SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin
1999; Springer Nature; Volume: 18; Issue: 24 Linguagem: Inglês
10.1093/emboj/18.24.7041
ISSN1460-2075
Autores Tópico(s)CRISPR and Genetic Engineering
ResumoArticle15 December 1999free access SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin Edward A. Sekinger Edward A. Sekinger Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author David S. Gross Corresponding Author David S. Gross Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Edward A. Sekinger Edward A. Sekinger Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author David S. Gross Corresponding Author David S. Gross Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Author Information Edward A. Sekinger1 and David S. Gross 1 1Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA, 71130-3932 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:7041-7055https://doi.org/10.1093/emboj/18.24.7041 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Previous work has suggested that products of the Saccharomyces cerevisiae Silent Information Regulator (SIR) genes form a complex with histones, nucleated by cis-acting silencers or telomeres, which represses transcription in a position-dependent but sequence-independent fashion. While it is generally thought that this Sir complex works through the establishment of heterochromatin, it is unclear how this structure blocks transcription while remaining fully permissive to other genetic processes such as recombination or integration. Here we examine the molecular determinants underlying the silencing of HSP82, a transcriptionally potent, stress-inducible gene. We find that HSP82 is efficiently silenced in a SIR-dependent fashion, but only when HMRE mating-type silencers are configured both 5′ and 3′ of the gene. Accompanying dominant repression are novel wrapped chromatin structures within both core and upstream promoter regions. Strikingly, DNase I footprints mapping to the binding sites for heat shock factor (HSF) and TATA-binding protein (TBP) are strengthened and broadened, while groove-specific interactions, as detected by dimethyl sulfate, are diminished. Our data are consistent with a model for SIR repression whereby transcriptional activators gain access to their cognate sites but are rendered unproductive by a co-existing heterochromatic complex. Introduction The expression state of a gene is influenced by its chromosomal position. Genes located within heterochromatin, the cytologically condensed form of chromatin, are susceptible to long-range, dominant repression, while genes located within euchromatin are not. Therefore, the location of a gene can dictate whether it is expressed or repressed. Such position-effect regulation is seen in eukaryotes as diverse as yeast, insects and mammals. In the budding yeast Saccharomyces cerevisiae, position effects have been identified at three distinct regions: the silent mating loci HMR and HML, the telomeres and the rDNA gene array. At each of these regions, repression is position dependent yet sequence independent. As such, pol II and pol III genes targeted to these regions are transcriptionally silenced (Brand et al., 1985; Schnell and Rine, 1986; Gottschling et al., 1990; Smith and Boeke, 1997). The cis-acting elements responsible for position-effect regulation are best understood for the HM loci. HMR and HML contain donor copies of the a and α genes, responsible for mating-type determination. The HM loci are maintained in a transcriptionally inactive state by flanking cis-acting elements, termed E and I, disposed to the left and right of each silent locus, respectively. These silencers each encompass a relatively short stretch of DNA (∼150 bp) and are located ∼1 kb from the genes they regulate. The most potent of these silencers, HMRE, is comprised of binding sites for three sequence-specific proteins: ORC (origin replication complex), RAP1 and ABF1 (reviewed in Loo and Rine, 1995). These proteins act by recruiting the Silent Information Regulatory (Sir) complex, composed of Sir2p, Sir3p and Sir4p (Rine and Herskowitz, 1987), which does not directly bind DNA. The same complex is also required for transcriptional repression at telomeres (Aparicio et al., 1991). Recruitment of the Sir complex is mediated by Sir1p, which binds directly to both Orc1p and Sir4p (Triolo and Sternglanz, 1996), and when tethered to the HMRE silencer can bypass the requirement for ORC (Fox et al., 1997). Interaction of Sir3p and Sir4p with RAP1 has also been demonstrated (Moretti et al., 1994; Cockell et al., 1995). Interestingly, repression of pol II genes targeted to the rDNA array requires only Sir2p (Smith and Boeke, 1997). In addition, the core histones H3 and H4 are required for silencing at both HM and telomeric loci (Kayne et al., 1988; Aparicio et al., 1991; Thompson et al., 1994). Their