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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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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.
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Figure 6: 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.

Mentions: The results described above suggested that Hmo1 and the +1 nucleosome determine the 5′- and 3′-border, respectively, of the PIC assembly zone. If this is so, Δhmo1 should disrupt the 5′-border of this zone, allowing ectopic PIC assembly on the IVR. An effect that induces ectopic PIC assembly could account for the upstream TSS shift in Δhmo1 cells. To test this possibility directly, ChIP analysis was conducted to determine the binding positions of several PIC components including TFIIB (Sua7), TFIIF (Tfg1), TFIIE (Tfa2) and TFIIH (Tfb3) on the RPS5 promoter in WT and Δhmo1 cells. The binding positions of these factors were shifted upstream in Δhmo1 cells (i.e. from position 8 to 7; Figure 6B, compare panels a, c, e, g with panels b, d, f, h, respectively). Our recent mapping analysis for core promoter elements in the RPS5 promoter revealed that the region corresponding to position 7 cannot bind PIC in the native context in the WT cells, although it can induce PIC assembly when artificially inserted into a different context (51). Therefore, the upstream shift of the PIC assembly site observed in Δhmo1 cells reflects the ectopic PIC assembly in this region (approximately position 7), where PIC intrinsically does not assemble in WT cells. In contrast, a similar upstream shift in the binding position of the PIC component (Sua7) was not observed in tfg1-E346A and Δrpb9 cells (Figure 6C), indicating that the upstream TSS shift in these mutants was caused by a post-PIC assembly defect. Therefore, we concluded that Hmo1 cooperates with the +1 nucleosome to direct PIC assembly to a site between the IVR and the +1 nucleosome by determining the 5′- and 3′-boundaries of a zone available for PIC assembly.Figure 6.


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)

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.
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Figure 6: 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.
Mentions: The results described above suggested that Hmo1 and the +1 nucleosome determine the 5′- and 3′-border, respectively, of the PIC assembly zone. If this is so, Δhmo1 should disrupt the 5′-border of this zone, allowing ectopic PIC assembly on the IVR. An effect that induces ectopic PIC assembly could account for the upstream TSS shift in Δhmo1 cells. To test this possibility directly, ChIP analysis was conducted to determine the binding positions of several PIC components including TFIIB (Sua7), TFIIF (Tfg1), TFIIE (Tfa2) and TFIIH (Tfb3) on the RPS5 promoter in WT and Δhmo1 cells. The binding positions of these factors were shifted upstream in Δhmo1 cells (i.e. from position 8 to 7; Figure 6B, compare panels a, c, e, g with panels b, d, f, h, respectively). Our recent mapping analysis for core promoter elements in the RPS5 promoter revealed that the region corresponding to position 7 cannot bind PIC in the native context in the WT cells, although it can induce PIC assembly when artificially inserted into a different context (51). Therefore, the upstream shift of the PIC assembly site observed in Δhmo1 cells reflects the ectopic PIC assembly in this region (approximately position 7), where PIC intrinsically does not assemble in WT cells. In contrast, a similar upstream shift in the binding position of the PIC component (Sua7) was not observed in tfg1-E346A and Δrpb9 cells (Figure 6C), indicating that the upstream TSS shift in these mutants was caused by a post-PIC assembly defect. Therefore, we concluded that Hmo1 cooperates with the +1 nucleosome to direct PIC assembly to a site between the IVR and the +1 nucleosome by determining the 5′- and 3′-boundaries of a zone available for PIC assembly.Figure 6.

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