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Hmo1 directs pre-initiation complex assembly to an appropriate site on its target gene promoters by masking a nucleosome-free region.

Kasahara K, Ohyama Y, Kokubo T - Nucleic Acids Res. (2011)

Bottom Line: In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift.The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s).This novel mechanism ensures accurate transcriptional initiation by delineating the 5'- and 3'-boundaries of the PIC assembly zone.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Biology, Graduate School of Nanobioscience, Yokohama City University, Yokohama 230-0045, Japan. k4kasaha@nodai.ac.jp

ABSTRACT
Saccharomyces cerevisiae Hmo1 binds to the promoters of ∼ 70% of ribosomal protein genes (RPGs) at high occupancy, but is observed at lower occupancy on the remaining RPG promoters. In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift. Analyses of chimeric RPS5/RPL10 promoters revealed a region between the RPS5 upstream activating sequence (UAS) and core promoter, termed the intervening region (IVR), responsible for strong Hmo1 binding and an upstream TSS shift in Δhmo1 cells. Chromatin immunoprecipitation analyses showed that the RPS5-IVR resides within a nucleosome-free region and that pre-initiation complex (PIC) assembly occurs at a site between the IVR and a nucleosome overlapping the TSS (+1 nucleosome). The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s). This novel mechanism ensures accurate transcriptional initiation by delineating the 5'- and 3'-boundaries of the PIC assembly zone.

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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.
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Figure 3: 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.

Mentions: In contrast, the feature of TSS shift was quite different in Δhmo1 and tfg1-E346A mutants (Figure 2). Primarily, tfg1-E346A caused an upstream TSS shift in all promoters tested (RPS5, ADH1, SPT15, HTB1, GAL1, GAL10, HIS3, HIS4, SNR7, SNR14, SNR19 and SNR20) [in this study, and (30)], while Δhmo1 shifted the TSS specifically in the Hmo1-enriched RPG promoters, e.g., RPS5, RPL32 (23), and RPL27B (Supplementary Figure S2A). Furthermore, it is noteworthy that transcription from −51A in the RPS5 promoter was markedly enhanced by tfg1-E346A, but not by Δhmo1 (Figure 2B, lanes 1–4). Conversely, TSSs around −220 (−215 and −225) were induced uniquely by Δhmo1 (Figure 2B, lanes 1–4). Similar but weaker effects were observed in Δhmo1, Δrpb9 and Δhmo1 Δrpb9 cells (Figure 3, lanes 1–4). These results suggested that the mechanism(s) underlying the TSS shift in the Δhmo1 cells might be different from that in the TFIIF/Pol II mutants. However, direct evidence will be required to confirm this possibility.Figure 2.


Hmo1 directs pre-initiation complex assembly to an appropriate site on its target gene promoters by masking a nucleosome-free region.

Kasahara K, Ohyama Y, Kokubo T - Nucleic Acids Res. (2011)

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.
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Figure 3: 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.
Mentions: In contrast, the feature of TSS shift was quite different in Δhmo1 and tfg1-E346A mutants (Figure 2). Primarily, tfg1-E346A caused an upstream TSS shift in all promoters tested (RPS5, ADH1, SPT15, HTB1, GAL1, GAL10, HIS3, HIS4, SNR7, SNR14, SNR19 and SNR20) [in this study, and (30)], while Δhmo1 shifted the TSS specifically in the Hmo1-enriched RPG promoters, e.g., RPS5, RPL32 (23), and RPL27B (Supplementary Figure S2A). Furthermore, it is noteworthy that transcription from −51A in the RPS5 promoter was markedly enhanced by tfg1-E346A, but not by Δhmo1 (Figure 2B, lanes 1–4). Conversely, TSSs around −220 (−215 and −225) were induced uniquely by Δhmo1 (Figure 2B, lanes 1–4). Similar but weaker effects were observed in Δhmo1, Δrpb9 and Δhmo1 Δrpb9 cells (Figure 3, lanes 1–4). These results suggested that the mechanism(s) underlying the TSS shift in the Δhmo1 cells might be different from that in the TFIIF/Pol II mutants. However, direct evidence will be required to confirm this possibility.Figure 2.

Bottom Line: In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift.The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s).This novel mechanism ensures accurate transcriptional initiation by delineating the 5'- and 3'-boundaries of the PIC assembly zone.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Biology, Graduate School of Nanobioscience, Yokohama City University, Yokohama 230-0045, Japan. k4kasaha@nodai.ac.jp

ABSTRACT
Saccharomyces cerevisiae Hmo1 binds to the promoters of ∼ 70% of ribosomal protein genes (RPGs) at high occupancy, but is observed at lower occupancy on the remaining RPG promoters. In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift. Analyses of chimeric RPS5/RPL10 promoters revealed a region between the RPS5 upstream activating sequence (UAS) and core promoter, termed the intervening region (IVR), responsible for strong Hmo1 binding and an upstream TSS shift in Δhmo1 cells. Chromatin immunoprecipitation analyses showed that the RPS5-IVR resides within a nucleosome-free region and that pre-initiation complex (PIC) assembly occurs at a site between the IVR and a nucleosome overlapping the TSS (+1 nucleosome). The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s). This novel mechanism ensures accurate transcriptional initiation by delineating the 5'- and 3'-boundaries of the PIC assembly zone.

Show MeSH
Related in: MedlinePlus