N-termini engage in direct contacts with Sir3p and Sir4p, which may facilitate horizontal templating of the Sir complex (Hecht et al., 1995). How SIR-mediated heterochromatin elicits its repressive effects is unknown. An appealing notion, supported by several lines of evidence, is that this specialized chromatin structure sterically hinders the access of sequence-specific regulatory factors, as well as components of the pre-initiation complex (PIC) to promoter DNA, thereby impairing transcription. In support of this concept is evidence that SIR induces the local formation of a specialized chromatin structure, inaccessible to restriction endonucleases in vitro (Loo and Rine, 1994) and the site-specific HO endonuclease in vivo (Nasmyth, 1982; Weiss and Simpson, 1998). The SIR-dependent chromatin is also refractory to DNA repair enzymes (Terleth et al., 1989) as well as ectopically expressed dam methyltransferase (Gottschling, 1992; Singh and Klar, 1992); however, the silenced chromatin at HMR and HML is fully permissive to homologous recombination that results in gene conversion of the MAT locus (Herskowitz et al., 1992). Moreover, Ty retrotransposon integration at the HM loci is enhanced by the Sir regulatory complex, as HMRE mutations that partially diminish silencing also partially reduce preferential Ty integration at these sites (Zou and Voytas, 1997). These latter observations raise the possibility that heterochromatin may act in a manner distinct from, or in addition to, strict steric interference. In this study we investigate the cis-acting requirements for silencing a transcriptionally potent, stress-inducible gene, and provide a detailed analysis of the distinctive nucleoprotein complex accompanying the silenced state. We find that dominant silencing requires a minimum of two properly positioned HMRE silencers and is accompanied by the appearance of novel, SIR-dependent, wrapped chromatin structures within the core and upstream promoter regions. Strong, sequence-specific interactions mapping to regulatory sites—including those for the principal activators, heat shock factor (HSF) and TATA-binding protein (TBP)—are preserved; nonetheless, groove-specific interactions are altered. Our results are consistent with a model for SIR repression whereby activators gain access to their target binding sites but where these proteins, once DNA bound, are rendered unproductive by the co-existing heterochromatic complex. Results HSP82 is conditionally silenced by SIR irrespective of HMRE orientation or dosage To explore the mechanistic basis for position-effect regulation, we used gene transplacement to target HMRE silencer elements to the HSP82 chromosomal locus in an effort to bring it under SIR control. As HSP82 regulatory elements and promoter chromatin structure have been extensively characterized (Szent-Gyorgyi et al., 1987; Gross, 1995; Erkine et al., 1999), the gene provides an ideal model system for such an investigation. In previous work we demonstrated that targeting an HMRE silencer upstream of HSP82 resulted in moderate, SIR-dependent silencing under non-inducing conditions. This repression was conditional, in that it was rapidly overridden under inducing conditions (Lee and Gross, 1993). Our previous study used relatively large HMRE fragments (0.35 or 4.9 kb) containing multiple potential binding sites for ORC, RAP1 and ABF1 (DeBeer and Fox, 1999; Hurst and Rivier, 1999), which were installed opposite their native orientation. To test whether a minimal HMRE fragment—comprising single binding sites for ORC, RAP1 and ABF1—could silence HSP82, and whether altering its orientation or increasing its dosage might increase the efficiency of silencing, we constructed a new series of HMRE/HSP82 alleles (illustrated in Figure 1). Figure 1.Physical map of the HSP82 locus and location of HMRE insertions. The heat shock gene is 95 kb distant and oriented towards the left telomere of chromosome XVI; it is flanked by YAR1 upstream and CIN2 downstream. HMRE elements (arrows; see inset) were targeted to unique restriction sites within the locus, at either ClaI (position −673, where +1 is the transcription start site) or MluI (position +2342, ∼50 bp 3′ of the transcription termination point), or both, in the orientations indicated. Stably positioned nucleosomes are indicated by continuous ovals, less precisely positioned nucleosomes by broken ovals, 'split' nucleosomes by half ovals, and DNase I hypersensitive, nucleosome-disrupted regions by horizontal lines (adapted from Erkine et al., 1995). Constitutively occupied sites within the HSP82 promoter are indicated by solid rectangles, inducibly bound sites by open rectangles (Gross et al., 1990; Giardina and Lis, 1995; Erkine et al., 1999). Download figure Download PowerPoint When targeted ∼700 bp upstream of the heat shock gene, a minimal HMRE element, oriented