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The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome

2000; Springer Nature; Volume: 19; Issue: 3 Linguagem: Inglês

10.1093/emboj/19.3.400

1460-2075

Charles Ducker,

Plant Molecular Biology Research

Article1 February 2000free access The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome Charles E. Ducker Charles E. Ducker Department of Biochemistry and Molecular Biology, 308 Althouse, Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Robert T. Simpson Corresponding Author Robert T. Simpson Department of Biochemistry and Molecular Biology, 308 Althouse, Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Charles E. Ducker Charles E. Ducker Department of Biochemistry and Molecular Biology, 308 Althouse, Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Robert T. Simpson Corresponding Author Robert T. Simpson Department of Biochemistry and Molecular Biology, 308 Althouse, Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Author Information Charles E. Ducker1 and Robert T. Simpson 1 1Department of Biochemistry and Molecular Biology, 308 Althouse, Pennsylvania State University, University Park, PA, 16802 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:400-409https://doi.org/10.1093/emboj/19.3.400 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In yeast α cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes. Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4. If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region. To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed. After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor. In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome. These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains. Introduction Saccharomyces cerevisiae exists as two haploid cell types: a and α (Herskowitz, 1989). Cell type is determined by the expression of master regulatory genes at the active mating type locus (MAT). In MATα cells, genes coding for Matα1p and Matα2p are transcribed. Matα1p is an activator for α cell-specific genes, and Matα2p is a repressor for a cell-specific genes. In MATa cells neither of these proteins are produced, resulting in the expression of the a cell-specific genes by default. Repression of the five a cell-specific genes in MATα cells is mediated by the binding of a complex of proteins at the nearly symmetrical 31 bp α2 operator, a sequence present ∼200 bp upstream of each of these genes (Johnson and Herskowitz, 1985). The repressor complex is comprised of a homodimer of the homeodomain protein Matα2p, which binds to the operator in a cooperative manner with a homodimer of the MADS box protein, Mcm1p (Sauer et al., 1988; Acton et al., 1997). Mcm1p binds to the center of the operator while the homodimer of Matα2p makes contact with the operator sequences flanking the Mcm1p binding site (Sauer et al., 1988; Zhong and Vershon, 1997; Tan and Richmond, 1998). This configuration requires the Matα2p dimer to straddle the Mcm1p dimer. In addition to Matα2p and Mcm1p, full repression also requires the non-DNA-binding proteins Ssn6p (Schulz and Carlson, 1987) and Tup1p (Lemontt et al., 1980; Keleher et al., 1992), which have been identified as general repressors in yeast (Keleher et al., 1992). It has been proposed that the role of Matα2p is to recruit Ssn6p and Tup1p, which in turn mediate repression of the a cell-specific genes (Keleher et al., 1992; Komachi et al., 1994; Tzamarias and Struhl, 1995). Both Ssn6p and Tup1p can mediate repression of transcription from a heterologous CYC1 promoter when targeted via a lexA–DNA-binding domain (Keleher et al., 1992; Tzamarias and Struhl, 1994). In these experiments, repression by Ssn6p was Tup1p dependent, whereas repression by Tup1p was found to occur in the absence of Ssn6p (Keleher et al., 1992; Tzamarias and Struhl, 1994). These data suggest that Tup1p may be the active repressor in the Ssn6p–Tup1p complex, and that Ssn6p may be required as a bridge between Tup1p multimers or Tup1p and other proteins. Tup1p has a mol. wt of 78 kDa and belongs to a family of proteins that are characterized by a region of amino acid residues having highly conserved tryptophanyl and aspartyl residues positioned approximately every 40 residues, termed WD40 or β-transducin motifs (Fong et al., 1986). This motif is now known to exist in a number of S.cerevisiae proteins, as well as extra sex combs and groucho in Drosophila (Schultz and Carlson, 1987; Williams and Trumbly, 1990; Komachi et al., 1994; Gutjahr et al., 1995). Tup1p contains seven WD40 repeats in the C-terminal half of the protein (Lemontt et al., 1980; Komachi and Johnson, 1997), which are thought to be involved in mediating protein–protein interactions. Different combinations