (100 pg) and a twofold dilution series of this amount was electrophoresed on a 7% SDSpolyacrylamide gel and electroblotted to nitrocellu- lose. The blot was then prepared for probing with anti-Sir3p serum diluted 1:lOOO (a generous gift from J. RINE) and for detection as described above. An estimate of the amount of Sir3p in strain A-GS3 carrying YLpFATlO was estimated by quantitating the amount of binding to Sir3p in the dilution series by densitometry.

Quantitative mating assay Tester strains (PTla and PT2m) and the strains to be tested were grown to mid-log phase (- 1 X 10' cells per ml) in liquid culture, then briefly sonicated. Dilutions of the strains were counted using a hemocytometer, and equal numbers (1 X lo5) of test cells and tester cells were mixed and pipetted onto sterile 25-mm nitrocellulose disks placed on prewarmed YEPD plates. The disks were incubated at 30" for 5 hr and then were transferred separately into 5 ml of sterile water and vortexed. The cell suspensions were pipetted into new tubes and centrifuged, and the pellets were resuspended in 1 ml of sterile water. Dilutions of each suspen- sion were plated on medium selective for diploids. At the same time that test and tester cells were mixed for mating, dilutions of the test strain culture were plated on YC (or medium selective for plasmid-bearing cells) to determine the number of viable cells. Mating efficiency was determined by dividing the number of diploids formed by the number of viable cells or by the number of plasmid-bearing cells in the case of plasmid-bearing strains.

Assay for telomere position effect: Two independent trans- formants for each plasmid were selected from each strain on synthetic medium, (hereafter called YC; ZAKLW and SCOTT 1982), lacking tryptophan (YC-trp) and then were streaked two successive times on medium lacking leucine (YC-leu) or on YC-trp. At least three colonies from the last restreak of each transformant were suspended in Hz0, and serial dilu- tions were made. The dilutions were plated on YC-leu (or YC- trp), YC-leu (or YGtrp) lacking uracil and YC-leu (or YC-trp) containing 5-fluoroorotic acid (5-FOA). To determine the percentage of cells displaying TPE, the number of colonies that formed on 5-FOA medium was divided by the number that formed on medium lacking 5-FOA. To ensure that the ability of cells to grow on 5-FOA was due to TPE instead of to loss of URA3 function, cells from each colony plated on 5- FOA were also plated on medium lacking uracil. In each case, the fraction of cells able to grow on medium lacking uracil was indistinguishable from that for cells able to grow on me- dium with uracil. The number of colonies on a plate was determined after 3-4 days of growth at 30". Median values for the fraction of 5-FOA-resistant (5-FOAR) cells from at least three colonies from each of two transformants (six colonies) was used as the value for the fraction of 5-FOAR cells as pre- sented in Tables 1-3. For TPE assays on cells carrying the RIFl disruption, three colonies were selected from each of two transformants grown on YC. Platings were the same as for cells carrying YEpFAT10-based plasmids, except that all media contained leucine. For TPE assays on cells overex-

pressing Sir3p by the GALZ,ZOpromoter, all media contained 3% galactose as the sole carbon source.

RESULTS

General methods for monitoring TPE: To monitor TPE, the URA3 gene was introduced next to the telo- mere on the left arm of chromosome VI1 (GOTTSCH- LING et al. 1990; Figure 1A). Expression of URA3 is nec- essary to support growth on medium lacking uracil. However, cells producing Ura3p cannot grow in the presence of 5-FOA, because it is converted into a toxic substance by the URA3 gene product (BOEKE et al. 1987). In strains with the URA3 gene at its normal loca- tion on chromosome V, 5-FOAR cells arise at a fre- quency of -lo", a frequency consistent with the spon- taneous mutation rate of URA3 (BOEKE et al. 1984). However, when the URA3 gene is near a telomere, ex- pression of the gene is repressed in many cells, such that a substantial fraction of these cells can form colonies on plates containing 5-FOA (GOTTSCHLING et al. 1990). Thus, the frequency of 5-FOAR cells can be used to monitor the fraction of cells in which a telomeric URA3 gene is transcriptionally repressed.

The fraction of 5-FOAR cells varies among different laboratory strains from to -9 X 10" (GOTTSCH- LING et al. 1990; unpublished results from this lab). Since the basis for these strain dependent differences is unknown, we examined the effects of the various conditions on TPE in three strains: WS105, 452, and 47925-1 11, hereafter called strains A, B, and C, respec- tively. With URA3 next to the telomere of chromosome VII-L in each strain, the basal level of 5-FOAR in the three strains was 5.5 X lo-* (strain A), 3.5 X 10" (strain B), and 1.0 X 10" (strain C) (Table 4).

In addition to differences between strains, the frac- tion of 5-FOAR cells also varies somewhat among indi- vidual colonies from the same strain. Therefore, for each condition tested, a minimum of three colonies from each of two independent transformants were tested for TPE. For each experiment, the median and range of values are presented for the six (or more) colonies assayed from each class of transformant (Ta- bles 1-4).