ORC-site-proximal (hsp82-102; see Figure 1), reduces HSP82 transcript levels by 30% under non-heat-shock conditions (Figure 2, lane 2 versus 1). This silencing is conditional, as it is lost upon heat shock (Figure 2, lane 4 versus 3; see also Figure 3) but it is re-established within 15 min of recovery (Figure 2, lane 6 versus 5). Our ability to assay the re-establishment of transcriptional repression is facilitated by the short half-life of HSP82 RNA (<1.7 min at 30°C; Lee and Gross, 1993), permitting rapid changes in transcription rates to be monitored. Notably, the repression seen is SIR dependent, as insertion of the HMRE fragment in a sir4− background has no effect on HSP82 expression (Figure 2, compare '−' lanes with corresponding HSP82+ lanes). Thus, the minimal silencer exerts a partial, conditional position effect upon HSP82. Figure 2.Position-effect regulation of HSP82 by an upstream silencer is weak and conditional irrespective of its orientation, while YAR1 is fully and unconditionally silenced by the same element. Total cellular RNA was isolated from isogenic sir4− and SIR+ strains ('−' and '+', respectively) bearing the indicated HMRE/HSP82 alleles. As a control, RNA was isolated from an HSP82+ strain (SLY101; lanes 13–15). Samples were isolated from non-shocked cultures (NHS), following heat shock at 39°C (HS), or following return to 30°C (Rec). HSP82 and YAR1 transcript levels were quantitated by PhosphorImager and internally normalized to those of ACT1. Download figure Download PowerPoint Figure 3.Polar HMRE/HSP82 alleles: summary of Northern analyses. HSP82 transcript levels of isogenic sir4− and SIR+ strains ('−' and '+', respectively) bearing the alleles indicated are graphically illustrated (values are means ± SEM; they are normalized to non-heat-shocked hsp82-201/sir4−, whose transcript level was assigned a value of 100). n, number of independent RNA isolations. Note that these data were derived from strains bearing either chromosomal or episomal copies of SIR4 (see Table I); no difference in expression levels was seen. Download figure Download PowerPoint It is possible that the relatively weak silencing observed with this and the constructs studied previously is attributable to their unnatural orientation with respect to the promoter (see, for example, Shei and Broach, 1995). To test this, we constructed an hsp82 allele with the HMRE silencer installed at the same site but in its native orientation (hsp82-101). However, as shown in Figure 2 (lanes 7–12), inverting the HMRE silencer had no effect: as above, SIR only partially represses non-induced expression levels; this repression is fully overridden by heat shock. A summary of multiple independent experiments is provided in Figure 3. To investigate whether silencer activity at HSP82 was additive, we constructed HMRE/HSP82 strains in which tandem HMRE modules separated by a 6 bp spacer were installed ∼700 bp upstream of the HSP82 coding region. Additive effects might be seen if, as suggested by a number of previous studies, silencers act to nucleate the assembly of a Sir complex whose constituent proteins are in limiting concentration within the nucleus (Renauld et al., 1993; Maillet et al., 1996; Marcand et al., 1996). However, the level of SIR repression mediated by dimeric constructs is similar to that seen with the monomeric constructs (Figure 3, compare hsp82-201 with hsp82-101). Moreover, as above, this is true irrespective of HMRE orientation. Taken together with previous findings showing equivalent repression mediated by HMRE elements positioned either 0.75 or 2.7 kb upstream of HSP82 (Lee and Gross, 1993), we conclude that SIR repression of the heat shock gene is independent of orientation, distance or dosage of 5′ silencers. YAR1 is fully and unconditionally silenced by a single, ectopic HMRE element The inability of HMRE, even in its natural orientation, to repress HSP82 significantly (Figure 2, lanes 7–12), contrasts markedly with its efficiency in silencing genes at either HMR or MAT (Brand et al., 1985; Shei and Broach, 1995). To test whether conditional silencing reflects an attribute of the heat shock promoter (such as its intrinsic strength), rather than an inability of HMRE to function efficiently at this locus (95 kb from the left telomere of chromosome XVI), we measured transcript levels of YAR1, a divergently transcribed gene located adjacent and centromere-proximal to HSP82 (see Figure 1). The principal YAR1 start site maps ∼50 bp downstream of the HMRE insertion site (Lycan et al., 1996). Nonetheless, HMRE per se has little effect on YAR1 transcription (Figure 2, compare lanes 1, 7 and 13). However, in a SIR+ context, quantitative silencing of YAR1 is seen independently of HMRE orientation or dosage (e.g. Figure 2, lanes 2 and 8). Thus, at this locus, SIR can fully and unconditionally silence one