of these repeats are required for repression of different sets of genes (Carrico and Zitomer, 1998). As an example, WD40 repeats 1 and 2 of Tup1p have been shown to interact directly with Matα2p (Komachi et al., 1994). The N-terminal 72 amino acid residues of Tup1p have been shown to interact with Ssn6p, and to be required for multimerization, but are not sufficient to repress transcription (Tzamarias and Struhl, 1994). Amino acids 73–386 have been defined as the Tup1p repression domain (Tzamarias and Struhl, 1994); this same part of the protein interacts with the N-terminal regions of histones H3 and H4 (Edmondson et al., 1996). Recently, Tup1p has been shown to exist as a trimer or a tetramer in complexes with one molecule of Ssn6p (Varanasi et al., 1996; Redd et al., 1997), which may be the functional oligomeric state of these proteins in the cell. Despite what is known about Tup1p, it is still unclear how it mediates repression of genes in S.cerevisiae. Given that Tup1p is involved in repression of a diverse group of genes, it appears likely that it interacts with a component common to many different promoters. Obvious potential targets include the basal transcription machinery and histones. There is evidence in support of both possibilities. Herschbach et al. (1994) observed modest repression of a naked DNA reporter construct in vitro after incubation with purified, recombinant Matα2p and cell extracts prepared from a yeast strain that overexpressed Ssn6p and Tup1p. From this result, these authors concluded that Tup1p may be mediating repression via interaction with the basal transcription machinery, as no chromatin assembly was likely to have occurred in the assay. A functional relationship has also been proposed between Ssn3p–Ssn8p, a part of the polymerase II holoenzyme mediator complex, and the Ssn6p–Tup1p complex (Kuchin and Carlson, 1998). Other experiments, however, suggest that Tup1p repression may be mediated through the organization of repressive chromatin. Previous work in our laboratory has shown that the binding of the Matα2p–Mcm1p complex to the α2 operator initiates the establishment of a chromatin domain starting at the α2 operator. In this chromatin domain, nucleosomes are positioned precisely over essential promoter elements and the entire coding region of the genes (Shimizu et al., 1991; Roth et al., 1992; Patterton and Simpson, 1994). The absence of this organized chromatin in a cells suggests that it is required for, or a result of, the gene being repressed in α cells. α cells expressing N-tail mutations in histone H4 show partial derepression of the a cell-specific genes (Roth et al., 1992). In these cells, the positions of nucleosomes are less well defined (Roth et al., 1992), again suggesting a relationship between organized chromatin and repression of the a cell-specific genes. Deletion of Tup1p results in the derepression of the a cell-specific genes and disruption of the positioned nucleosomes adjacent to the α2 operator. This perturbation of the chromatin structure does not appear to be due solely to the transcriptional activity of the derepressed gene (Cooper et al., 1994). One caveat to the chromatin organization hypothesis is the observation that the haploid-specific genes do not appear to show the extent of chromatin organization seen with other genes repressed in a Tup1p-dependent manner. This may reflect the ability of Tup1p to confer repression via multiple mechanisms, as is suggested by the two lines of evidence presented above. However, repression of these genes is clearly chromatin dependent despite the apparent lack of an organized chromatin domain (Huang et al., 1997). To elucidate further the precise mechanism by which Tup1p mediates repression of the a cell-specific genes, we have determined the extent of spreading and stoichiometry of Tup1p with the in vivo assembled chromatin of STE6. We reason that if the role of Tup1p is solely to mediate interactions between the basal transcription machinery and the promoter, then the protein will be present in a limited copy number at the promoter. However, if Tup1p is involved in either establishing or maintaining a repressive chromatin structure by interacting with the histone tails, then it will be present in multiple copies possibly spread along the entire nucleosomal domain of the gene. In order to distinguish between these two possibilities, we have used chromatin immunoprecipitation techniques to investigate the extent of Tup1p spreading. To determine the stoichiometry of Tup1p to STE6 chromatin we have developed a minichromosome affinity purification (MAP) technique, which allows the isolation of the gene as in vivo packaged chromatin. Central to the MAP technique is the fact that episomal DNA in S.cerevisiae is stably packaged