Extra telomeres, but not internal tracts of telomeric DNA, substantially reduce WE To determine if the addition of extra telomeres affects TPE, a linearized version of the multicopy plasmid YEpFATlO (Figure 1B) was used. YEpFATlO contains the TRPl gene and a promoter-defective allele of LEU2 called leu2-d (RUNGE and ZAKIAN 1989). With this vector, plasmid copy number can be controlled by the growth medium. When cells are grown in synthetic medium (hereafter called YC) lacking tryptophan (YC-trp), the plasmid is present in 20-50 copies per cell whereas cells typically have 100-200 copies of the plasmid when grown in

A Yeast Telomere-Specific Factor 71

TABLE 1

Extra Cl-a/TGl-s DNA as telomeres reduces TPE

Fraction FOAR cells"

Strain A Circular vector (-leu)

Linear plasmid (- trp)

Linear plasmid (-leu)/

Internal tract plasmid (-trp)

Internal tract plasmid (-leu)

Strain B Circular vector (-leu)

Linear plasmid (-trp)

Linear plasmid (-leu)

Internal tract plasmid (- trp)

Internal tract plasmid (-leu)

Strain C Circular vector (-leu)

Linear plasmid (- trp)

Linear plasmid (-leu)/

Internal tract plasmid (-trp)

Internal tract plasmid (-leu)

2.0 x 10"

1.0 X 10-~

2.0 x lo-"

(1.0 X 10"-4.0 X 6)

(3.0 X 10-6-2.0 X 8)

(1.4 X 10-"-7.0 X 6) 6.4 X 10"

(4.7 X 10-'-6.9 X lo-'; 6) 7.2 X 10"

(2.5 X 10"-9.9 X 10"; 6)

2.1 x 10"

1.0 X

2.7 X

(1.2 X 10-'-3.3 X lo-'; 6)

(4.0 X 10-5-l.l X 10"; 6)

(8.5 X 10-"-4.6 X 6) 6.1 X 10"

(3.9 X 10"-7.4 X 10"; 6) 7.2 X 10"

(4.6 X 10-'-9.8 X 10"; 6)

8.8 X 10" (1.6 X 10-2-1.8 X 10"; 6)

4.0 X (2.0 X 10-7-1.4 X 10"; 8)

(1.1 X 10-5-2.1 X 6) 8.0 X 10"

(7.6 X 10-'-8.3 X 10"; 6) 6.5 X 10"

(3.0 X 10-'-8.8 X lo-'; 6)

6.0 X lo4

Total Cl-.&/ Copy No./cellb TG-3 (kb)'

ND' NA'

35 (30-40)

62 (60-65)

55 (50-60)

160 (145-175)

41

70

28

61

ND NA

50

85

60 (58-62)

120 (1 10- 130)

(46-54)

(70-95)

53

92

30

50

ND NA

50

50

60

130

(47-53)

(45-60)

(55-65)

(126-134)

53

53

30

53

Values are the median fraction of 5-FOAR cells with the range of values and the number of colonies tested in parentheses. bThe average plasmid copy numbers per cell with ranges in parentheses. 'The total amount of C1-&/TG1-3 DNA in cells carrying the internal tract plasmid or the linear plasmid was estimated by

measuring the average lengths of telomeres and plasmid copy numbers, both determined by Southern hybridization. Not determined. Not applicable.

/Conditions and strains in which the linear plasmid was rearranged.

medium lacking leucine (YC-leu) (RUNGE and ZAKIAN 1989). The linearized version of YEpFATlO, YLpFATlO, hereafter referred to as "the linear plasmid" (Figure l D ) , was introduced into each strain carrying URA3 at the telomere of chromosome WJL. As a control, the effect of the circular vector alone was also monitored in each strain. For each strain growing in YC-trp, the addition of -70 to -100 extra telomeres, that is, -35- -50 copies of the linear plasmid, reduced the fraction of cells exhibiting TPE (Table 1). Compared with the fraction of 5-FOAR cells for the same strain carrying the circular vector control, the median reduction in TPE was -200-fold in strain A and -2000-fold in strains B and C (Table 1). At these copy numbers, the linear

vector has little or no effect on telomere length (RUNGE and Z m 1989, Figure 2). When cells were grown in YC-leu, where the copy number of the linear plasmid is expected to increase, telomeres grew longer in all three strains (Figure 2). In YC-leu medium, TPE was reduced even further in strains A and B compared with cells grown in YC-trp (10,000- and 8,000-fold, respec- tively, compared with cells carrying the circular vector control; Figure 3) . In strain C, however, the linear plas- mid copy number did not increase (Table 1 ) and TPE actually increased in cells grown in YC-leu compared with cells grown in YC-trp, although TPE was still - 100- fold reduced compared with cells carrying the circular vector control.

