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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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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).
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Figure 4: 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).

Mentions: Δhmo1 caused an upstream TSS shift in the Hmo1-enriched RPS5 promoter, but not in the Hmo1-limited RPL10 promoter (23). Therefore, to identify the RPS5 promoter region responsible for this shift in Δhmo1 cells, we constructed a series of chimeric promoters in which the UAS, Core or IVR of RPS5 and RPL10 were mutually exchanged (Figure 4A). These modified promoters were integrated into the ADE2 chromosomal locus in WT or Δhmo1 cells. Primer extension analysis for these strains clearly revealed that the IVR of RPS5 was required for an upstream TSS shift in Δhmo1 cells (Figure 4B) while the UAS and Core of RPS5 can be exchanged for those of RPL10 without blocking the TSS shift.Figure 4.


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)

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).
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Figure 4: 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).
Mentions: Δhmo1 caused an upstream TSS shift in the Hmo1-enriched RPS5 promoter, but not in the Hmo1-limited RPL10 promoter (23). Therefore, to identify the RPS5 promoter region responsible for this shift in Δhmo1 cells, we constructed a series of chimeric promoters in which the UAS, Core or IVR of RPS5 and RPL10 were mutually exchanged (Figure 4A). These modified promoters were integrated into the ADE2 chromosomal locus in WT or Δhmo1 cells. Primer extension analysis for these strains clearly revealed that the IVR of RPS5 was required for an upstream TSS shift in Δhmo1 cells (Figure 4B) while the UAS and Core of RPS5 can be exchanged for those of RPL10 without blocking the TSS shift.Figure 4.

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