Domains of Gene Silencing Near the Left End of Chromosome III in Saccharomyces cerevisiae

Corresponding author: Xin Bi

Communicating editor: L. PILLUS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Saccharomyces cerevisiae the HM loci and regions adjacent to the telomeres are transcriptionally silent. HML is situated 11 kb from the left telomere of chromosome III. I have systematically examined gene silencing along this 11-kb chromosomal region. I found that silencing extends at least 1.1 kb beyond HML, indicating that the HML E silencer acts on both sides. Moreover, I obtained evidence indicating that a 0.71-kb sequence near the E silencer acts as a barrier to the spread of silencing and coincides with the left boundary of the silent HML domain. I also showed that silencing at the telomere is limited to an 2-kb domain. On the other hand, an 7-kb region between HML and the telomere is not silenced by HML or the telomere. These results provide a clear example of organization of the eukaryotic genome into interspersed domains with distinct potentials for gene expression.

TELOMERES and the HML and HMR loci in Saccharomyces cerevisiae are transcriptionally silent. These silent loci are established and maintained through combined actions of cis-acting and trans-acting factors (LUSTIG 1998 ). The cis-acting elements include silencers flanking each HM locus and the TG_1-3 telomeric repeats (Fig 1A). The trans-acting proteins include histones, the Sir2p-Sir4p proteins, and silencer/telomere-binding proteins Rap1p, Abf1p, and origin recognition complex (ORC). It was proposed that silencer/telomere-binding proteins recruit a complex of Sir2p/Sir3p/Sir4p, which then propagates sequentially along neighboring nucleosomes to form a silent chromatin similar to metazoan heterochromatin that represses gene expression.

View larger version (52K): In this window In a new window Download PPT slide   Figure 1. Silencing by the HML E silencer on both sides. (A) Schematic representation of chromosome III highlighting its telomeres and the HM loci. Tandem arrowheads, telomeric TG1-3 repeats; CEN, centromere; TEL III-L and TEL III-R, the left and right telomeres of chromosome III, respectively; ACS, ARS consensus sequence, which is the binding site for ORC; ARS, autonomous replication sequence. The HML E and I silencers and HMR E and I silencers are shown. The open rectangle in HML E is an uncharacterized silencer element (MAHONEY et al. 1991 ). (B) The pattern of transcriptional silencing around HML E. Left, strains used. HML E and its surrounding sequences are shown. Solid rectangle with arrow, the URA3 gene. Strain C is DMY2 as a control. Strains 1 and 2 have URA3 inserted close to the E silencer within HML in two orientations. Strains 3–10 have URA3 inserted 0.18, 0.79, 1.1, and 1.8 kb from HML E in both orientations, respectively. Construction of these strains is detailed in MATERIALS AND METHODS. Right, growth phenotypes. Cells of each strain were grown to late log phase and serial dilutions (10-fold) were spotted on test plates and allowed to grow for 3 days. SC, synthetic complete medium; -Ura, SC medium lacking uracil; FOA, SC medium containing 5-FOA.

The spread of silencing from its initiation site (silencer) was originally thought to be a continuous process with silencing gradually decreasing along the path (RENAULD et al. 1993 ). However, recent studies on telomeric silencing indicate that the spread of silencing is more complex. Internal to the telomeric TG_1-3 repeats are the middle repetitive sequences X and Y' (LOUIS 1995 ). Each telomere has an X repeat and none or up to four tandem copies of the Y' repeats (Fig 3A). Both the X and Y' repeats were shown to influence telomeric silencing (RENAULD et al. 1993 ; PRYDE and LOUIS 1999 ). The potency of silencing at telomeres containing both X and Y' (Y' telomeres, Fig 3A) is different from silencing at telomeres that have only X (X telomeres) or silencing at artificial telomeres lacking both X and Y' (truncated telomeres).