72 E. A. Wiley and V. A. Zakian

TABLE 2

Long internal tracts of Cl-&TGl-s DNA slightly reduce TPE

Strain B Total Cl-.&/

Fraction FOAK cells" Copy no./cell" TGI-8 (kb)'

Circular vector 5.6 X 10" 95 NA'

Internal triple tract' 1.0 x 10" 115 107 (3.4 X 10"-8.9 X 10"; 6) (90-105)

(8.0 X 10-'-4.7 X 10"; 6) (105-130)

The median fraction of 5-FOAR cells with the range of values and the number of colonies tested in

The average plasmid copy numbers per cell with ranges in parentheses. 'The total amounts of telomeric DNA present per cell are also presented. 'Not applicable. 'Vector with 828 bp of Cl-&/TGl-s DNA.

parentheses.

Prompted by the disparity between strain C and the others, the structure of the linear plasmid was exam- ined. Most linear plasmid molecules were rearranged in strain C grown on YC-leu medium. These re- arrangements resulted in either an increase in plasmid size consistent with end-toend fusion of plasmid mole- cules or in plasmid circularization (data not shown). Since these rearrangements reduced the number of ex- tra telomeres, they probably explain why growth in YC- leu did not cause a further reduction in TPE in this strain. Some linear plasmid molecules were also larger in strain A grown on YC-leu, but the size and structure of the linear plasmid was as expected in all three strains grown on YCtrp and in strain B grown on YGleu (data not shown).

Similar experiments were conducted with cells car- rying the circular plasmid YEpFAT10.3, which contains a 276-bp tract of C1-&TG1-3 DNA, a plasmid hereaf-

ter called "the internal tract plasmid" (Figure 1B). In each strain, for cells grown in either YC-trp or YC- leu, the structure of the internal tract plasmid was indistinguishable from that of the starting plasmid (data not shown). Southern hybridization established that the presence of the internal tract plasmid caused an increase of -200 bp in the lengths of chromosomal telomeres in each strain for cells grown in YC-leu (Fig- ure 2). In contrast to the reduction in TPE observed with the linear plasmid, the presence of the internal tract plasmid caused a small increase in TPE in each strain (Table 1, Figure 3).

These data suggest that a factor that is important for TPE can be titrated from chromosomal telomeres by extra telomeres but not by internal tracts of C,-.& TG1-8 DNA. Since the amount of C1-.&TGl-s DNA in YC-leu-grown cells with the internal tract plasmid was comparable to or greater than that in YC-trp-grown cells

TABLE 3

Overexpression of Si3p does not restore wild-type levels of TPE to cells carrying extra telomeres

Fraction FOARcells" Copy no./cell

Strain A Wild type' 1.4 X 10" NA'

Circular vector 9.4 x 22

Linear plasmid 5 X 20

(1.2 X 10-*-1.7 X 10"; 6)

(8.5 X 10-3-l.l X 6) (18-25)

(<1 X 10-7-8 X 6) (18-22) Strain A-GS

Wild type 1.1 x 10-1 NA

Circular vector 7.0 X lo-' 30

Linear plasmid 4.1 X 27

(9.0 X 10-'-1.2 X 10"; 6)

(6.4 X 10"-8.2 X lo-'; 6) (25- 35)

(5.0 X 10-7-l.2 X 6) (22-32)

"The median fraction of 5-FOAK cells with the range of values and the number of colonies tested in

'Wild type refers to the untransformed strain. 'Not applicable.

parentheses. All data are for cells growing with galactose as the sole carbon source.

A Yeast Telomere-Specific Factor

TABLE 4

Expression of RaplABBp reduces telomere position effect

Fraction FOAK cells''

Strain A No plasmid 5.5 x 10-2

YEpFATl 0 2.0 x 10"

FATRAPARRF 8.0 x IO"

FATRAPARB 2.0 x lo-"

RIF1::TRPI 7.0 x 10-1

(5.0 x 10"-6.0 x 10-2; 6 )

(1.0 X 10"-4.0 X IO-'; 6)

(7.0 x 10-2-2.1 x 10-1; 6)

(1.0 x 10-"-3.0 x 6)

(6.4 X 10-'-7.3 X lo-'; 6) FAT-SIR3 2.1 x 10"

(4.8 x 10-"2.9 x lo- ' ; 6) FATABBSIRS 2.3 x 10"

(3.2 X 10"-4.3 X lo-': 6 ) Strain R

No plasmid 3.5 x IO"

YEpFAT I O 2.1 x 10"

FATRAPARBF 5.8 x 10"

FATRAPARB 3.0 x

RIF1::TRPI 8.2 x 10"

(3.3 x 10-I-4.0 x 10-I; 6)

(1.2 X 10-'-3.3 X lo-'; 6)

(5.0 X 10-'-6.5 X 10"; 6)

(5.0 x 10-"-8.0 x 10P; 12)

(6.5 X 10-'-9.5 X IO-'; 6) Strain C

No plasmid 1.0 x 10"

YEpFATl0 8.8 x 10"

FATRAPARRF 3.1 x 10"

FATRAPARB 2.5 x 10"l

RIF1::TRPI 7.9 x 10"

(5.0 x 10-"2.0 x 10-1; 6)

(1.6 X 10"-1.8 X IO-'; 6)

(1.7 X 10"-4.0 X lo-'; 6)

