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

Abstract

In yeast alpha 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 6. Western blot analysis of affinity-purified SALT₆ and SALT₁ minichromosomes, isolated from α cells, and probed with anti-Tup1p antibodies. (A) A representative analysis of the SALT₆ minichromosome showing 14 Tup1p molecules per SALT₆ minichromosome. Replicates (n = 3) of the SALT₆ analysis each performed with a new minichromosome preparation show 14.1 ± 0.3 copies of Tup1p per SALT₆ minichromosome. (B) A representative analysis of the SALT₁ minichromosome showing less than one Tup1p molecule per SALT₁ minichromosome. In replicates (n = 3) of this analysis, each performed with a new minichromosome preparation, Tup1p is present in <1 copy per SALT₁ minichromosome.

Fig. 7. Model for Tup1p-mediated repression. Tup1p is recruited to the STE6 gene by Matα2p and interacts with the H3/H4 tails forming a scaffold, which extends from the α2 operator to the 3′ end of the gene.