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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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Pol II-bound enhancer selection procedure and features.(A) Workflow of the enhancers (390) and control promoters (4618) selection based on ChIP-seq of H3K36me3, H3K4me3, H3K4me1, and Pol II. Details of procedure and number of regions isolated at each step (E1-4 and P1-3) are indicated in ‘Materials and methods–Selection of enhancers and promoters using Pol II’. (B) Plot of H3K4me3/me1 mean values ratios of selected intergenic regions at step E2 (in red), promoter regions at step P1 (in blue), and Hg19 RefSeq annotated promoters (in black). To stringently select isolated promoters and intergenic regions shown in (A) and attribute their putative enhancer and control promoter status, a threshold was defined (in dashed green line). (C) Nonoriented profiling of epigenetic marks associated with putative enhancers (in red) and control promoters (in blue) selected at steps P3 and E4 of procedure described in (A) and centered on the main Pol II peak as in Figure 4A. (D) Boxplots of H3K4me3 (363/4325 genes plotted) and H3K4me1 (375/4259 genes plotted) signals at putative enhancers (in red) and control promoters (in blue). Nonparametric Mann-Whitney-Wilcoxon test gave p-values <10−152. (E) Tissue specificity analysis of the genes associated with putative enhancers (closest genes on each side of the isolated genomic loci) compared to genes of HGU133 array (whole genes, see ‘Materials and methods–Tissue specificity analysis’). The isolated tissues are ranked by p-values (indicated on the left) from top to bottom. This analysis indicates that both WT (CD19) and Raji human B-cells are among the most significant tissues thus validating the putative enhancer regions identified in our analysis and as described in mouse lymphocytes (Li et al., 2012).DOI:http://dx.doi.org/10.7554/eLife.02105.018
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fig4s1: Pol II-bound enhancer selection procedure and features.(A) Workflow of the enhancers (390) and control promoters (4618) selection based on ChIP-seq of H3K36me3, H3K4me3, H3K4me1, and Pol II. Details of procedure and number of regions isolated at each step (E1-4 and P1-3) are indicated in ‘Materials and methods–Selection of enhancers and promoters using Pol II’. (B) Plot of H3K4me3/me1 mean values ratios of selected intergenic regions at step E2 (in red), promoter regions at step P1 (in blue), and Hg19 RefSeq annotated promoters (in black). To stringently select isolated promoters and intergenic regions shown in (A) and attribute their putative enhancer and control promoter status, a threshold was defined (in dashed green line). (C) Nonoriented profiling of epigenetic marks associated with putative enhancers (in red) and control promoters (in blue) selected at steps P3 and E4 of procedure described in (A) and centered on the main Pol II peak as in Figure 4A. (D) Boxplots of H3K4me3 (363/4325 genes plotted) and H3K4me1 (375/4259 genes plotted) signals at putative enhancers (in red) and control promoters (in blue). Nonparametric Mann-Whitney-Wilcoxon test gave p-values <10−152. (E) Tissue specificity analysis of the genes associated with putative enhancers (closest genes on each side of the isolated genomic loci) compared to genes of HGU133 array (whole genes, see ‘Materials and methods–Tissue specificity analysis’). The isolated tissues are ranked by p-values (indicated on the left) from top to bottom. This analysis indicates that both WT (CD19) and Raji human B-cells are among the most significant tissues thus validating the putative enhancer regions identified in our analysis and as described in mouse lymphocytes (Li et al., 2012).DOI:http://dx.doi.org/10.7554/eLife.02105.018

Mentions: Many groups including ours have shown that highly active and tissue-specific enhancers are transcribed by Pol II in various tissues (De Santa et al., 2010; Kim et al., 2010; Koch et al., 2011; Natoli and Andrau, 2012). These enhancers can also be hallmarked by the occurrence of H3K4me1high/H3K4me3low epigenetic marks combination (Koch et al., 2011; Pekowska et al., 2011). To investigate if Tyr1P can be detected at enhancers, we first isolated intergenic regions (IGRs) with stringent criteria in B-cells using Pol II, H3K4me1, and me3 signals. These were further discriminated from noncoding promoters using the relative ratio of H3K4me1/me3 (Pekowska et al., 2011; Li et al., 2012), and from both unannotated coding and some long intergenic noncoding genes using the absence of H3K36me3 that marks gene bodies (Guttman et al., 2009). Using these criteria, we isolated 390 B-cells enhancers (Figure 4—figure supplement 1A–D). Our selection was further validated using tissue-specificity analyses (Figure 4—figure supplement 1E) indicating IGRs associated with genes specific to B-cells. We next performed profiling of the various Pol II isoforms at these enhancers. As before (Koch et al., 2011), we observed that these IGRs were associated with Ser5P (Figure 4A) but not with Ser2P Pol II (not shown) as well as with short transcripts (reflecting paused transcription) and a discrete nucleosome depleted region. Consistent with early elongating Pol II at enhancers, we found signal for both Ser7P and Tyr1P at these IGRs. Importantly, Tyr1P appeared more bound to enhancers as compared to promoters and total Pol II (Figure 4B,C, Figure 4—figure supplement 2), suggesting that Tyr1 is more phosphorylated than Ser5 or Ser7 at enhancers and represent a hallmark of these essential areas of the genome. Additionally, Tyr1P also displayed the best correlation with Pol II at isolated enhancers (Figure 4D). Finally, using an independent selection for active enhancers based on H3K27ac brought very similar results (Figure 4—figure supplement 3). Together, our investigations showed that Tyr1P is a strong signature of Pol II-transcribed active enhancers associated with tissue-specific gene expression.10.7554/eLife.02105.017Figure 4.Tyr1P is a hallmark of enhancers relative to Pol II and promoters signal.