(2.0 x 10-"9.0 x lo-.*; 6 )

(6.3 X 10"-9.0 X IO- ' ; 6)

"The median fraction of 5FOAK cells with the range of values and the number of colonies tested in parentheses.

carrying extra telomeres (linear plasmid; Table l ) , their inability to compete for this hypothetical factor was not a consequence of there being less CI-.&/TGI-l DNA in cells carrying the internal tract plasmid compared with those with the linear plasmid. One explanation for these data is that there is a terminus binding factor that is important for TPE, that binds to and is titrated by extra telomeres but not by internal tracts of Cl-&/

I t has recently been shown that -890 bp of Cl-&/ TGI-3 DNA can repress transcription of an acljacent gene at an internal site on a chromosome whereas -280 or -550 bp of CI-:AA/TGI-:l DNA cannot (STAL'ENHA- GEN and ZAKIAN 1994). These data caused us to consider the possibility that the putative terminus binding factor

TGI -:,DNA.

plasmid linear

z y 1 2 1 2 - t r p I - leu

2.0 - 1.6 - 1.0 -

internal tract 0

plasmid F - t r p -leu U

1 2 1 2 y

FI(:L.RF. 2.-Extra Cl-:A/TG-:, seqwncc incrc-;tsc.s thc length of the telomere on chromosome 1 W L . Two intlcprn- dent transformants from strain A with circul;u- v c ~ t o r (Wp FATIO) or linear plasmid or the vector with a 27CFbp ( ; l - : W

TG,-:% tract (internal tract plasmid). Two transformants with each plasmid were grown for 1 0 0 generations o n Y(;-lcu o r YGtrp. DNA was digcstcd with I ' d , sul?jcctetl t o clrctrophorc- sis on a 1.0% agarose gel. and transfcrrrd to a nylon n w n - brane. The b l o t was probed with the Ifindlll fragmcnt of URA3 (Figure IA).

might bind internal tracts of CI-:A/TGI-:l DNA i f the internal tracts were long enough. To test this possibility, a plasmid related to YEpFATlO carrying 828 hp of Cl-:&TGl-3 DNA, hereafter called "the triple tract plasmid" was used (YEpFAT.8; Figure 1C; SIAL'ENI I;{-

GEN and ZAKIAN 1994). The triple tract plasmid or the vector alone as a control were transformed into strain B. Cells were grown in YGleu, and individual colonies were assayed for the fraction of .~-FoA~ cells.

Strain B carrying the triple tract plasmid contained -107 kb of CI-:A/TGI-:r DNA when grown i n YC-leu (Table 2, STALXNHAGEN and ZAKIAN 1994). This amount was considerably more than the amount of CI-:A/ TGl-3 DNA for YGtrp grown cells carrying extra te- lomeres (linear plasmid; Table 1). Nonetheless, com- pared with control cells, the triple tract plasmid re- duced the fraction of 5-FOAK cells only -5-fold (Table 2), whereas the linear plasmid reduced TPE -8000- fold (YGleu; Table 1). Thus, if long internal tracts of CI-:A/TGI-:I DNA compete for the putative terminus binding protein, they do so much less effectively than telomeres. Alternatively, the small reduction in TPE as- sociated with the presence of the triple tract plasmid could be due to the ability of the internal C,-:+A/TG,-:I tracts to compete with chromosomal telomeres for Raplp, Raplp interacting factors or both. Since Raplp is an abundant protein, estimated to be present in 5000 copies/cell (BIJCHMAN P/ nl. 1988), it is probably not limiting in most situations. However, using an estimate of one Raplp binding site ever), -40 bp i n Cl-:&/TGl-:I DNA (LONGTINE P/ d . 1989; WANG and ZAKIAN 1990), the triple tract plasmid introduced enough potential Raplp binding sites that Raplp might start to be lim- iting in vivo in cells carrying the plasmid, and thereby reduce the fraction of cells exhibiting TPE.

74 E. A. Wiley and V. A. Zakian

I I

strain A : strain B : strain C 1

0.1 FIGURE 3.-The median value and ranges

strains with different plasmids or the RlFl dis-

plasmid were grown in YC-leu medium except o.ooo1 for strain C carrying the linear plasmid, where

cells were grown in YC-trp. Arrows indicate an o.00001 increase or a reduction in the fraction of 5-

FOA' cells compared with cells with control o.ooooo1 vector. The control for FATRAPABB was FA-

TRAPABBF; the control for the linear plas- o.oooooo1 mid and the internal tract plasmid was the

circular vector (YEpFATlO). Data are from Tables 1 and 2.

0.01 for the fraction of 5-FOA' cells in all three

0.001 ruption. In each case, cells containing the

w circular vector 0 linear plasmid (extra telomeres)

FATRAPABB internal tract plasmid - Arif l

Taken together, these data suggest that at least one factor that interacts with chromosomal telomeres and is important for TPE can be titrated effectively from chromosomal telomeres by extra telomeres but not by short or long internal tracts of CI-I/\/TG1-J DNA.