and maintained as chromatin. The resulting minichromosomes provide a unique model system for studying the relationship between gene regulation and chromatin. Yeast minichromosomes have been used to study how nucleosome positioning affects the function of specific cis-acting elements (Simpson, 1990; Patterton and Simpson, 1994), chromatin remodeling upon transcription factor binding (Xu et al., 1998), and chromatin structure effects on DNA repair (Smerdon and Thoma, 1990; Smerdon et al., 1990; Bedoyan et al., 1992; Suter et al., 1997). Minichromosomes have also been used to study the interactions of trans-acting factors with DNA in chromatin (Morse et al., 1992; Morse, 1993), as well as twisting constraints on DNA in nucleosomes (Morse et al., 1987). Yeast minichromosomes have also been used extensively to study the factors involved in repression of the a cell-specific genes in α cells (Roth et al., 1990, 1992; Shimizu et al., 1991; Cooper et al., 1994; Patterton and Simpson, 1994). Chromatin immunoprecipitation experiments using Tup1p antibodies show Tup1p to be associated with the STE6 chromatin along the entire coding region of the gene. To investigate the stoichiometric relationship between Tup1p and the STE6 nucleosomal array we employed the MAP technique. Using this technique we isolated three minichromosome constructs containing different fragments of the STE6 gene, and determined the stoichiometric relationship between Tup1p and the positioned nucleosomes present on these constructs in vivo. The results of these experiments led us to propose that Tup1p may exert its repressive effect on the STE6 gene in α cells by forming a scaffold along the length of the chromatin domain associated with the gene. Results Tup1p spreads over the entire STE6 chromatin domain The currently accepted model for repression of the a cell-specific genes in α cells proposes that Tup1p initiates repression after being recruited to the gene by the DNA-binding protein Matα2p (Keleher et al., 1992; Tzamarias and Struhl, 1994, 1995). Edmondson et al. (1996) have demonstrated that the repression domain (Tzamarias and Struhl, 1994) of Tup1p interacts with the tails of histones H3 and H4 in vitro, suggesting a direct connection between Tup1p repression and chromatin organization. To this end Cooper et al. (1994) have shown that a TUP1 deletion has a disorganizing effect on the chromatin of the a cell-specific gene STE6 in α cells. These findings all suggest that Tup1p may facilitate repression by interacting with chromatin directly. To address this possibility chromatin immunoprecipitations (ChIP) were performed. Tup1p antibodies were used to immunoprecipitate formaldehyde-cross-linked, sonicated chromatin from wild-type a and α cells. The precipitated DNA was visualized by PCR (Figure 1). At the top of Figure 1 is a schematic diagram of the STE6 gene showing the positions of the fragments amplified in the PCRs. Each fragment is 150–200 bp long and identified as the position of the 5′ base of each fragment relative to the start site of transcription. The first panel shows the STE6 fragments amplified from a cell ChIPed material. Comparing this signal to the input for this cell type shows only a uniform background amplification from the ChIPed material. The next panel shows the STE6 fragments amplified from the α cell ChIPed material. Comparing the signal from a cells to α cells it is clear that the fragments between the α2 operator and the 3′ end of the gene are precipitated from α cells, indicating that Tup1p is spread along the entire chromatin domain of the gene. No PCR product is obtained from the ChIPed DNA when primers outside the STE6 gene (−711 and +4460) are used. These results show that Tup1p spreads unidirectionally from the α2 operator and ends at the 3′ end of the gene, exactly the direction of positioned nucleosomes in the STE6 chromatin domain (Y.Tsukagoshi and R.T.Simpson, in preparation). Figure 1.Chromatin immunoprecipitations of soluble genomic chromatin using Tup1p antibodies. At the top is a schematic diagram of the STE6 gene showing the positions of the fragments amplified in the PCRs to visualize the precipitated DNA. Eight pairs of PCR primers were used to generate the STE6 fragments. Each pair of primers amplifies a fragment ∼150–200 bp long and is labeled with the position of the 5′ base of each fragment relative to the start site of transcription. The panel labeled a ChIP is material amplified from Tup1p immuno- precipitates from a cells. The panel labeled a input is material amplified from the input for the a cell ChIPs. The panel labeled α ChIP is material amplified from Tup1p immunoprecipitates from α cells. The panel labeled α input is material amplified from the input for the α cell ChIPs. To the right of the