Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells
- Université Aix-Marseille, France
- Centre National de la Recherche Scientifique (CNRS) UMR6102, France
- Inserm U631, France
- Institut de Génétique Moléculaire de Montpellier (IGMM), CNRS-UMR5535, France
- Helmholtz Center Munich, Center of Integrated Protein Science Munich, Germany
- Centre Nacional D'Anàlisi Genòmica, Spain
Figures
Figure 1 with 2 supplements
Y1F mutations of the CTD heptads yield a truncated Pol IIB Rpb1.
(A) Rpb1-Y1F mutant was expressed after removal of tetracycline and in the presence of endogenous Rpb1. Probing with Rpb1 Ab reveals both endogenous and recombinant Rpb1 whereas HA reveals only …
Figure 1—figure supplement 1
Tyr1P is expressed in various human and mouse cell lines.
(A) Western blot analyses of antibody recognition in mouse and human cell lines of Rpb1, CTD (8WG16), and CTD isoforms including Tyr1P (3D12). MEF, mouse embryo fibroblasts; Raji, Burkitt-Lymphoma; …
Figure 1—figure supplement 2
Sequence of the CTD heptads for the Tyr1 to Phe mutant (Y1F).
Amino-acid composition of the C-terminal domain of the Y1F mutant (as described in the ‘Materials and methods–Construction of the CTD Y1F mutant’) used for phenotypic and western blot analyses (Figur…
Figure 2 with 7 supplements
CTD Tyrosine 1 is phosphorylated mainly at TSS and is dominant in antisense transcription.
(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 …
Figure 2—figure supplement 1
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 …
Figure 2—figure supplement 2
Pol II and CTD PTMs correlate positively with expression.
Based on microarray expression data, three groups of genes with low (L, 3414 genes), medium (M, 1238 genes), and high (H, 1007 genes) expression were used to profile Pol II isoforms and short ssRNA …
Figure 2—figure supplement 3
Examples of Tyr1P binding patterns at genic locations.
EIF1B and SNHG8 are mainly bound by Tyr1P (3D12) at TSS as for RPL22L1 gene of Figure 2B.
Figure 2—figure supplement 4
Average profiling of Pol II and phospho-isoforms at genic and promoter locations using wide relaxed threshold selections.
(A) Composite and TSS focused average profiling of ChIP-seq data as in Figure 2C,D, for a selection threshold of 0 as described in Figure 2—figure supplement 1B, at coding genes locations for Pol II …
Figure 2—figure supplement 5
Ser2P average profile at genic locations and examples of Tyr1P signal at promoter locations.
(A) Ser2P average profile on 1415 genes selected on mean values distribution shown in Figure 2—figure supplement 1B and represented as for Figure 2C. (B) Examples of Tyr1P (and other isoforms, short …
Figure 2—figure supplement 6
Tyr1P presents a specific pattern of phosphorylation along genes compared to Pol II.
(A) Genome-wide profiling of Pol II (N20) and CTD isoforms (as in Figure 2) for different classes of binding levels indicate a distribution of Tyr1P more prominent at promoters vs gene bodies as …
Figure 2—figure supplement 7
Tyr1P specific antibodies with distinct peptide recognition patterns show similar genome-wide profiling at TSS.
(A) CTD peptide recognition patterns of 3D12 and 8G5 Tyr1P Abs used in this study. Note that 8G5 shows a wider range of peptide recognition compared to 3D12. (B) Specificity and reactivity of mAbs …
Figure 3 with 2 supplements
Tyr1 is preferentially phosphorylated in antisense orientation on a particular subset of genes.
(A) Heatmaps of Tyr1P (3D12), Pol II, Ser5P, Ser7P, nucleosome midpoints (positioning) and short strand specific RNAs (red for AS and blue for S signal) at promoters with a significant level of Pol …
Figure 3—figure supplement 1
Three classes of Pol II-bound promoters ordered by Tyr1P location in human Raji cells.
(A) Heatmaps of a selection of Pol II-bound promoters for ssRNAs, nucleosome and AT, GC contents ordered by Tyr1P (3D12) maximum signal from the most upstream to the most downstream of the annotated …
Figure 3—figure supplement 2
CTD isoforms and nucleosome distribution around Pol II upstream of TSSs in class I promoters.