View larger version (46K): In this window In a new window Download PPT slide   Figure 2. A sequence that limits the spread of silencing by the HML E silencer. (A) A 0.71-kb sequence to the left of HML functions as a barrier to silencing. Strains 9 and 10 are described in Fig 1B. Strain 9' was derived from strain 9 by replacing the 0.71 LB fragment (1.1–1.8 kb to the left of E, solid box) with a 0.77-kb sequence from the ORF of ADE2 (+1139 to +1908, hatched box). Strain 10' was similarly derived from strain 10. Growth phenotypes of these strains are shown. (B) The LB sequence can also function as a barrier to silencing when inserted to the right of HML. In strains a, b, c, and b', the direction of the HML I sequence was inverted. The inverted silencer is denoted as I'. Strain b' was derived from strain b by inserting the LB sequence between I' and URA3. Growth phenotypes of these strains are shown.

View larger version (38K): In this window In a new window Download PPT slide   Figure 3. Limited silencing at the left telomere of chromosome III. (A) Various types of telomeres in yeast. Tandem arrowheads, telomeric TG1-3 repeats. The Y' and X repeats are shown. (B) Left, strains used. Solid diamond, the ACS sequence in the X repeat at TEL III-L. Right, growth phenotypes.

Within the X and Y' repeats there are elements that appear to block the spread of silencing (named STARs for subtelomeric antisilencing regions) and elements that enhance silencing (named proto-silencers; FOUREL et al. 1999 ; PRYDE and LOUIS 1999 ). STARs are sequences containing multiple sites for Tbf1p and/or Reb1p (FOUREL et al. 1999 ). Proto-silencers include individual binding sites for Abf1p or ORC (FOUREL et al. 1999 ; PRYDE and LOUIS 1999 ; LEBRUN et al. 2001 ). Individual Rap1p-binding sites can also act as proto-silencers (BOSCHERON et al. 1996 ). A proto-silencer is not able to initiate silencing on its own but can cooperate with a silencer at a distance to exert silencing that is stronger than that brought up by the silencer alone (BOSCHERON et al. 1996 ). These findings suggest that the strength and extent of silencing depend on the interplay of silencers, proto-silencers, and insulators (like STAR; also see below) and can be discontinuous.

In addition to STARs, sequences having the ability to hinder the spread of silencing were found in regions flanking the HMR locus (DONZE et al. 1999 ). The upstream activating sequence (UAS) of the TEF1 or TEF2 gene can also block the spread of silencing (BI and BROACH 1999 ). These elements, referred to as heterochromatin barriers, are functionally similar to chromatin boundary or insulator elements in higher cells (SUN and ELGIN 1999 ; BI and BROACH 2001 ). In an earlier study of HML, we found that silencing is uniformly high between the HML E and I silencers but decreases sharply beyond HML I (BI et al. 1999 ). Interestingly, this is not because there is a barrier to silencing near HML I, but is because HML I initiates silencing in only one direction (toward the -genes). Therefore, there is more than one mechanism for creating the boundaries of silenced genomic domains.

To gain more insight into the domain organization of the eukaryotic genome, I systematically examined the silencing profile across an 11-kb region between the HML locus and the left telomere of chromosome III in this study. My results, together with our earlier characterization of silencing around the HML I silencer, provide a complete map of domains of gene silencing near the left end of chromosome III.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmid pMB21 consists of pRS404 (SIKORSKI and HIETER 1989 ) into which the SIR3 and SUP4-o genes have been inserted at the SacI + BamHI and BamHI sites, respectively. The following plasmids were constructed by inserting sequences of chromosome III into the polylinker of pUC19. Plasmid pUC26 contains the BamHI-HML-BamHI fragment corresponding to region 9666–16263 of chromosome III (base pair coordinates are from the complete sequence of chromosome III). Plasmid pUC26-B was derived from pUC26 by deleting its BstBI fragment. pXB156 contains the sequence 7197–9666 of chromosome III as a SphI-EcoRI fragment. pXB163 contains the sequence 4200–6873 as a HindIII-MfeI fragment. pXB171 has the sequence 870–3694 as a SacI-SphI fragment. pXB188 has the sequence 8766–11294 as a HindIII-EcoRI fragment. pAR61 contains the sequence 14838–16263 as a HindIII-BamHI fragment. The ClaI-XhoI fragment in pXB188 was replaced by a 770-bp fragment of ADE2 (see text), resulting in pXB190.

