Genetic interaction between HMO1 and TFG1. (A) Effect of Δhmo1 and/or tfg1-E346A on growth. Growth of Δtfg1 and Δtfg1 Δhmo1 cells carrying the plasmid encoding TFG1 (WT) or tfg1-E346A was analysed as described in Figure 1A. (B) Effect of the Δhmo1 and/or tfg1-E346A mutation on the TSS in RPS5 and ADH1 promoters. Yeast strains were as indicated in (A). Cells were grown at 25°C, and total RNA (15 µg) was prepared and analysed by primer extension as described in Figure 1B. (C) Results shown in (B) were quantified and the data are summarized as described in Figure 1C.

Mapping of the promoter region required for Hmo1 binding and upstream TSS shift in Δhmo1 cells. (A) Schematic diagram depicting chimeric RPS5/RPL10 promoters, RPS5 promoters lacking one of three segments (UAS, IVR or Core), or a promoter containing the RPL27B-IVR. The designation indicated at the left is an abbreviation of each promoter construct. For instance, ‘5-5-5’ at the top denotes a construct that contains RPS5-UAS, RPS5-IVR, and RPS5-Core (+16 bp of 5′ region of RPS5 ORF). All modified promoters were fused to His3MX6 by PCR and then integrated into the ADE2 locus with an accompanying deletion of an ∼1.2-kb DNA region encoding the N-terminal portion of Ade2. The regions amplified by PCR in the ChIP assays are underlined and labelled ‘a’, ‘b’ or ‘c’. The primer TK10595, used for primer extension, is indicated with an arrow. (B) The promoter region required for the upstream TSS shift in Δhmo1 was analysed by primer extension, as described in Figure 1B. The TSSs of the chimeric promoters, described in (A), were examined in Δhmo1 (odd-numbered lanes; Δ) or WT cells (even-numbered lanes; W). TSSs at −36 and −87 in the RPS5-Core and −21 in the RPL10-Core are marked with asterisks (* and **) and dagger (†), respectively. (C) Hmo1 binding to the test promoters described in (A) was analysed in vivo by ChIP assays. The strains carrying modified promoters and expressing Hmo1-FLAG were grown in YPD medium to mid-log phase at 25°C. Cross-linked chromatin was prepared and immunoprecipitated with an anti-FLAG antibody (0.1 µg) and Dynabeads Protein G. Immunoprecipitation was also conducted using an anti-Myc antibody (1 µg) as a negative control (indicated as ‘−’). Panels 1 and 4 summarize the results for the promoters that contain the RPS5-Core (region ‘a’ is amplified). Panel 2 summarizes the results for the promoters that contain the RPL10-Core (region ‘b’ is amplified). Panel 3 summarizes the results for the promoters that contain the RPS5-UAS (region ‘c’ is amplified). (D) Simultaneous binding of more than two Hmo1 molecules to an RPS5-IVR was tested by sequential ChIP analysis. The strains expressing Hmo1-FLAG and/or Hmo1-Pk were grown in YPD medium to mid-log phase at 25°C, cross-linked chromatin was prepared, and the samples were subjected to sequential immunoprecipitation using an anti-FLAG antibody (first) and an anti-Pk antibody (second). PCR was conducted for the RPS5-IVR (region ‘5’ in Figure 5A). The result is reliable because amplification occurred only when immunoprecipitation was conducted using both antibodies against chromatin from yeast cells expressing both of the different Hmo1 species (strain 3).

Δhmo1 shifts the PIC assembly site upstream in the RPS5 promoter. (A) Schematic diagram depicting the endogenous RPS5 promoter. (B) Analysis of the effect of Δhmo1 on the position of PIC assembly on the RPS5 promoter. The exact positions of several PIC components including TFIIB (Sua7-Pk), TFIIF (Tfg1-Pk), TFIIE (Tfa2-Pk) and TFIIH (Tfb3-Pk) on the RPS5 promoter were determined in WT and Δhmo1 cells by ChIP analysis as described in Figure 5A. The upper and lower panels for each component indicate the results in WT and Δhmo1 cells, respectively. The numbers under each panel (‘4’–‘10’) represent the position of regions amplified in the RPS5 promoter as depicted in Figure 5A. (C) Analysis of the effect of Δrpb9 or tfg1-E346A on the position of PIC assembly on the RPS5 promoter. The TFIIB binding site on the RPS5 promoter was determined in WT, Δrpb9 and tfg1-E346A cells by ChIP analysis as described in (A), except that immunoprecipitation was conducted by using an anti-Sua7 polyclonal antibody. (D) A model for the proposed role of Hmo1 in Hmo1-enriched RPG promoters (DBD, DNA binding domain; AD: Activation domain). See text for description of the model.

