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Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Eyboulet F, Wydau-Dematteis S, Eychenne T, Alibert O, Neil H, Boschiero C, Nevers MC, Volland H, Cornu D, Redeker V, Werner M, Soutourina J - Nucleic Acids Res. (2015)

Bottom Line: The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II.We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin.This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Institut de Biologie et de Technologies de Saclay (iBiTec-S), CEA, CNRS, Université Paris Sud, F-91191 Gif-sur-Yvette cedex, France.

No MeSH data available.


Related in: MedlinePlus

Global destabilization of PIC assembly in med17-444 and -504 mutant strains. The density of sequence tags in Med15, Rad3, Kin28 and TBP ChIP-seq experiments was calculated for promoter regions of Pol II-transcribed genes. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. The density of sequence tags in the Pol II ChIP-seq experiments was calculated for the Pol II-transcribed genes. In all, 3852 Pol II-enriched genes were used for these analyses. Tag densities were normalized relative to qPCR data on a set of selected genes. Each point on the plot corresponds to one promoter region or one ORF. Promoter regions correspond to intergenic regions in tandem or in divergent orientation, excluding intergenic regions encompassing Pol III-transcribed genes. A linear regression (red line) for ChIP-seq density in mutant versus ChIP-seq density in WT and an R2 correlation coefficient are indicated. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-444 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-504 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
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Figure 5: Global destabilization of PIC assembly in med17-444 and -504 mutant strains. The density of sequence tags in Med15, Rad3, Kin28 and TBP ChIP-seq experiments was calculated for promoter regions of Pol II-transcribed genes. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. The density of sequence tags in the Pol II ChIP-seq experiments was calculated for the Pol II-transcribed genes. In all, 3852 Pol II-enriched genes were used for these analyses. Tag densities were normalized relative to qPCR data on a set of selected genes. Each point on the plot corresponds to one promoter region or one ORF. Promoter regions correspond to intergenic regions in tandem or in divergent orientation, excluding intergenic regions encompassing Pol III-transcribed genes. A linear regression (red line) for ChIP-seq density in mutant versus ChIP-seq density in WT and an R2 correlation coefficient are indicated. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-444 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-504 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).

Mentions: The ChIP results on the selected class II genes showed the most pronounced effects on Pol II, TBP and Kin28 occupancy for the med17-444 mutant whereas Rad3 occupancy remained unaffected in this mutant (Figures 2 and 3). Genome-wide analysis of the med17-444 mutant revealed a global 1.8-fold decrease (the slope of regression line, shown in red, is equal to 0.55) in Med15 Mediator occupancy with a high correlation coefficient (R2 equal to 0.93) (Figure 5A). As a consequence, the presence of other PIC components like Pol II, TFIIH and TBP was also globally reduced to a different extent. A global decrease in genome-wide occupancy for Pol II and the Kin28 TFIIK subunit (2.6- and 2.7-fold, respectively) was observed. The TBP association to chromatin was also globally affected with a 1.6-fold decrease. The Rad3 TFIIH core subunit binding was slightly affected (1.2-fold decrease), consistent with the ChIP results on the ADH1, PYK1 and PMA1 gene promoters. Taken together, the genome-wide results on the med17-444 mutant confirmed a central role for the Med17 Mediator subunit in PIC formation and suggested that the Mediator tail module destabilization on the chromatin could lead to a global change in the in-vivo binding of several PIC components but only marginally affected Rad3 occupancy.


Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Eyboulet F, Wydau-Dematteis S, Eychenne T, Alibert O, Neil H, Boschiero C, Nevers MC, Volland H, Cornu D, Redeker V, Werner M, Soutourina J - Nucleic Acids Res. (2015)

Global destabilization of PIC assembly in med17-444 and -504 mutant strains. The density of sequence tags in Med15, Rad3, Kin28 and TBP ChIP-seq experiments was calculated for promoter regions of Pol II-transcribed genes. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. The density of sequence tags in the Pol II ChIP-seq experiments was calculated for the Pol II-transcribed genes. In all, 3852 Pol II-enriched genes were used for these analyses. Tag densities were normalized relative to qPCR data on a set of selected genes. Each point on the plot corresponds to one promoter region or one ORF. Promoter regions correspond to intergenic regions in tandem or in divergent orientation, excluding intergenic regions encompassing Pol III-transcribed genes. A linear regression (red line) for ChIP-seq density in mutant versus ChIP-seq density in WT and an R2 correlation coefficient are indicated. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-444 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-504 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
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Related In: Results  -  Collection

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Figure 5: Global destabilization of PIC assembly in med17-444 and -504 mutant strains. The density of sequence tags in Med15, Rad3, Kin28 and TBP ChIP-seq experiments was calculated for promoter regions of Pol II-transcribed genes. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. The density of sequence tags in the Pol II ChIP-seq experiments was calculated for the Pol II-transcribed genes. In all, 3852 Pol II-enriched genes were used for these analyses. Tag densities were normalized relative to qPCR data on a set of selected genes. Each point on the plot corresponds to one promoter region or one ORF. Promoter regions correspond to intergenic regions in tandem or in divergent orientation, excluding intergenic regions encompassing Pol III-transcribed genes. A linear regression (red line) for ChIP-seq density in mutant versus ChIP-seq density in WT and an R2 correlation coefficient are indicated. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-444 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-504 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
Mentions: The ChIP results on the selected class II genes showed the most pronounced effects on Pol II, TBP and Kin28 occupancy for the med17-444 mutant whereas Rad3 occupancy remained unaffected in this mutant (Figures 2 and 3). Genome-wide analysis of the med17-444 mutant revealed a global 1.8-fold decrease (the slope of regression line, shown in red, is equal to 0.55) in Med15 Mediator occupancy with a high correlation coefficient (R2 equal to 0.93) (Figure 5A). As a consequence, the presence of other PIC components like Pol II, TFIIH and TBP was also globally reduced to a different extent. A global decrease in genome-wide occupancy for Pol II and the Kin28 TFIIK subunit (2.6- and 2.7-fold, respectively) was observed. The TBP association to chromatin was also globally affected with a 1.6-fold decrease. The Rad3 TFIIH core subunit binding was slightly affected (1.2-fold decrease), consistent with the ChIP results on the ADH1, PYK1 and PMA1 gene promoters. Taken together, the genome-wide results on the med17-444 mutant confirmed a central role for the Med17 Mediator subunit in PIC formation and suggested that the Mediator tail module destabilization on the chromatin could lead to a global change in the in-vivo binding of several PIC components but only marginally affected Rad3 occupancy.

Bottom Line: The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II.We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin.This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Institut de Biologie et de Technologies de Saclay (iBiTec-S), CEA, CNRS, Université Paris Sud, F-91191 Gif-sur-Yvette cedex, France.

No MeSH data available.


Related in: MedlinePlus