The decrease in TPE by extra telomeres is not spe- cific to URA3 nor to the VII-L telomere: To test whether the decrease in TPE caused by the presence of extra telomeres was specific to the chromosome v71-L telo- mere or to the URA3 gene, a wild-type copy of ADE2 was placed near the chromosome VR telomere in a derivative of strain B in which ADE2 was disrupted at its normal locus. On medium containing low amounts of adenine (YC low ade) , cells that fail to express ADE2 produce red colonies (ROMAN 1956). Therefore, cells with ADE2 near a telomere give rise to two types of colonies, red colonies with white sectors (ADE2 re- pressed switching to ADE2 expressed) and white colo- nies with red sectors (ADE2 expressed switching to ADE2 repressed) (GOTTSCHLING et al. 1990; KYRION et al. 1993; Figure 4A).

To test the effect of extra telomeres on telomeric ADE2 expression, strain B carrying M E 2 at the telo- mere of chromosome VR was transformed with either a linear plasmid or with the circular vector control. Transformants with the vector control gave rise to red colonies with white sectors and white colonies with red sectors (Figure a), while transformants with the linear plasmid gave rise to only white colonies with very few FIGURE 4.-Extra telomeres reduce TPE on ADE2 at the or no red sectors (Figure 4B). When the linear plasmid chromosome V-R telomere. Cells carrying a circular derivative was lost, TPE was restored as detected by the presence Of YEPFAT1o lacking Leu2-d, ca11ed El'T (A) Or a linearized

of red colonies with white sectors and by an increased trp low adenine medium. Cells in which ADE2 is expressed version of that plasmid called YLpT (B) were grown on YG

number of red sectors in the mostly white colonies (data are white; cells in which ADE2 is repressed are red, The arrOw not shown). These results demonstrate that the reduc- points to a very small patch of red.

A Yeast Telomere-Specific Factor 7.5

str. A-GS3 + vx,*b9 linear plas. 6' tQ I 8-e!* '

g7-&.

kSir3p

69 - - c -

FIGURE 5.-Overexpression of Sir3p in strain A-GS3. Total proteins extracted from strain A and strain A-GS3 with the linear plasmid and grown in medium containing galactose were subjected to Western blot analysis to compare levels of Sir3p. Two transformants of strain A-GSS carrying the linear plasmid, designated (1) and (2), and one transformant of strain A carrying the linear plasmid were compared. Wild- type levels of Sir3p were undetectable with the antisera used, even when 16 times more total protein was loaded compared with the total protein loaded from strain A-GSS in a dilution experiment. The -60-kD band that cross-reacts with the Sir3p antiserum is seen even in a sir3A strain as noted previously by others using the same antiserum (PALIADINO et al. 1993).

tion of TPE caused by extra telomeres is not specific to one gene nor to one telomere.

Overexpression of Sir3p does not restore wild-type levels of TPE to cells carrying extra telomeres: Of the genes known to modi9 TPE (APARACIO et al. 1991), the product of SIR3 behaves as if it is limiting, since overexpression of Sir3p increases TPE (RENAULD et al. 1993). We tested the possibility that the reduction in TPE observed with the addition of extra telomeres was due to their titrating Sir3p. A DNA fragment containing the CALI, 10promoter upstream of SIR3was integrated into the LYS2 locus of strain A, to produce strain A- GS3. Growth of strain A-GS3 with or without the linear plasmid on medium containing galactose caused a 2 10- fold increase in the amount of Sir3p over wild-type lev- els as estimated by Western analysis of serial dilutions of total protein from strain A-GS3 compared with the amount of Sir3p in strain A grown in YGtrp galactose and carrying the linear plasmid, as described in MATERI- ALS AND METHODS (Figure 5, data not shown). Since cells with the linear plasmid had only two to three times more telomeres than control cells (Table l ) , a mini- mum 10-fold increase in the level of Sir3p should be sufficient to suppress the reduction in TPE by extra telomeres if this reduction were caused by titration of Sir3p.

The fraction of 5-FOA' cells in individual colonies grown on YGtrp medium containing galactose was de- termined for both strain A and strain A-GS3 carrying the circular vector control (YEpFATlO) or extra telo- meres (the linear plasmid). As expected (RENAULD et nl. 1993), overexpression of Sir3p increased TPE in both strains (Table 3). Also, as expected, extra telomeres virtually eliminated TPE for galactose-grown cells

(strain A; fraction FOA' cells -1 X lo-', Table 3). In the presence of excess Sir3p and extra telomeres, the fraction of FOAK cells was -4 x lo-" (strain A-GS3; Table 3). That is, despite the large increase in the amount of SirSp, TPE was still reduced - 1,000-fold in cells containing extra telomeres compared with control cells (Figure 6, right). These data suggest that even if extra telomeres titrate Sir3p from chromosomal teltr meres, there must be at least one other factor important for TPE that is also titrated by extra telomeres. Consis- tent with the idea that extra telomeres do not reduce TPE by titrating SiAp, extra telomeres have very little effect on the mating efficiency of strain A, whereas mat- ing efficiency was reduced -10,000-fold in a sir3A de- rivative of strain A (data not shown).