gene while being simultaneously overridden in the opposite direction by another. Dominant repression of HSP82 can be established by bracketing the gene with silencers The foregoing results argue against a simple model in which the dosage of cis-acting silencers is proportional to the extent of SIR repression. However, it is possible that functional cooperativity between HMRE elements is more effectively achieved by positioning them 5′ and 3′ of the promoter, permitting looping or other topological interactions to take place between them (Hofmann et al., 1989). To investigate this possibility, we constructed a strain whose hsp82 allele is bracketed by two HMRE silencers, one 5′ of the promoter and the other 3′ of the transcription termination point, thereby positioning them ∼3 kb apart (hsp82-1001; Figure 1). The ability of this strain to silence HSP82 was analyzed by Northern blotting as above. In marked contrast to hsp82 alleles bearing two 5′ silencers (Figure 3), the bracketed allele is strongly silenced: 11-fold under non-inducing conditions, 2-fold following heat shock and 20-fold following a 15min recovery from heat shock (Figure 4). As expected, the effect of bracketing is independent of silencer orientation (Figure 4, compare hsp82-1001 with hsp82-1002). However, in contrast to the polar constructs discussed above, silencing is enhanced by increasing HMRE dosage. In strains bearing either the hsp82-2001 or hsp82-2002 allele, non-induced and recovery transcript levels are virtually eliminated (down 50- to 100-fold), while induced transcript levels are also significantly repressed (4-fold). Figure 4.Bracketed HMRE/HSP82 alleles: summary of expression data. Methodology, symbols and abbreviations are as in Figure 3. Samples were normalized to non-heat-shocked hsp82-2001/sir4−. Download figure Download PowerPoint Downstream HMRE elements silence HSP82 inefficiently The preceding experiments indicate that monomeric or dimeric HMRE sequences integrated 3′ of the HSP82 coding region, in combination with identical sequences integrated at the 5′ end, impose dominant SIR repression. One interpretation, as discussed above, is that 5′ and 3′ silencers functionally cooperate to mediate this effect. Alternatively, it is possible that the potent silencing seen is due exclusively to the presence of the 3′ silencer(s). This might be the case if propagation of the Sir complex along chromatin requires contiguous nucleosomes (Hecht et al., 1995), absent within the promoter but present within the transcription unit of HSP82 (Szent-Gyorgyi et al., 1987; see Figure 1). To test the repressive activity of 3′ silencers alone, we constructed alleles bearing single or dimeric copies of HMRE installed at the +2342 MluI site (termed hsp82-301 and hsp82-401, respectively; Figure 1). While virtually complete, SIR-dependent silencing of the neighboring CIN2 gene was seen at each allele, there was no discernable silencing of HSP82 (data not shown). We conclude that the dominant silencing characteristic of the bracketed alleles requires both 5′ and 3′ silencers. SIR repression is overridden in a progressive, not quantum, fashion The foregoing experiments indicate that when subjected to maximally inducing conditions, the HSP82 promoter partially overrides SIR repression, even when bracketed by silencers (Figure 4). However, HSP82 can be induced to submaximal levels that correlate with the intensity of the applied stress (E.A.Sekinger and D.S.Gross, unpublished observations). Thus, it is formally possible that silencing remains near-absolute until a threshold temperature is reached, with transcription detectable only once this threshold is breached. However, we have found that an incremental increase in the severity of heat shock is accompanied by a corresponding decrease in SIR-mediated repression (data not shown). Diminished repression is unlikely to reflect thermolability of the SIR complex for two reasons. First, HMRE-mediated silencing of non-stress-responsive genes (YAR1, CIN2, a1) is not affected by heat shock (Figure 2 and data not shown). Secondly, a similar inverse relationship between fold repression of HSP82 and severity of stress is elicited by an inhibitor of oxidative phosphorylation, 2,4-dinitrophenol (DNP; data not shown). We conclude that there is no threshold stress level; rather, the extent to which HSP82 overrides SIR repression correlates with the severity of the stress. SIR repression is accompanied by the de novo assembly of a specialized chromatin structure at HSP82 Previous work has shown that SIR repression at other loci is accompanied by a heterochromatic-like structure inaccessible to DNA repair and modifying enzymes (Terleth et al., 1989; Gottschling, 1992; Singh and Klar, 1992) and endonucleases (Nasmyth, 1982; Loo and Rine, 1994; Weiss and Simpson, 1998). To investigate whether dominant SIR