Promoter structure. The promoter used in these experiments has two copies of a URS1-containing fragment from the IME2 promoter upstream of the CYC1 promoter (UAS, TATA elements T₁ and T₂, and mRNA initiation site [bent arrow] indicated), and it drives expression of a LacI-LacZ fusion gene. As described previously, LacZ expression is repressed in a manner dependent on the URS1 elements Ume6, Sin3, and Rpd3 (, ). The region upstream of this promoter contains sequences from the URA3 gene, which serves as the plasmid marker. Shown below the promoter structure are the regions (typically 300 bp, with the upstream and downstream boundaries being defined by a pair of PCR primers) that are analyzed by the chromatin immunoprecipitation procedure. The regions labeled URS1 and LacZ are analyzed in Fig. and , whereas the upstream (U) and downstream (D) regions analyzed in Fig. are defined by the approximate number of base pairs from the center of the URS1 elements to the center of the indicated region.

Acetylation status of individual lysines of histone H4. Cross-linked and fragmented chromatin preparations from wild-type (+), rpd3 (R), sin3 (S), and ume6 (U) strains were immunoprecipitated with the antibodies to acetylated histone H4 isoforms of lysines (K) 5, 8, 12, and 16 or were analyzed prior to immunoprecipitation (Total). Recovered DNA was analyzed by quantitative PCR; for each determination, the reaction mixture contained primers both for the region corresponding to URS1 and for the region corresponding to the LacZ structural gene (Fig. ). Because individual PCRs are internally controlled, the relative level of histone acetylation in the URS1 is defined with respect to the level of histone acetylation within the LacZ region. These data are quantitated in Table and expressed as the URS1/LacZ ratio of band intensities of the PCR fragments; the absolute level of band intensities reflects the amount of input DNA in each reaction mixture and is irrelevant to the analysis.

Acetylation status of histone H3. Cross-linked and fragmented chromatin preparations from wild-type (+), rpd3 (R), sin3 (S), and ume6 (U) strains were immunoprecipitated with the antibodies to generally acetylated (Ac) histone H3 or to nonacetylated (UnAc) H3; as a control, the analysis was performed prior to immunoprecipitation (Total). Recovered DNA was analyzed by quantitative PCR as described in the legend to Fig. .

Mapping the domain of localized histone deacetylation. Cross-linked and fragmented chromatin preparations from wild-type (+), rpd3 (R), sin3 (S), and ume6 (U) strains were immunoprecipitated with the antibodies to histone H4 acetylated at lysine 5, and recovered DNA was analyzed by quantitative PCR. For each determination, the reaction mixture contained primers both for the indicated promoter (or flanking) region and for the internal control region corresponding to the LacZ structural gene (Fig. ). Because individual PCRs are internally controlled, the relative level of histone acetylation in the indicated region is defined with respect to the level of histone acetylation within the LacZ region. These data are quantitated in Table and expressed as the ratio of band intensities of the PCR fragments; the values are normalized to that obtained with the wild-type strain, which is defined as 1.0. The absolute level of band intensities reflects the amount of input DNA in each reaction mixture and is irrelevant to the analysis.

Theoretical approach for determining the extent of the actual domain. (A) The diagrammed situation contains an actual domain of 1 bp (X) located at position 0 (shown within a region that extends from −400 to +400). Horizontal lines below the coordinate scale indicate 400-bp chromosomal DNA fragments that contain position 0; there are 400 such fragments. The subset of DNA fragments that are detectable as 300-bp PCR fragments (defined by the central position of the PCR fragment) are indicated by the shaded boxes. (B) The graphs represent various situations in which the length of the chromosomal fragments (350 to 550 bp) is indicated; in all cases, the PCR fragments are 300 bp. The number of distinct chromosomal DNA fragments containing position 0 that can be detected by 300-bp PCR fragments (y axis) is shown as a function of the central position of the PCR fragment (x axis). For any PCR fragment (as defined by the location of the central base pair), the number of distinct chromosomal DNA fragments is directly related to the expected experimental signal. This approach assumes that chromosomal fragmentation is random with respect to nucleotide position.

Creation of a repressive chromatin domain by targeted recruitment of the Sin3-Rpd3 histone deacetylase complex. The Ume6 repressor binds URS1 (shown as occurring in the context of a nucleosomal template) and recruits the Sin3-Rpd3 corepressor complex to the promoter. As a consequence, histones H3 and H4 (lysines 5 and 12 and to a lesser extent lysine 16) are deacetylated (“Ac” does not appear) over a range of one to two nucleosomes from the site of recruitment. Thick arrows indicate sites of frequent histone deacetylation, whereas dashed arrows indicate histone tails that are infrequently modified. Nucleosomes further downstream and upstream are not specifically deacetylated (Ac). This region of local histone deacetylation is defined with respect to the promoter analyzed in this paper; it includes the UAS element but probably ends upstream of the TATA elements (T). Analogous regions of other Sin3-Rpd3 repressed promoters might vary in length and position. The TATA elements are indicated in the spacer region for clarity of the figure; there is no information on the nucleosomal position of these TATA elements in vivo. Although transcriptional repression is associated with the generation of a domain of localized histone deacetylation, the figure is not intended to suggest any particular mechanism of repression (e.g., inhibiting access of activators, TFIID, or the Pol II holoenzyme or inhibiting the communication between these components).