Expression of the carboxyl terminus of Raplp re- duces telomere position effect: In previous work, a de- letion derivative of the R A P 1 gene was made by digcst- ing a plasmid containing R A P 1 with BnmHl and then religating (CONRAD et (11. 1990). Elimination of the 1.4 kb fragment between the two BnmHI sites in IMP1 crc- ates an in-frame deletion. The deleted gene can poten- tially encode a 348 amino acid polypeptide (called RaplABBp), which lacks most of the DNA binding do- main of Rapl p (HENRY et (11. 1990; Figure l R). As ex- pected, RaplABBp does not bind telomeric DNA in an in vitro gel shift assay (CONRAD PI d . 1990).

The gene encoding Rapl ABBp was carried on Ep- FAT10 (CONRAD et a1. 1990; Figure 1 R ) . This plasmid containing the deletion derivative of RAP1 will be re- ferred to as FATRAPABB. Two plasmids were used as controls for FATRAPABB. One plasmid was the vector alone, YEpFATlO. The other was a version of FA- TRAPABB in which the RnmHI site had been filled so as to create a frameshift mutation of RAPIABB, hereafter called FATRAPABBF (CONRAD et n1. 1990).

YEpFATlO, FATRAPABB and FATRAPABBF were introduced separately into the three strains. Cells car- rying FATRAPABB and grown in YC-leu produced large amounts of a protein of the proper size to be RaplABBp, as determined by Western analyses using a Raplp antiserum (data not shown). In each of the strains studied here, expression of Rap1 ABBp caused the chromosome VII-L telomere to lengthen by -200 bp compared with the controls (Figure 7), consistent with results shown previously for a different strain (CONRAD et (11. 1990). Consistent with this preL' '1011s

study, overexpression of full-length Raplp caused telo- meres to become more heterogeneous in length (FA- TRAP1 ; Figure 7).

Individual colonies carrying the different plasmids and grown on YGleu were assayed for TPE by determin- ing the fraction of 5-FOA' cells (Table 4). In all three strains, the two control plasmids had little or no effect on the fraction of 5-FOAR cells compared with cells without plasmid. In contrast, FATRAPABB substantially

76 E. ,.\. M'ilcy and \'. A . %;d&m

RaplABBp + 8 linear plasmid + Sir3p overexpression Sir3p overexpression

(glucose grown) (galactose grown) I

1

0.1

0.01

0.001

0.0001

0.00001

0.000001

0.0000001

a I T

1

0.1

0.01

0.001

0.0001

0.00001

0.000001

1 $. 40,OOOX 8

8 I 0.0000001 8 ~20,000x

YEpFATlO (vector Control) YEpFATlO (Vector Control)

E FAT-SIR3

0 FATABB-SIR3

linear plasmid (extra telomeres)

reduced TPE in each strain. When compared with cells carrying FATRAPABBF, an -40,000-fold decrease was observed with strain A, -200-fold with strain B and

LL

1.0 - Q

E a

z U t-

1 2

m m m m 4 P n n a a U L T

FIGLIKE 7.-Expression of Rap1 ABBp makes chromosome VII-I. telomeres longer. Strain A was transforlned with E p FATlO, FATRAPABIZ, FATRAPABBF antl. for comparison IO previous results, with FATRAPI. Two indrprndent tranfor- mants for each plasmid were analyzed after 40 gcnel-ations of growth on Y<;ler~. DNA was digested with /'$/I, suhjecttd t o electrophoresis in a 1.0%) agarose gel, antl transferred t o a nylon membrane. The b l o t was prol)etl with the Hind111 frag- ment from URA3 (Figurc ]A). Size markers ;u'e in khp.

FI(X:KI; 6.-Thc Incdian K I I U C and rangcs li)r the lracrion of .5-FOAK cclls in strain A and strain AGSJ (ovcrcx- pressing SirJp) with various plasmids. Arrows indicate an increase o r ;I rctluc- tion in the fraction of .'-FOXK crlls compared with adjacent controls. Cells on the lrft were grown in glucose me- dium and on the right in galactose me- dium. Data are from Tables 3 antl 4.

-1200-fold with strain C (Figure 3). These results sug- gest that there is at least one factor that is important for TPE that interacts with the carboxyl terminus o f Raplp and that can he titrated away from chromosomal telomeres by expressing large amounts of a non-DNA binding version of Raplp. By the colony color assay for ADi:'2 expression described previously in this paper, expression o f Rap1 ABBp also reduced TPE on A f X 2 (data not shown).