Genetic interaction between HMO1 and RPB1. (A) Effect of Δhmo1 and/or rpb1 mutations on growth. Δrpb1 and Δrpb1 Δhmo1 cells carrying a plasmid encoding the RPB1 (WT) or rpb1 mutant (N445S, R344A) were spotted onto YPD (yeast extract, peptone, dextrose) plates at three dilutions and grown for 3 days (30°C, 35°C and 37°C) or 4 days (25°C). (B) Effect of Δhmo1 and/or rpb1 mutations on the TSS in RPS5 and ADH1 promoters. The strains described in (A) were grown at 25°C. Total RNA (15 µg) was prepared and analysed by primer extension. The positions of several TSSs are indicated on the left (RPS5) or right (ADH1). TSSs are numbered relative to the A (+1) of the start codon, ATG (−22, −26, −36, −71, −87, −133, −215 and −225 for the RPS5 promoter and −27 and −38 for the ADH1 promoter). TSSs at −36 and −87 in the RPS5 promoter and −27 and –38 in the ADH1 promoter are marked with asterisks (* and **) and daggers († and ‡), respectively. (C) Results shown in (B) were quantified by densitometry. The number in the upper right corner of each panel corresponds to the lane number in (B). Values were normalized to the strongest peak in each panel (i.e. strongest peak set to a value of 1). The horizontal axis represents the position of each band within the region shown in (B). Note that the regions shown in (B) and (C) are identical. Asterisks (* and **) and daggers († and ‡) correspond to the bands at −36 and −87 (RPS5), and those at −27 and −38 (ADH1), respectively, as shown in (B). Part of the scanned region was enlarged and is shown in the inset to highlight the differences.

Effects of artificial recruitment of TBP/TFIID to upstream regions of the core promoter on upstream TSS shift in Δrpb9 and/or Δhmo1 cells. (A) Schematic diagram depicting ectopic TATA elements inserted into the RPS5 promoter. RPS5 promoters with or without an ectopic TATA element at −125 (TATA1) or −165 (TATA2) were fused to a mini-CLN2 reporter gene and inserted into a plasmid. A 16-bp DNA fragment (5′-CTTGCTGTCAGCGATC-3′) was inserted between the RPS5 promoter and mini-CLN2 (indicated with a grey square). This sequence was used to discriminate between mRNA produced from the endogenous RPS5/CLN2 or from mini-CLN2. The primer, TK9911, used for the primer extension is indicated with an arrow. (B) Effect of Δhmo1 and/or Δrpb9 on the TSS in RPS5 promoters, with or without an ectopic TATA element, was analysed by primer extension. The plasmid containing one of the three RPS5 promoters (original, TATA1 or TATA2) was transformed into WT, Δhmo1, Δrpb9 and Δhmo1 Δrpb9 cells. Transformants were selected on YPD medium containing aureobacidin A (0.2 µg/ml). Total RNA (15 µg) from these strains, which were grown in the same medium at 25°C, was analysed by primer extension as described in Figure 1B. (C) Results shown in (B) were quantified and summarized as described in Figure 1C.

Identification of exact binding sites for Hmo1, nucleosome and PIC in Hmo1-enriched and Hmo1-limited promoters. (A) Exact binding sites for Hmo1, nucleosome and PIC were identified in several promoters by high-resolution ChIP analyses. The schematic diagrams depicting the endogenous promoter regions tested are indicated above the panels, which summarize the results for each promoter (RPS5, RPL27B, RPL10 and HMO1). These promoters were divided into UAS, IVR and Core based on information for certain promoter elements (see Supplementary Data). The grey arrows represent the direction of the ORF. The black bars closely aligned to the entire promoter (and part of the ORF) of each gene represent the regions amplified by PCR in ChIP assays. ChIP analyses were conducted as described in Figure 4C, except that chromosomal DNA was fragmented to an average size of 100–200 bp. The ChIP results for Hmo1 (+Pk tag) and histone H3 are indicated in the upper panels (a, c, e, g) with a solid line or broken line, respectively, while results for TFIIB (Sua7-Pk) are indicated in the lower panels (b, d, f, h). The numbers indicated under the panels (‘1’–‘36’) correspond to the regions depicted in the schematic diagram. (B) The binding profiles of histone H3 on the RPS5 promoter were analysed in WT and Δhmo1 cells by ChIP analysis as described in (A).