STE6 results is a control for amplification from both cell types. The RNR2 gene is a DNA damage-responsive gene, which is repressed by Tup1p. As with the STE6 fragment, labeling of this fragment begins 530 bp upstream of the start site of RNR2 transcription. In this experiment RNR2 should be repressed in both cell types and so associated with Tup1p in both cell types. Equal amplification from both a and α cell immunoprecipitated material is observed. Download figure Download PowerPoint To the right of the STE6 result is a control for amplification from both cell types. The RNR2 gene is a DNA damage-responsive gene, which is repressed by Tup1p (Elledge et al., 1993). It should be repressed in both cell types in this experiment and so should be associated with Tup1p in both cell types. As expected the ChIP results show equal amplification from both a and α cell material immunoprecipitated with Tup1p antibodies. Minichromosome affinity purification Knowing the stoichiometry of a protein associated with a gene in vivo helps significantly when considering its role in the control of DNA function. For non-DNA-binding proteins, there are two problems with a biochemical approach to this question. First, there is a small amount of material associated with any given single-copy gene in the genome. Second, it has not been possible to isolate any gene from the rest of the genome as intact chromatin. Using the MAP technique provides a way to overcome these hurdles. MAP provides a method of isolating a large quantity of potentially any gene as intact chromatin, and quantification of associated non-DNA-binding proteins that have been identified by other techniques. The MAP protocol is derived from that previously developed by Dean et al. (1989). The method takes advantage of the high affinity interaction between the Escherichia coli lac repressor protein and its operator sequence. We have designed the procedure as an affinity matrix system to be carried out in low pressure chromatography columns. The affinity ligand for this system was created by attaching a chitin-binding domain (CBD) to a lac repressor–β-galactosidase fusion protein (lacI–Z). The lacI–Z fusion gene was cloned from a mutant E.coli strain, BMH 72-19-1 (Müller-Hill and Kania, 1974), into the pTyb2 cloning and expression vector (New England Biolabs), which fuses the intein-CBD to the C-terminus of the protein. The lacI–Z–intein-CBD fusion protein is expressed in E.coli and isolated directly by binding to a chitin matrix. The charged resin is used as an affinity matrix to bind minichromosomes containing the lac operator. The large size of the fusion protein provides a spacer between the DNA-binding portion of the molecule and the matrix. This minimizes potential steric hindrance during binding of the episomes in the isolation procedure. Equally important in the design of a MAP experiment is the proper location for the lac operator in the episomal DNA. For the TAL vector a position between two elements of the autonomously replicating sequence, which is constitutively nuclease sensitive and thus judged likely to be nucleosome free, was utilized, as discussed in detail in Materials and methods. The MAP procedure begins with 1 l of yeast cells being grown to an OD600 of 1–1.5. The cells are spheroplasted, and lysed with a motor-driven pestle. The minichromosomes are allowed to diffuse passively from the nuclei for 3–4 h on ice. Intact nuclei and other cellular debris are subsequently removed by centrifugation. The supernatant, which contains the minichromosome, is loaded onto a 1 ml (packed bed volume) chitin–agarose column charged with the lacI–Z–intein-CBD fusion protein. After washing, the minichromosomes are released following an overnight incubation at 4°C in minichromosome binding buffer (mbb) containing 30 mM dithiothreitol (DTT). The DTT causes an autocatalytic cleavage of the intein moiety leaving the intein-CBD fragment bound to the matrix and the lacI–Z bound minichromosome free in solution. Minichromosomes are washed off the column in mbb containing 300 mM NaCl. A typical purification profile, monitoring minichromosome DNA, is shown in an ethidium bromide-stained agarose gel and by Southern blot analysis in Figure 2. Quantification of the Southern blot (Figure 2B) was performed using ImageQuant software (Molecular Dynamics). Comparison of the signal generated in lanes 1 and 7 to lanes 2 and 8 shows 40–60% of the minichromosomes being released from the nuclei. Of the material loaded onto the column >95% is retained (compare lanes 2 and 8 to lanes 3 and 9). Finally, >90% of the minichromosomes released from the nuclei and retained on the column can be recovered in the eluate (lanes 5 and 11). The ethidium-stained agarose gel (Figure 2A) was imaged and quantified