The 1.1-kb BglII-URA3-BglII fragment of plasmid pFL44 (CHEVALLIER et al. 1980 ) was used in constructing a series of plasmids for the insertion of URA3 at different sites between the HML locus and the left telomere of chromosome III. URA3 was inserted at the ClaI site of pUC26-B in both orientations to make plasmids pCB5 and pCB6, at the Bsu36I site to make pCB7 and pCB8, at the AgeI site to generate pCB2 and pCB3, and at the XhoI site to make pCB1 and pCB4, respectively. URA3 was inserted at the ClaI site of pXB156 in both orientations to make plasmids pXB157 and pXB158, at the SalI site to make pXB159 and pXB160, and at the BglII site to make pXB161 and pXB162. URA3 was inserted at the ClaI site of pXB156 in both orientations to make plasmids pXB157 and pXB158, at the SalI site to make pXB160, and at the BglII site to make pXB161 and pXB162. URA3 was inserted at the EcoRI site of pXB163 to make pXB164-I, at the BamHI site to make pXB165-I and -II, at the XhoI site to make pXB166-I, and at the BglII site to make pXB167-II, respectively. URA3 was inserted at the KpnI site of pXB171 to make plasmids pXB175-I and -II, at the SalI site to make pXB185 and pXB186, at the BglII site to make pXB183, at the BamHI site to make pXB176-I and -II, and at the MfeI site to make pXB181 and pXB184, respectively. URA3 was inserted at the ClaI site of pXB190 to make pXB191 and pXB192. URA3 was inserted at the EcoRV site of pAR61 to make pMB22-a (BI et al. 1999 ). The sequence 9304–10014 of chromosome III was inserted at the SnaBI site of pMB22-a to make pXB193. The above plasmids are listed in Table 1.

Strains used in this study, except YXB83-I, YXB85-I, YXB87-I, YXB195, and YXB195', were derived from strain DMY2 (MATa ura3-52 leu2-3,112 ade2-1 lys1-1 his5-2 can1-100 sir3::LUE2; MAHONEY and BROACH 1989 ) using a two-step procedure. DMY2, a sir3^- strain, was first transformed to Ura⁺ using a chromosome III sequence bearing a URA3 insertion. The resultant strain was then converted to SIR3⁺ by integrating the pMB21 plasmid at the TRP1 locus in the genome. For example, DMY2 was first transformed to Ura⁺ with BamHI plus SpeI-digested pCB5 DNA, resulting in strain YXB150s. YXB150s was then rendered SIR3⁺ using pMB21 to yield YXB150. Other strains listed in Table 1 were similarly constructed with corresponding plasmids (also in Table 1). Strain YXB77 was derived from DMY2 by inverting the orientation of the HML I silencer (BI et al. 1999 ). Strains YXB83-I, YXB85-I, and YXB87-I were derived from YXB77 by first inserting the URA3 gene at coordinates 14441, 15410, and 16263, respectively, and then integrating SIR3 at TRP1 using pMB21 (BI et al. 1999 ). YXB195 was similarly derived from YXB77 using first pXB193 and then pMB21. All strain constructions were confirmed by Southern analysis.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The HML E silencer functions on both sides:
The URA3 gene is essential for yeast pyrimidine biosynthesis. This gene has proved to be a convenient reporter for studying transcriptional silencing. URA3 expression can be assessed by cell viability on medium containing 5-fluoroorotic acid (5-FOA) or medium lacking uracil. Ura3p converts 5-FOA to a toxic metabolite, so that cells with basal level URA3 expression are sensitive to 5-FOA (BOEKE et al. 1987 ). Depletion of uracil from the medium activates the Ppr1p transactivator, which increases the expression of URA3 to a higher activated level (LOSSON and LACROUTE 1981 ). Therefore, while cell growth on 5-FOA medium indicates repression of the basal level transcription of URA3, growth on uracil minus (-Ura) medium reflects activated expression of URA3. When URA3 is inserted inside the HML or HMR locus, both its basal and activated expressions are strongly silenced. Thus cells with URA3 at HM loci are able to grow on 5-FOA medium due to their lack of basal expression of URA3. Meanwhile, these cells are not able to grow on -Ura medium since the activated expression of URA3 is also repressed in them (BI et al. 1999 ). On the other hand, telomeric silencing can repress only basal expression but not activated expression of URA3 (APARICIO and GOTTSCHLING 1994 ). Therefore, cells with the URA3 gene inserted near the telomere can grow on both 5-FOA medium and -Ura medium (APARICIO and GOTTSCHLING 1994 ). In this article, URA3 silencing is generally referred to as the repression of its basal expression unless otherwise specified.

