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Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells.

Descostes N, Heidemann M, Spinelli L, Schüller R, Maqbool MA, Fenouil R, Koch F, Innocenti C, Gut M, Gut I, Eick D, Andrau JC - Elife (2014)

Bottom Line: Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation.Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5' associated) Pol II in mammalian cells, in contrast to what was described in yeast.Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, Marseille, France Centre National de la Recherche Scientifique (CNRS) UMR6102, Marseille, France Inserm U631, Marseille, France Institut de Génétique Moléculaire de Montpellier (IGMM), CNRS-UMR5535, Montpellier, France.

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Reproducibility of ChIP-seq experiments and selection of relevant signals used for analyses.(A) Correlation plots of biological replicates (for all but H3K36me3 i.e., a technical replicate) of ChIP-seq experiments used in this study at gene locations (‘Materials and methods–Correlation of biological replicates and cross-correlation’). Spearman correlation coefficient is indicated on the top left of the plots. (B) Distribution and threshold of background-subtracted signal used for profiling of significantly bound gene (Total, i.e., whole genic regions) in Figure 2, Figure 2—figure supplement 5A, and Figure 2—figure supplement 7C. The mean values used for distribution were computed on [TSS-1000 bp:TES+2000 bp] (TSS: transcription start site; TES: transcription end site). Note that the thresholds were set to the mean of the second Gaussian of the distribution (‘Materials and methods–Gene selection and average binding profiles’). Numbers of genes selected for Pol II, Ser2P, Ser5P, Ser7P, Tyr1P 3D12, and Tyr1P 8G5 are 1521, 1536, 1543, 2382, 2652, and 2608, respectively. (C) Distribution and threshold of Pol II significantly bound promoters (TSS) as in (B). The selection is used in Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2. 2044 genes were selected based on their mean values on TSS −/+ 500 bp.DOI:http://dx.doi.org/10.7554/eLife.02105.007
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fig2s1: Reproducibility of ChIP-seq experiments and selection of relevant signals used for analyses.(A) Correlation plots of biological replicates (for all but H3K36me3 i.e., a technical replicate) of ChIP-seq experiments used in this study at gene locations (‘Materials and methods–Correlation of biological replicates and cross-correlation’). Spearman correlation coefficient is indicated on the top left of the plots. (B) Distribution and threshold of background-subtracted signal used for profiling of significantly bound gene (Total, i.e., whole genic regions) in Figure 2, Figure 2—figure supplement 5A, and Figure 2—figure supplement 7C. The mean values used for distribution were computed on [TSS-1000 bp:TES+2000 bp] (TSS: transcription start site; TES: transcription end site). Note that the thresholds were set to the mean of the second Gaussian of the distribution (‘Materials and methods–Gene selection and average binding profiles’). Numbers of genes selected for Pol II, Ser2P, Ser5P, Ser7P, Tyr1P 3D12, and Tyr1P 8G5 are 1521, 1536, 1543, 2382, 2652, and 2608, respectively. (C) Distribution and threshold of Pol II significantly bound promoters (TSS) as in (B). The selection is used in Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2. 2044 genes were selected based on their mean values on TSS −/+ 500 bp.DOI:http://dx.doi.org/10.7554/eLife.02105.007

Mentions: (A) Co-immunoprecipitation with specific CTD isoforms in Raji B-cells reveals Tyr1P (3D12) association with Ser5P and Ser7P but not with Ser2P and Thr4P. (B) ChIP-seq example illustrating Tyr1P (3D12) association around the promoter of RPL22L1 gene. (C) Composite average profiling of ChIP-seq data at coding genes locations for Pol II (1433 genes), Tyr1P (3D12, 2462 genes), Ser5P (1464 genes), and Ser7P (2186 genes) in Raji B-cells and based on selections described in Figure 2—figure supplement 1B. Less stringent selections with more genes gave equivalent profiling (Figure 2—figure supplement 4A). (D) Profiling of Pol II, Tyr1P (3D12), Ser5P, Ser7P, nucleosomes midpoint and short strand specific RNAs (ssRNAs) around TSS locations with same selections described in (C). (E) Boxplots on 3201 genes without outliers showing mean levels of Pol II (2986 genes), Tyr1P (2964 genes), Ser5P (2909 genes), and Ser7P (2948 genes) ChIP-seq signal on regions representing each transcription orientation. The p-values (parametric two sided paired t test) of the difference of AS vs S signal are Pol II = 0.5, Tyr1p=3.4 × 10−15, Ser5p=0.6, Ser7p=3.5 × 10−2.


Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells.

Descostes N, Heidemann M, Spinelli L, Schüller R, Maqbool MA, Fenouil R, Koch F, Innocenti C, Gut M, Gut I, Eick D, Andrau JC - Elife (2014)

Reproducibility of ChIP-seq experiments and selection of relevant signals used for analyses.(A) Correlation plots of biological replicates (for all but H3K36me3 i.e., a technical replicate) of ChIP-seq experiments used in this study at gene locations (‘Materials and methods–Correlation of biological replicates and cross-correlation’). Spearman correlation coefficient is indicated on the top left of the plots. (B) Distribution and threshold of background-subtracted signal used for profiling of significantly bound gene (Total, i.e., whole genic regions) in Figure 2, Figure 2—figure supplement 5A, and Figure 2—figure supplement 7C. The mean values used for distribution were computed on [TSS-1000 bp:TES+2000 bp] (TSS: transcription start site; TES: transcription end site). Note that the thresholds were set to the mean of the second Gaussian of the distribution (‘Materials and methods–Gene selection and average binding profiles’). Numbers of genes selected for Pol II, Ser2P, Ser5P, Ser7P, Tyr1P 3D12, and Tyr1P 8G5 are 1521, 1536, 1543, 2382, 2652, and 2608, respectively. (C) Distribution and threshold of Pol II significantly bound promoters (TSS) as in (B). The selection is used in Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2. 2044 genes were selected based on their mean values on TSS −/+ 500 bp.DOI:http://dx.doi.org/10.7554/eLife.02105.007
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2s1: Reproducibility of ChIP-seq experiments and selection of relevant signals used for analyses.(A) Correlation plots of biological replicates (for all but H3K36me3 i.e., a technical replicate) of ChIP-seq experiments used in this study at gene locations (‘Materials and methods–Correlation of biological replicates and cross-correlation’). Spearman correlation coefficient is indicated on the top left of the plots. (B) Distribution and threshold of background-subtracted signal used for profiling of significantly bound gene (Total, i.e., whole genic regions) in Figure 2, Figure 2—figure supplement 5A, and Figure 2—figure supplement 7C. The mean values used for distribution were computed on [TSS-1000 bp:TES+2000 bp] (TSS: transcription start site; TES: transcription end site). Note that the thresholds were set to the mean of the second Gaussian of the distribution (‘Materials and methods–Gene selection and average binding profiles’). Numbers of genes selected for Pol II, Ser2P, Ser5P, Ser7P, Tyr1P 3D12, and Tyr1P 8G5 are 1521, 1536, 1543, 2382, 2652, and 2608, respectively. (C) Distribution and threshold of Pol II significantly bound promoters (TSS) as in (B). The selection is used in Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2. 2044 genes were selected based on their mean values on TSS −/+ 500 bp.DOI:http://dx.doi.org/10.7554/eLife.02105.007
Mentions: (A) Co-immunoprecipitation with specific CTD isoforms in Raji B-cells reveals Tyr1P (3D12) association with Ser5P and Ser7P but not with Ser2P and Thr4P. (B) ChIP-seq example illustrating Tyr1P (3D12) association around the promoter of RPL22L1 gene. (C) Composite average profiling of ChIP-seq data at coding genes locations for Pol II (1433 genes), Tyr1P (3D12, 2462 genes), Ser5P (1464 genes), and Ser7P (2186 genes) in Raji B-cells and based on selections described in Figure 2—figure supplement 1B. Less stringent selections with more genes gave equivalent profiling (Figure 2—figure supplement 4A). (D) Profiling of Pol II, Tyr1P (3D12), Ser5P, Ser7P, nucleosomes midpoint and short strand specific RNAs (ssRNAs) around TSS locations with same selections described in (C). (E) Boxplots on 3201 genes without outliers showing mean levels of Pol II (2986 genes), Tyr1P (2964 genes), Ser5P (2909 genes), and Ser7P (2948 genes) ChIP-seq signal on regions representing each transcription orientation. The p-values (parametric two sided paired t test) of the difference of AS vs S signal are Pol II = 0.5, Tyr1p=3.4 × 10−15, Ser5p=0.6, Ser7p=3.5 × 10−2.

Bottom Line: Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation.Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5' associated) Pol II in mammalian cells, in contrast to what was described in yeast.Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, Marseille, France Centre National de la Recherche Scientifique (CNRS) UMR6102, Marseille, France Inserm U631, Marseille, France Institut de Génétique Moléculaire de Montpellier (IGMM), CNRS-UMR5535, Montpellier, France.

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