using the Eagle Eye II still imaging system (Stratagene). From this analysis we determined that the minichromosomes are 50–70% pure as judged by comparing the plasmid band to other DNA contaminants. Figure 2.Minichromosome affinity purification fractions. (A) SALT10 isolation fractions from both a and α cells, resolved on a 1% agarose gel stained with ethidium bromide. All samples on the gel represent 1% of the total starting volume. Supercoiled minichromosome (scSALT10), relaxed minichromosome (rSALT10), and genomic DNAs are indicated to the right of the figure. Lanes 1 and 7 contain whole-cell extracts. Lanes 2 and 8 contain chromatography input; all DNA that has diffused from the nuclei. Lanes 3 and 9 contain the column flow-through; DNA not bound by the matrix. Lanes 4 and 10 contain the three washes combined. Lanes 5 and 11 contain the eluted DNA. Lanes 6 and 12 contain the column strip, which shows DNA bound non-specifically. (B) Southern blot of the agarose gel probed with an oligo specific to the lac operator. The flow chart at the bottom is a general schematic of the isolation procedure. Download figure Download PowerPoint Multiple copies of Tup1p associate with STE6 in vivo From the ChIP results it is clear that Tup1p associates with the entire STE6 chromatin domain. In order to assess how many copies of Tup1p are associated with the repressive chromatin structure, we created a series of minichromosomes containing fragments of the STE6 gene. The parent minichromosome in this series is composed of the ALT backbone (see Materials and methods) with 5273 bp of STE6 and flanking DNA inserted into HSR B. This minichromosome was called SALT24 (STE6/ARS1/Lac-operator/TRP1, see Figure 3). The acronym SALT24 was used to signify the DNA components and the 24 nucleosomes associated with the transcriptionally repressed STE6 gene in α cells (Y.Tsukagoshi and R.T.Simpson, in preparation). The precisely positioned array of nucleosomes begins 15 bp downstream of the α2 operator, extends through the coding region and ends abruptly 70 bp downstream of the termination codon of the gene. Although the mechanism of termination of the organized chromatin is not known, it ends as precisely as it begins, forming a discrete domain (Y.Tsukagoshi and R.T.Simpson, in preparation). In order to ensure the inclusion of all regulatory sequences in the 'STE6 insert' in SALT24, extensive sequences were included both upstream (690 bp) and downstream (800 bp) of the coding region of the gene. It is important to remember that the organized chromatin domain does not spread to the ends of the STE6 inserted sequences—only the coding region and ∼200 bp of 5′ flanking sequences out to the α2 operator have positioned nucleosomes. Figure 3.Minichromosome constructs. In the center is the unaltered TRP1/ARS1 minichromosome, showing the positions of the nucleosomes and nuclease-hypersensitive sites. The arrow represents the direction of transcription of the TRP1 gene. In the expanded box at the bottom is a blow-up of the ARS1 region of the minichromosome showing the placement of the lac operator. Bases in bold are those shared between the B2 element and the lac operator. Expanded at the top are the STE6 inserts for the four minichromosomes used in this study. All four fragments were cloned into HSR B at the EcoRI site. Gaps in the STE6 insert represent the extent of sequence removed (not drawn to scale). Indicated are the α2 operator, the start site for transcription, and the ARS consensus sequence (ACS) present at the 3′ end of the gene. Download figure Download PowerPoint Initial attempts at MAP using SALT24 showed that this construct did not diffuse from the nuclei of either a or α cells. This might result from the minichromosome being sequestered to a location in the nuclei where its diffusion was impaired. However, it seemed unlikely that it would be sequestered in both cell types, and more likely that it was simply too large for passive diffusion out of the nuclei. To test this possibility we generated a smaller construct; SALT10 was created by deleting 2384 bp from the middle of the STE6 coding region (Figure 3). Functional and structural characterization of the SALT10 minichromosome revealed that the remaining portion of the STE6 gene accurately reflected the features of the whole gene. Thus, primer extension analysis of mRNA isolated from strains carrying the SALT10 minichromosome showed that the construct is transcribed in a cells and fully repressed in α cells, identical to the behavior of the parent gene (data not shown). The predicted nucleosomal array for SALT10 consists of two nucleosomes from the 5′ end of the gene and eight from the 3′ end of the gene. Indirect end-label mapping of micrococcal nuclease digests of SALT10 isolated from α cells