We have previously demonstrated that HML I defines the right (centromere-proximal) boundary of the silent HML domain by initiating silencing in only one direction (BI et al. 1999 ). To define the left (telomere-proximal) boundary of HML, we examined the level of expression of URA3 inserted at various locations around the HML E silencer (Fig 1B, left). Consistent with our earlier findings, both the basal and activated expression of URA3 located within HML was strongly repressed as evidenced by robust cell growth on FOA medium and reduced cell growth on -Ura medium (Fig 1B, right, strains 1 and 2). However, unlike silencing around the I silencer, the basal expression of URA3 inserted 180 bp away from the HML E silencer was silenced as evidenced by cell viability on FOA medium (strains 3 and 4). Activated URA3 expression in strain 4 was not silenced as indicated by its robust growth on -Ura medium, a situation similar to telomeric silencing. However, activated expression of URA3 in strain 3 was reduced to a level comparable to that of URA3 in strains 1 and 2 where URA3 was inserted inside of HML (compare growth of strains 2 and 3 on -Ura medium). When URA3 was inserted farther left of the E silencer, its activated expression was not silenced (Fig 1B, strains 5–8 on -Ura medium) whereas its basal expression was still silenced (strains 5–8 on FOA medium). Note that since the URA3 fragment was 1 kb, the promoter of URA3 in strain 7 was actually 2 kb from E. These results indicate that, in contrast to the HML I silencer, the HML E silencer can initiate silencing that spreads at least as far as 2 kb outside of HML bracketed by the E and I silencers. However, the strength of silencing to the left of HML E is generally weaker than that within HML and resembles telomeric silencing.

A sequence that limits the spread of silencing by the HML E silencer:
As shown in Fig 1B, silencing of the basal expression of URA3 decreased slightly as it was inserted farther and farther from the E silencer (strains 3–8, FOA). However, there was a sharp drop in silencing in strains 9 and 10 compared to strains 7 and 8. URA3 in strain 10 was 710 bp farther from the E silencer than that in strain 8. I named the 710-bp sequence (9304–10014 of chromosome III) LB for left boundary of HML. Was the difference in silencing between strain 8 and strain 10 simply the result of a distance effect, in other words, a gradual decrease of silencing as a function of distance from HML E? To test this possibility, I substituted the LB sequence in strains 9 and 10 with a 770-bp stuffer sequence from the open reading frame (ORF) of the ADE2 gene, resulting in strains 9' and 10', respectively (Fig 2A). Although the distance between URA3 and HML E in strain 9' was comparable to that in strain 9, silencing of URA3 was much stronger in 9' compared to that in 9 (Fig 2B, FOA). The same was true for strain 10' vs. strain 10 (Fig 2B). The ADE2 sequence has previously been shown to be neutral in terms of gene silencing or activation (BI and BROACH 1999 ). Therefore, the sharp difference in silencing between 8 and 10 was not the result of a distance effect. It is possible that LB is a strong activating sequence that can overcome silencing by HML and thus renders URA3 inserted nearby resistant to silencing. However, this is unlikely since URA3 to the right of LB in strains 7 and 8 was still silenced (Fig 1B, FOA).