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)

Pol II-bound enhancer selection procedure and features.(A) Workflow of the enhancers (390) and control promoters (4618) selection based on ChIP-seq of H3K36me3, H3K4me3, H3K4me1, and Pol II. Details of procedure and number of regions isolated at each step (E1-4 and P1-3) are indicated in ‘Materials and methods–Selection of enhancers and promoters using Pol II’. (B) Plot of H3K4me3/me1 mean values ratios of selected intergenic regions at step E2 (in red), promoter regions at step P1 (in blue), and Hg19 RefSeq annotated promoters (in black). To stringently select isolated promoters and intergenic regions shown in (A) and attribute their putative enhancer and control promoter status, a threshold was defined (in dashed green line). (C) Nonoriented profiling of epigenetic marks associated with putative enhancers (in red) and control promoters (in blue) selected at steps P3 and E4 of procedure described in (A) and centered on the main Pol II peak as in Figure 4A. (D) Boxplots of H3K4me3 (363/4325 genes plotted) and H3K4me1 (375/4259 genes plotted) signals at putative enhancers (in red) and control promoters (in blue). Nonparametric Mann-Whitney-Wilcoxon test gave p-values <10−152. (E) Tissue specificity analysis of the genes associated with putative enhancers (closest genes on each side of the isolated genomic loci) compared to genes of HGU133 array (whole genes, see ‘Materials and methods–Tissue specificity analysis’). The isolated tissues are ranked by p-values (indicated on the left) from top to bottom. This analysis indicates that both WT (CD19) and Raji human B-cells are among the most significant tissues thus validating the putative enhancer regions identified in our analysis and as described in mouse lymphocytes (Li et al., 2012).DOI:http://dx.doi.org/10.7554/eLife.02105.018
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fig4s1: Pol II-bound enhancer selection procedure and features.(A) Workflow of the enhancers (390) and control promoters (4618) selection based on ChIP-seq of H3K36me3, H3K4me3, H3K4me1, and Pol II. Details of procedure and number of regions isolated at each step (E1-4 and P1-3) are indicated in ‘Materials and methods–Selection of enhancers and promoters using Pol II’. (B) Plot of H3K4me3/me1 mean values ratios of selected intergenic regions at step E2 (in red), promoter regions at step P1 (in blue), and Hg19 RefSeq annotated promoters (in black). To stringently select isolated promoters and intergenic regions shown in (A) and attribute their putative enhancer and control promoter status, a threshold was defined (in dashed green line). (C) Nonoriented profiling of epigenetic marks associated with putative enhancers (in red) and control promoters (in blue) selected at steps P3 and E4 of procedure described in (A) and centered on the main Pol II peak as in Figure 4A. (D) Boxplots of H3K4me3 (363/4325 genes plotted) and H3K4me1 (375/4259 genes plotted) signals at putative enhancers (in red) and control promoters (in blue). Nonparametric Mann-Whitney-Wilcoxon test gave p-values <10−152. (E) Tissue specificity analysis of the genes associated with putative enhancers (closest genes on each side of the isolated genomic loci) compared to genes of HGU133 array (whole genes, see ‘Materials and methods–Tissue specificity analysis’). The isolated tissues are ranked by p-values (indicated on the left) from top to bottom. This analysis indicates that both WT (CD19) and Raji human B-cells are among the most significant tissues thus validating the putative enhancer regions identified in our analysis and as described in mouse lymphocytes (Li et al., 2012).DOI:http://dx.doi.org/10.7554/eLife.02105.018
Mentions: Many groups including ours have shown that highly active and tissue-specific enhancers are transcribed by Pol II in various tissues (De Santa et al., 2010; Kim et al., 2010; Koch et al., 2011; Natoli and Andrau, 2012). These enhancers can also be hallmarked by the occurrence of H3K4me1high/H3K4me3low epigenetic marks combination (Koch et al., 2011; Pekowska et al., 2011). To investigate if Tyr1P can be detected at enhancers, we first isolated intergenic regions (IGRs) with stringent criteria in B-cells using Pol II, H3K4me1, and me3 signals. These were further discriminated from noncoding promoters using the relative ratio of H3K4me1/me3 (Pekowska et al., 2011; Li et al., 2012), and from both unannotated coding and some long intergenic noncoding genes using the absence of H3K36me3 that marks gene bodies (Guttman et al., 2009). Using these criteria, we isolated 390 B-cells enhancers (Figure 4—figure supplement 1A–D). Our selection was further validated using tissue-specificity analyses (Figure 4—figure supplement 1E) indicating IGRs associated with genes specific to B-cells. We next performed profiling of the various Pol II isoforms at these enhancers. As before (Koch et al., 2011), we observed that these IGRs were associated with Ser5P (Figure 4A) but not with Ser2P Pol II (not shown) as well as with short transcripts (reflecting paused transcription) and a discrete nucleosome depleted region. Consistent with early elongating Pol II at enhancers, we found signal for both Ser7P and Tyr1P at these IGRs. Importantly, Tyr1P appeared more bound to enhancers as compared to promoters and total Pol II (Figure 4B,C, Figure 4—figure supplement 2), suggesting that Tyr1 is more phosphorylated than Ser5 or Ser7 at enhancers and represent a hallmark of these essential areas of the genome. Additionally, Tyr1P also displayed the best correlation with Pol II at isolated enhancers (Figure 4D). Finally, using an independent selection for active enhancers based on H3K27ac brought very similar results (Figure 4—figure supplement 3). Together, our investigations showed that Tyr1P is a strong signature of Pol II-transcribed active enhancers associated with tissue-specific gene expression.10.7554/eLife.02105.017Figure 4.Tyr1P is a hallmark of enhancers relative to Pol II and promoters signal.

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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