using MAP shows 10 positioned nucleosomes, confirming this prediction (Figure 4A). Although these positioned nucleosomes map slightly differently than their counterparts in the genomic copy of the full-length gene (Y.Tsukagoshi and R.T.Simpson, in preparation) they are precisely positioned in α cells and lose this positioning in a cells. These results demonstrate that the STE6 gene fragment in the SALT10 minichromosome contains all of the necessary regulatory elements to properly control expression and establish the characteristic chromatin structure observed in the genomic copy of the gene. Furthermore, proteins necessary for repression and organized chromatin structure, Matα2p, Mcm1p, Tup1p and Ssn6p, are not limiting, even with a multicopy minichromosome. Figure 4.Indirect end-label mapping of the chromatin structure of the SALT10 and SALT6 minichromosomes. (A) The cleavage pattern obtained by MNase digestion of SALT10 chromatin (CH), MAP isolated from a and α cells. (B) The cleavage pattern obtained by MNase digestion of SALT6 chromatin (CH) also, MAP isolated from a and α cells. (M) indicates the λ-HindIII/φX174-HaeIII marker (NEB). The purified MNase-cleaved DNA was subsequently digested to completion with FspI and electrophoresed on a 1.5% agarose gel, transferred to a membrane and probed with an [α-32P]dCTP random prime labeled fragment. The inferred positions of the α2 operator, ARS consensus sequence and nucleosomes (depicted as ovals) are shown to the left of the gels. The schemes at the top of the gels represent the STE6 inserts in the two constructs showing the location of the FspI site and the direction of mapping indicated by the heavy arrow. Download figure Download PowerPoint In MAP isolations using the SALT10 minichromosome, a similar diffusion efficiency was observed for a and α cell nuclei (Figure 2). Determination of the quantity of Tup1p associated with the minichromosomes from the two cell types using silver-stained SDS–polyacrylamide gels was not possible due to the level of background contaminants in these samples. Therefore, we probed Western blots with antibodies against Tup1p (see Materials and methods). The SDS–polyacrylamide gels run for the Western blots contained 1% of the total isolated material. This is the same amount of material run on the agarose gel (Figure 2), where the concentration of isolated minichromosome was quantified using the Eagle Eye II still imaging system (Stratagene) and ImageQuant software (Molecular Dynamics). Figure 5A shows a representative Western blot containing MAP isolated SALT10 minichromosomes and a graded set of standards constructed using recombinant Tup1p expressed in E.coli. Densitometry of the blot (Figure 5C) shows a ratio of 24 Tup1p molecules per SALT10 minichromosome isolated from α cells. Statistical analysis of seven replicates of this experiment, each performed with a new minichromosome preparation, shows 24.2 ± 0.5 copies of Tup1p per SALT10 minichromosome. Figure 5.Western blot analysis of affinity-purified SALT10 mini- chromosomes probed with anti-Tup1p antibodies. (A) Lane 1, a mock isolation from a cells containing no minichromosomes; lane 2, ALT isolated from α cells; lane 3, SALT10 with the Mcm1p binding site of the STE6 α2 operator mutated, designated GG::CC, isolated from a cells; lane 4, wild-type SALT10 isolated from a cells; lane 5, SALT10 with the Mcm1p binding site of the STE6 α2 operator mutated, designated GG::CC, isolated from α cells; lane 6, wild-type SALT10 isolated from α cells; lanes 7–13, a titration series of E.coli expressed recombinant Tup1p. Each lane in the titration series represents the indicated molar ratio of rTup1p to SALT10 minichromosomes. (B) A Coomassie-stained 10% SDS–polyacrylamide gel showing the rTup1p used for the standard series in the Western blot in (A) and broad range standards (Bio-Rad). (C) Densitometry of the Western blot analysis shows 24 Tup1p molecules per nucleosome. Replicates (n =7) of the SALT10 analysis, each performed with a new minichromosome preparation, show 24.2 ± 0.5 copies of Tup1p per SALT10 minichromosome. Download figure Download PowerPoint Controls for the specificity of the Tup1p signal appear in Figure 5A, lanes 1–5. Lane 1 contains a mock isolation performed with a cells containing no minichromosomes. As expected no Tup1p signal is detected in this sample, indicating that Tup1p does not associate with the column matrix. Lane 2 contains the ARS1/Lac-operator/TRP1 (ALT) minichromosome isolated from α cells. Again, there is no Tup1p signal detected with the minichromosome lacking the STE6 insert. Lanes 3 and 5 contain a derivative of the SALT10 minichromosome isolated from a and α cells, respectively, bearing a mutation in the Mcm1p binding site which blocks Mcm1p binding (Sm