Titration of Sir3p but not Riflp can explain the de- crease in TPE associated with Rapl ABBp expres- sion: R F I encodes a nonessential protein that interacts with the carboxyl terminus of Raplp in vivo (HARDY PI nl. 1992). To test if the decrease in TPE resulting from expression of the carboxyl terminus of Raplp were due to titrating Riflp from telomeres, I W l was disrupted in strains A, €3 and C . In each strain, disruption of fUFI caused a 200-300-bp increase in telomere length (Fig- ure s), as well as a 1.3-8-fold increase in the fraction of .i-FOAK cells (Table 4, Figure 3), consistent with an earlier study showing that TPE is somewhat increased i n the absence of Riflp (KWION PI nl. 1993). Thus, the ohsenfed reduction in TPE associated with expression of Rapl ABBp cannot he explained by removal of Riflp from chromosomal telomeres, and long telomeres are

h Yeast Telomere-Sprcific Factor ”

/ /

2.0-

1.6-

I

FIGI’KI. X.-Disruption o f IW:l and expression of RaplpABBp i n the same cell has an additive effect o n teltr mere length. YEpFAT4ARR. a plasmid similar t o FA- TMPARR that carries lilbt3 instead o f TIU’I, was trans- formed i n t o strain A w i t h o r w i t h o u t a IUFI disruption. Cells with plasmid were grown in YC-leu. Telomcrr lengths from three intlcpendent transformants w i t h a IUFI disruption ( ~ l : : ’ / ~ l U ’ l ) on ly , YEpFAT4ABR only, or w i t h Kp- FAT4ARR and 1UFI::TIWl were compared. DNA was tli- gested with Xlrol, subjected t o electrophoresis i n a 1.1 ’%I a p - rose gel, transferred to a nylon membrane, and probed w i t h a fragment containing (;,-:A. The strongly hybridizing band in each lane contains the terminal restriction fragment from -70% of the chromosome ends. Higher molecular weight hands represent individual telomeres.

not in themselves sufficient to reduce TPE. Moreover, in cells with a IUFl disruption, expression of Rapl ABRp had an additive effect on telomere length (Figure 8). Therefore, the increase in telomere length observed with expression of Rapl ABRp is also not due to removal of Riflp from telomeres.

Since SirSp is limiting for TPE, we tested the possibil- i ty that the decrease in TPE that resulted from expres- sion of the carboxyl terminus of Raplp were due t o titrating SirSp from telomeres. SirSp was overexpressed in cells producing RaplABBp by placing S I R 3 on FA- TRAPABR to make FATARRSIRS. As a control, SIIU was also placed on YEpFATl0 to make FAT-SIRS. FATARRSIRS and FAT-SIRS were introduced sepa- rately into strain A. Western analysis of total proteins established that cells carrying FATARRSIRS produced the same amount of RaplARRp as cells carrying FA- TRAPARR and also showed that SirSp was overex- pressed in cells carrying FATABRSIRS or FAT-SIRS (data not shown). FAT-SIRS and FATABB-SIRS both caused an -10-fold increase in the fraction of FOAK cells compared with cells carrying YEpFATlO (Table 4, Figure 6, left). Therefore, overexpressing SirSp com- pletely suppressed the reduction in TPE associated with Rapl ARRp. These results suggest that the reduction in TPE observed with expression of RaplARRp is due to titration of SirSp. Alternatively, high levels of SirSp might bypass Rapl ARBp expression.

DIS<:uSSIOS

This study describes several conditions that affect TPE. Extra telomeric DNA introduced as -iO to - 100 extra telomeres ( -35 to “50 linear plasmids/ccll) suk stantially reduced TPE on h4o different genes at two different telomeres i n three different strains (Table 1, Figure 4). At this copy nambcr, extra telomeres had little or no effect on telomere length (Figure 2; R ~ * x ; E and ZAKIAS 1989). M’e propose that extra telomeres decrease TPE by titrating telomere-interacting fictors away from chromosomal telomeres. I n contrast, intro- duction of circular plasmids containing extra C , -:A/ TG,-:, DNA as internal tracts the size of normal telo- meres carlsed telomrres to lengthen (Figure 2) but ac- trlally increased TPE (Table 1). Introduction ofintcrnal tracts that are three times longer than normal telomeres only slightly reduced TPE (Table 2, S-~A\~I-SIIA(;ES and ZAKIAS 1994). The disparity bctwccn the cffcct ofinter- nal C,-:&/TGI-:, tracts 71.7. telomeres on TPE was not a trivial consequence of there being less telomeric DNA i n cells carrying the circular plasmid. Therefore, the different effects of the linear plasmid 71s. the internal tract plasmid on TPE must be a consequence o f the nature of the added CI-:&TGI-:I DNA, i r . , telomeres r)s. internal tracts, rather than of the quantity of C,-& TG,-:, DNA. These data suggest that there is at least one factor that is important for TPE that has a higher affinity for DNA termini than for internal (:,-:A/ TGI-:, tracts.

As shown previously ( C o x ~ ~ \ n P/ 01. 1990). exprcssion of large amounts of a version o f Raplp that cannot bind DNA, Rap1 ARRp, caused telomeres to lengthen (Figure 6) and, as shown here, substantially reduced TPE in three different strains and on two different genes: URA3 (Table 4) and AIX2 (data not shown). M‘c propose that this phenotype results from the titration away from chromosomal telomeres of one or more lim- iting factors that interact with the carboxyl terminus o f Raplp. The importance of the Raplp carboxyl terminus for TPE has also been establishecl by demonstrating that cells expressing carboxyl terminal truncations of Rapl p have greatlv reduced TPE ( KWIOS P/ nl. 1993). Moreover, the effects on telomere length suggest that there is at least one factor in addition to Riflp that interacts with the carboxyl terminus of Raplp to limit telomere length (Figure 8).