An alternative explanation for the difference in silencing between strains 8 and 10 is that LB serves as a barrier to the spread of silencing. If this is the case, LB may also function at ectopic loci, similar to other known barrier elements such as STAR (reviewed in BI and BROACH 2001 ). To test this hypothesis, I examined if LB could act as a barrier to silencing when it is inserted to the right of the HML locus. We have previously shown that when the direction of the HML I silencer was inverted, silencing could spread 1.4 kb to the right of HML I (BI et al. 1999 ; Fig 2B, strains a–c, FOA). URA3 inserted 0.5 kb to the right of the inverted HML I silencer experiences significant silencing (strain b in Fig 2B, FOA). Silencing of URA3 inserted 1.4 kb to the right of HML I was also readily detectable albeit at a reduced level (compare strains c and b in Fig 2B, FOA). LB was inserted between the inverted HML I silencer and URA3 in strain b to make strain b' (Fig 2B). Little or no silencing of URA3 was detected in strain b' (Fig 2B, FOA). This was probably not the result of a distance effect since URA3 in strain b' was 0.15 kb closer to the HML I silencer than URA3 in strain c, yet silencing was readily detectable in strain c. These results were consistent with the hypothesis that the 710-bp LB sequence (9304–10,014) acted as a barrier to the spread of silencing.

A limited domain of silencing at TEL III-L:
The left telomere of chromosome III (TEL III-L) belongs to the group of telomeres that contain only the X repeat (X telomeres, Fig 3A; LOUIS 1995 ). Unlike the Y' repeat, the X repeat is not a discrete element and ranges from 0.3 to 3 kb in size. In fact, X consists of at least five small repeats, not all of which are present in every X. An 475-bp repeat called the core X is present at most telomeres. Given the wide variation of X and the fact it can affect silencing, it is not surprising that silencing at X telomeres varies significantly from telomere to telomere (PRYDE and LOUIS 1999 ). Silencing at TEL III-L has not been examined before.

As part of the effort to characterize silencing domains near the left end of chromosome III, I systematically examined silencing near TEL III-L. URA3 was inserted 1.1, 1.8, 2.3, or 3 kb from the end of chromosome III (or 10.1, 9.4, 8.9, or 8.2 kb from the HML E silencer), respectively (Fig 3B, left). Silencing was strong in strains 27 and 28 (Fig 3B, FOA). Strains 24–26 exhibited reduced but readily detectable silencing (Fig 3B, right). However, little or no silencing was detected in strains 22 and 23. These results indicate that silencing at TEL III-L is limited to a domain no larger than 2 kb.

A large silencing-free domain between HML and TEL III-L:
Silencers and proto-silencers are able to cooperate at a distance (the longest distance tested being 7 kb) in maintaining silencing (BOSCHERON et al. 1996 ; DONZE et al. 1999 ; FOUREL et al. 1999 ; PRYDE and LOUIS 1999 ). Moreover, this cooperation is not abolished by a barrier(s) to silencing positioned between them (FOUREL et al. 1999 ; PRYDE and LOUIS 1999 ). HML is only 11 kb from the left end of chromosome III, and a known ACS, a proto-silencer, is located 10 kb from HML E (Fig 3B, left). I decided to test if the HML silencers and the telomeric silencer/proto-silencers cooperate in any way to influence the expression of genes between them. URA3 was inserted at various locations between HML E and TEL III-L in strains 9–23, respectively (Fig 4A). There was little or no growth of any of these strains on medium containing 5-FOA, indicating that a large domain (7 kb) between HML and the telomere was free of silencing (Fig 4B). These results argue against any long-distance cooperation between HML and the telomere.

View larger version (52K): In this window In a new window Download PPT slide   Figure 4. A large unsilenced domain between HML and TEL III-L. (A) Strains used. (B) Growth phenotypes.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Using URA3 silencing as an indicator, I have systematically analyzed domains of gene expression between the HML locus and the left telomere of chromosome III in S. cerevisiae. As illustrated in Fig 5, my results indicate that there is a limited (2 kb) silent domain near the telomere and a 5-kb well-defined silent domain at HML. A large, continuous, unsilenced domain (7 kb) lies between the two silent domains.

View larger version (10K): In this window In a new window Download PPT slide   Figure 5. Silencing profile in chromosomal domains between the left telomere and HML on chromosome III. The X repeat (box with X) and the LB sequence (solid bar) are shown. See text for description.