repression of HSP82 is likewise accompanied by a novel chromatin structure, we performed a nucleotide resolution DNase I footprinting analysis. Spheroplast lysates isolated from SIR+ and sir4− strains were digested with DNase I and the HSP82-specific pattern analyzed by amplified primer extension (AMPEX). Such an analysis reveals the presence of a distinctive, SIR-dependent chromatin structure at hsp82-2001, evident under all three expression states and extending over the entire upstream regulatory region (Figure 5). In the absence of SIR, chromatin-specific protections are primarily restricted to HSEs 1–3 and TATA (Figure 5, open bars, compare lanes 7–12 with lanes 5 and 6). In the presence of SIR, protection over HSE1 is retained and is in fact extended ∼70 bases downstream, encompassing both URS1 and HAP2/3/5 sites (Figure 5, solid bars). Punctuating the cleavage profile of each strand is a prominent DNase I hypersensitive site flanking HSE1 (Figure 5, arrows). These hypersensitive sites, coupled with the intervening protection, are consistent with the sequence-specific binding of HSF (McDaniel et al., 1989; Gross et al., 1990), see below. Additional chromatin-specific protections map to the core promoter, where the TATA-associated upper-strand footprint appears extended to at least position −50 in the SIR+ strain (Figure 5B, solid bar, lanes 13–18). Figure 5.A novel, SIR-dependent chromatin structure is formed over the HSP82 promoter as revealed by DNase I genomic footprinting. Isogenic SIR+ and sir4− strains bearing the hsp82 alleles indicated were grown to early log phase in rich medium at 30°C, heat shocked for 20 min at 39°C, then downshifted to 30°C for 15 min. Spheroplast lysates were isolated from appropriate aliquots [NHS, HS and Rec (N, H and R), respectively], digested with DNase I, genomic DNA extracted, and lower- and upper-strand cleavage profiles revealed by AMPEX using primers −342→ −315 and +26→ −11, respectively. (A) Lower-strand analysis. (B) Upper-strand analysis. Note that all DNA samples are derived from hsp82-2001 strains except lanes 19 and 20 (hsp82-1001), and lanes 21 and 22 (hsp82-201) of (B). Dots, SIR-specific cut sites; arrows, location of DNase I hypersensitive sites marking the upstream and downstream flanks of HSE1; open bars, sequence-specific protection in a sir4− background; solid bars, sequence-specific protection in an SIR+ background. C, T, A and G, dideoxy sequence ladders (sequence of the footprinted strand is shown). DNA (D), naked genomic DNA digested with DNase I and processed identically to chromatin samples. Depicted below each lane is an RNA analysis of the same sample. HSP82 transcript levels relative to those of ACT1, quantitated by PhosphorImager, are indicated. Note that three different gels are depicted in (B): lanes 1–12 and 23–24 are derived from gel 1; lanes 13–18 are from gel 2; and lanes 19–22 are from gel 3. Download figure Download PowerPoint There are at least two other striking attributes revealed by DNase I. First, a series of nine chromatin-specific cleavages spaced at either 10–12 or 20–25 nt intervals, and spanning ∼150 bp of the core promoter and 5′ end of the transcription unit (dots), is evident in the lower-strand analysis (Figure 5A). Such a pattern may indicate the presence of a wrapped structure. A similar, although more regular pattern of cleavages is seen in genetically inactivated hsp82 promoter mutants lacking the high-affinity HSF binding site, HSE1. These mutants exhibit a near-perfect 10–11 nt cleavage periodicity spanning −120 to +28, possibly reflecting the presence of a rotationally phased nucleosome (Gross et al., 1993). Secondly, the SIR-dependent chromatin structure appears to be metastable, since it is cleaved quite differently at high levels of digestion (Figure 5A, lanes 18, 22 and 26). The lower-strand hypersensitive site and accompanying footprints are lost; in their place is a ladder of uniformly spaced cleavages ∼10 nt apart, mapping principally to the core promoter. Thus, DNase I genomic footprinting reveals the presence of a novel, SIR-dependent structure compatible with sequence-specific DNA binding proteins. To determine whether the SIR-dependent chromatin structure was also recognized by micrococcal nuclease (MNase), which recognizes distinct attributes of DNA structure (Gross and Garrard, 1988), we performed MNase genomic footprinting (Figure 6). The most prominent finding is a ladder of chromatin-specific cleavages between −112 and −225 (Figure 6, bracketed), which exhibits a distinctive 10 nt periodicity on both strands (stars); this ladder may even extend to −260 (dots), thereby spanning 148 bp. The structure detected by MNase appears to be an intrinsic feature of the UAS region since a majority of the cut sites are also seen in the sir4− strain, yet are greatly accentuated in the SIR+ background. Interestingly, this cleavage ladder, which may reflect the presence of a wrapped structure over the upstream promoter, is far less prominent in the heat-shocked SIR+ strain (Figure 6, compare lane 11 with either 10 or 12; and lane 23 with either 22 or 24), more closely resembling the cleavage profile of the sir4− strains. Also notable are broad regions of SIR-dependent protection on the upper strand mapping to the intergenic region between HSP82 and YAR1 (Figure 6, solid bars), the significance of which is unclear. In contrast, the prominent regions of MNase protection evident within the core promoter and coding region of the recovered sir4− strain (Figure 6, lane 8) suggest the presence of translationally positioned nucleosomes (dotted ovals). While such nucleosomes may accompany the transcriptional down-regulation seen during recovery from heat shock, it is notable that no evidence for discrete ∼160 nt regions of protection can be found in the SIR+ samples despite far lower transcript levels (Figure 6, compare lane 8 with either 10 or 12). Finally, strong MNase protection is seen at position −183 on the lower strand (Figure 6, lanes 10–12), a site of intense DNase I cleavage under all three transcription states (Figure 5A). Thus, the MNase analysis detects structures distinct from those recognized by DNase I, yet both argue for the presence of a specialized, SIR-dependent chromatin structure upstream of HSP82. Figure 6.MNase genomic footprinting reveals the presence of a novel, SIR-dependent chromatin structure upstream of HSP82. Isogenic SIR+ and sir4− strains bearing the double bracketed HMRE/HSP82 allele (hsp82-2001) were cultivated under non-shocked (N), heat shocked (H) or recovery (R) conditions, and nuclei isolated. Nuclei were digested with MNase, genomic DNA purified, and strand-specific footprints revealed by AMPEX as in Figure 5. As a control, deproteinized genomic DNA (D) was digested with MNase and analyzed similarly. Bottom panel, Northern analysis of RNA isolated from the same cultures used for nuclear isolation. Stars, cleavages novel to (or strongly enhanced in) SIR+ nuclei spaced at an ∼10 nt periodicity; these span the UAS region of each strand (brackets). Dots, cleavages extending the region of 10 nt cutting periodicity (not chromatin specific). Ovals, inferred positions of nucleosomes in recovered sir4− samples. Filled bars, regions of SIR-dependent protection. T, TATA box; H, HAP2/3/5 consensus sequence; U, URS1; 1, 2, 3, HSEs 1–3. Download figure Download PowerPoint The novel chromatin structure is unique to dominantly silenced alleles Is the distinctive chromatin structure seen at the double bracketed hsp82-2001 allele functionally linked to dominant silencing, or is it also present at partially and conditionally silenced HMRE/HSP82 alleles? To address this question, we digested nuclei purified from strains carrying either the hsp82-201 or hsp82-1001 allele with DNase I, and analyzed the resultant cleavage profile by AMPEX as above. Both of these alleles bear two silencers, yet only one is efficiently repressed (Figures 3 and 4). Therefore, if chromatin is the cause and not the consequence of this transcriptional silencing, the signature features of the DNase I genomic footprint—the hypersensitive site at position −155 and the extended UAS and core promoter footprints—will be seen at hsp82-1001 but not at hsp82-201. Such an expectation is dramatically confirmed, as shown in Figure 5B (compare lanes 19 and 20 with lanes 21 and 22). Interestingly, the cleavage profile of the polar construct (Figure 5B, lanes 21 and 22) is novel, and resembles neither that of the sir4− control (lanes 7–12) nor that of the SIR-repressed bracketed alleles (lanes 13–20). SIR alters but does not obviate methylation protection at heat shock and TATA elements in vivo DNase I and MNase genomic footprinting suggest that stable SIR repression of HSP82 is associated with novel chromatin structures localized to both upstream and core promoter regions. The DNase I analysis additionally suggests that sequence-specific interactions are preserved in an SIR+ context and may in fact be strengthened and broadened, particularly in alleles subject to robust silencing. To investigate the effect of the SIR repressive complex on protein–DNA interactions in more detail, we conducted dimethyl sulfate (DMS) in vivo footprinting. Cells bearing an hsp82 allele bracketed with single silencers were reacted with DMS during the final 2 min of cultivation, and genomic DNA was isolated and subjected to AMPEX. As illustrated in Figure 7, HSE1 is strongly occupied in a sir4− background, even under non-inducing conditions (indicated by protection of guanines at −161, −162 and −174; lane 6). This is consistent with previous work demonstrating that S.cerevisiae HSF binds
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