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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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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.
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Figure 1: 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.

Mentions: In a previous study, we showed that Δhmo1 caused an upstream TSS shift in Hmo1-enriched RPGs and suppressed the temperature sensitive growth of some sua7 mutants (e.g. sua7-R78C, -E62K), which caused a downstream TSS shift in many class II genes (23). To determine whether the suppressive effect of Δhmo1 is specific to sua7 mutants, we tested for a genetic interaction between HMO1 and RPB1, which encodes the largest Pol II subunit. The mutations, rpb1-N445S (42) and rpb1-R344A (43), are in or near to the active centre of Pol II, and cause a downstream TSS shift. As previously reported (42,43), both rpb1 mutants showed significant growth defects at all temperatures tested, and no growth at 37°C (Figure 1A). In contrast, Δhmo1 cells showed less severe growth defects at high temperature than at low temperature. Importantly, Δhmo1 suppressed the growth defect of the rpb1 mutant at 37°C (Figure 1A), as observed for the sua7 mutants (23).Figure 1.


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)

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.
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Related In: Results  -  Collection

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Figure 1: 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.
Mentions: In a previous study, we showed that Δhmo1 caused an upstream TSS shift in Hmo1-enriched RPGs and suppressed the temperature sensitive growth of some sua7 mutants (e.g. sua7-R78C, -E62K), which caused a downstream TSS shift in many class II genes (23). To determine whether the suppressive effect of Δhmo1 is specific to sua7 mutants, we tested for a genetic interaction between HMO1 and RPB1, which encodes the largest Pol II subunit. The mutations, rpb1-N445S (42) and rpb1-R344A (43), are in or near to the active centre of Pol II, and cause a downstream TSS shift. As previously reported (42,43), both rpb1 mutants showed significant growth defects at all temperatures tested, and no growth at 37°C (Figure 1A). In contrast, Δhmo1 cells showed less severe growth defects at high temperature than at low temperature. Importantly, Δhmo1 suppressed the growth defect of the rpb1 mutant at 37°C (Figure 1A), as observed for the sua7 mutants (23).Figure 1.

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