The transition between silenced and unsilenced regions on the centromere-proximal side of HML was shown to coincide with the HML I silencer (BI et al. 1999 ). However, no sequence with the ability to block the spread of silencing resides at or near this transition point. Rather, the boundary appears to derive from the fact that the HML I silencer promotes silencing to one side of the silencer but not the other. In this study I showed that the left boundary of HML silencing does not coincide with the HML E silencer. Instead, it lies 1.1 kb away from the silencer. Therefore, HML E silences sequences on both sides. Previous studies have shown that the HMR E silencer also functions on both sides (LOO and RINE 1994 ; DONZE et al. 1999 ). What determines the directionality of a silencer? The four yeast silencers each contain a unique combination of two or three binding sites for the Abf1p, Rap1p proteins and ORC (Fig 1A). An additional nonessential weak Rap1p site is also present in the HML I silencer and an 80-bp sequence in HML E other than its Rap1p and ORC sites is also involved in silencing but has not been well characterized (MAHONEY et al. 1991 ; BOSCHERON et al. 1996 ). A silencer-binding protein most likely has different affinities to different silencers. All these may contribute to the directionality of a silencer. Interestingly, Abf1p, a multifunctional protein, has been shown to contribute to chromatin organization at the replication origin ARS1, the RPS28A promoter, as well as a silenced region (VENDITTI et al. 1994 ; REIMER and BUCHMAN 1997 ; LASCARIS et al. 2000 ). This raises the possibility that Abf1p bound to a silencer may help establish the direction of the formation of silent chromatin at the HM loci. We are testing if a single component of a silencer like the Abf1p site or the concerted action of all components is responsible for the observed directionality of a silencer. We are also looking into an alternative possibility that directionality of a silencer is determined by its chromosomal context.

The HMR silent domain is demarcated by two barrier elements (DONZE et al. 1999 ). In this article I report evidence indicating that a 710-bp sequence 1.1 kb to the left of the HML E silencer acts as a barrier to silencing and may serve as the LB of the HML domain. Substitution of LB with a stuffer sequence results in an expansion of the silent domain. When inserted to the right of HML, LB also functioned as a barrier to silencing. LB consists of the upstream regulatory sequence (URS; -397 to -1) and part of the ORF (+1 to +313) of the gene YCL069W of unknown function. Our preliminary deletion analysis indicates that sequences from both the UAS and the ORF of YCL069W contribute to the silencing-barrier function of LB (X. BI, unpublished results). Further deletion analysis is underway to determine the minimal sequence(s) required for the barrier function of LB, which may help delineate the mechanism of function of LB as a barrier to silencing. We have previously shown that TEF2-UAS can function as a barrier to silencing, and the three Rap1p-binding sites in TEF2-UAS are essential for barrier function (BI and BROACH 1999 ). The UAS of YCL069W bears sites for various transcriptional activators including Gcn4p, Gcr1p, and Swi5p. We are currently testing if any of these factors is involved in the barrier function of the LB sequence.

Silencing at TEL III-L is limited. It extends to only 2 kb from the TG_1-3 repeats or only 1 kb from the ACS site in the X repeat (Fig 3B). This is consistent with results from studies of silencing at other X telomeres similar to TEL III-L (PRYDE and LOUIS 1999 ). The rate of distance-dependent decrease in silencing at TEL III-L (Fig 3B) is comparable to that observed at X telomeres such as TEL III-R (PRYDE and LOUIS 1999 ). Is the decrease of silencing at an X telomere the result of a simple distance effect or the action of a barrier element? At the X telomeres, sequences internal to the X repeats are quite diverse and yet there is a similar decrease of silencing over these sequences. This is consistent with the hypothesis that the decrease is a distance effect and argues against the existence of a common barrier sequence near all the X telomeres. However, it does not rule out the possibility that a distinct barrier sequence at each X telomere restricts silencing.

	ACKNOWLEDGMENTS

I thank Chris Brown and Su Cai for assistance. This work was supported by a start-up fund from University of Nebraska-Lincoln and grant GM-62484 from the National Institutes of Health.

Manuscript received July 31, 2001; Accepted for publication January 21, 2002.

	LITERATURE CITED

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