SirSp appears to be limiting for TPE (RESAULI) r / 01. 1993). Overexpressing SirSp completely suppressed the reduction in TPE caused bv RaplARRp. I n contrast, TPE was eliminated in most cells carrying extra telo- meres (FOAK -1OF.’) even in the presence of excess SirSp. These results suggest that SirSp interacts with the carboxyl terminus of Raplp in 71i710and that Rap1 ARRp reduces TPE by titrating SirSp. However, extra telo- meres must reduce TPE by titrating a factor other than

78 E. A. Wiley and V. A. Zakian

Sir3p. This titrated factor is unlikely to be Raplp. Raplp is an abundant protein (BUCHMAN et al. 1988), and it displays no preference for telomeres over internal tracts of C1-&TG1-3 DNA in an in vitro binding assay (BER- MAN et al. 1986). In addition, strains with temperature sensitive RAP1 alleles that have reduced or no DNA binding activity by in vitro gel shift assays, have short telomeres when grown at semi-permissive temperatures (CONRAD et al. 1990; LUSTIG et al. 1990). These results suggest that when cells are limited for Raplp, telomere length decreases. Therefore, it is unlikely that Raplp is limiting in cells with extra telomeres.

A protein with a high affinity for DNA termini could serve functions in vivo similar to those proposed for the heterodimer that binds tenaciously to the ends of Oxytricha DNA (see Introduction). In strains A and C, when cells were grown under conditions selecting for higher copy number of the linear plasmid, that plasmid was rearranged. These rearrangements served to reduce the number of telomeres introduced by the linear plas- mid in the cell. These results are consistent with the possibility that the terminus binding protein is essential for telomere function. Alternatively, the observed re- arrangements may occur spontaneously and be selected for in medium lacking leucine because they eliminate TPE on the h 2 - d gene. However, the linear plasmid rearrangements were also observed in a sir2A derivative of strain C (data not shown) even though TPE is elimi- nated in such strains (APARACIO et al. 1991). Moreover, the presence of the linear plasmid at lower copy num- ber substantially reduced TPE on chromosomal telo- meres. Therefore, we favor a model in which the termi- nus binding protein is required directly for telomere function. With either model, it should be possible to identify the gene that encodes this protein by searching for genes whose over-expression restores TPE to cells with extra telomeres.

How might a protein with a high affinity for DNA termini affect TPE? The terminus binding protein might confer TPE by mediating telomere interactions with the nuclear scaffold (DELANGE 1992). Such interac- tions could create torsional constraints on the DNA that would prevent the formation or progression of a transcription complex. Alternatively, a terminus bind- ing protein could enhance TPE by affecting the rate of switching between the on and off expression states of telomere-linked genes, such that in its absence more time is spent with the gene on. It has been proposed that expression state switching results from competition between the formation of a stable transcription com- plex and the formation of heterochromatin at the telo- mere (GOTTSCHLING et al. 1990; A~ARACIO et al. 1991). The terminus binding protein and Sir3p could be in- volved in chromatin condensation in a manner similar to that proposed for some PEV modifiers in Drosophila. For example, HP-1 is a component of heterochromatin

in Drosophila that enhances PEV in three copies and suppresses in one copy (reviewed in REUTER and SPIERER 1992). PEV is also sensitive to the presence of extra Y chromosomes, which bind heterochromatin proteins.

Taken together, these results suggest that the fraction of cells exhibiting TPE in yeast can be reduced or en- hanced by altering the levels of factors that interact directly or indirectly with telomeric DNA. In addition, TPE can be substantially reduced by increasing the number of substrates, i.e., DNA termini, for such pro- teins. These results provide yet another parallel be- tween TPE in yeast and PEV in Drosophila.

We thank B. BONVEN and M. CONRAD for carrying out preliminary experiments, S. GASSER, J. RINE, V. SCHULZ and D. SHORE for reagents, and A. LUSTIG for sharing data prior to publication. We also thank S. HENIKOFF, K. RUNGE, L. SANDELL, V. SCHULZ, and J. STAVENHAGEN for their critical reading of the manuscript. This work was supported by grant GM43265 from the National Institutes of Health (NIH). E.A.W. was also supported through the Pathology Department of the University of Washington by an NIH training grant AGO0057, Genetic Approaches to Aging Research.

LITERATURE CITED

APARACIO, 0. M., B. L. BILLINGTON and D. E. GOITSCHLING, 1991 Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cereuisiae. Cell 66: 1279-1287.

BERMAN, J., C. Y. TACHIBANA and B.-K TYE, 1986 Identification of a telomere-binding activity from yeast. Proc. Natl. Acad. Sci. USA 83: 3713-3717.

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Communicating editor: D. BOTSTEIN