(A) 3D plots of Tyr1P, Ser5P, Ser7P and nucleosomes midpoints (MP) maximum signal locations as compared to Pol II ChIP-seq maxima for genes of group 1 of Figure 3A. Only genes with a significant …
Figure 4 with 3 supplements
Tyr1P is a hallmark of enhancers relative to Pol II and promoters signal.
(A) Average profiling of Pol II, Tyr1P (3D12), Ser5P, Ser7P, nucleosomes occupancy, and short ssRNAs. 390 active putative enhancers (red) and 4618 control promoters (blue) were detected in human …
Figure 4—figure supplement 1
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 …
Figure 4—figure supplement 2
Examples of Tyr1P enhancer association upstream or downstream of CXCR4, DUSP2, and IER5 genes.
As in Figure 4, light orange and blue rectangles highlight enhancer and promoter locations with higher H3K4me3 at promoters and higher H3K4me1 at enhancers. Relative amount of Tyr1P is higher at …
Figure 4—figure supplement 3
Selection of enhancers using H3K27ac also shows a dominance of Tyr1P on active and tissue specific enhancers.
(A) Workflow of a complementary selection of enhancers (927/2598 active), and control promoters (5946/6057 active) based on ChIP-seq of H3K36me3, H3K4me3, and H3K4me1. H3K27ac was used to extract …
Tables
Table 1
Summary of ChIP conditions and bioinformatics treatment for each experiment (NR = not relevant, NA = not available)
| ChIP antibodies and conditions used (* For ChIP-QPCR) | Peak detection | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Experiment | Antibody (clone) | Origin | Reference Antibody | Number of cells | Antibody/Beads | Washes (RIPA/TE) | Replicates Number | Tags Not Aligned/Multiple Alignment (× 106) | Tags Used (× 106) | Lanes Number | Extension Size (bp) | Threshold | Max Gap |
| Pol II | Total (N-20) | Rabbit polyclonal | Santa Cruz (sc-899x) | 1 × 108 | 20 µg/200 µl | 8x/1x | 1 | 8.93 | 19.83 | 1 | 176 | 80 | 350 |
| 2 | 17.94 | 33.02 | 2 | 166 | |||||||||
| 3 | 16.54 | 28.48 | 1 | 156 | |||||||||
| H3K4me1 | H3K4me1 | Rabbit polyclonal | Abcam (ab8895) | 5 × 106 | 2 µg/20 µl | 8x/1x | 1 | 9.35 | 7.83 | 1 | 176 | 60 | 700 |
| 2 | 7.59 | 20.93 | 1 | 226 | |||||||||
| H3K4me3 | H3K4me3 | Rabbit polyclonal | Abcam (ab8580) | 5 × 106 | 2 µg/20 µl | 8x/1x | 1 | 7.12 | 2.61 | 1 | 186 | 50 | 400 |
| 2 | NA | 14.14 | 1 | 123 | |||||||||
| H3K36me3 | H3K36me3 | Rabbit polyclonal | Abcam (ab9050) | 2 × 107 | 8 µg/80 µl | 8x/1x | 1 | NA | 21.21 | 1 | 196 | 40 | 1000 |
| 2 | NA | 5.57 | 1 | 316 | |||||||||
| H3K27ac | H3K27ac | Rabbit polyclonal | Ab4729 | 5 × 106 | 2 µg/20 µl | 5x/1x | 1 | 5.33 | 52.50 | 1 | 197 | 100 | 750 |
| Tyr1P | Tyr1P (3D12) | Rat monoclonal | Mayer et al. (2012) | 1 × 108 | 10 µg/100 µl | 5x/1x | 1 | 12.30 | 15.56 | 1 | 206 | NR | NR |
| 2 | 9.98 | 15.55 | 1 | 276 | |||||||||
| Tyr1P (8G5) | Rat monoclonal | This article | 1 × 108 | 10 µg/100 µl | 5x/1x | 1 | 30.26 | 28.78 | 1 | 187 | NR | NR | |
| Ser2P | Ser2P (3E10) | Rat monoclonal | Chapman et al. (2007) | 2 × 108 | 80 µg/400 µl | 5x/1x | 1 | 9.31 | 11.28 | 1 | 192 | NR | NR |
| 2 | 9.85 | 15.94 | 1 | 286 | |||||||||
| Ser5P | Ser5P (3E8) | Rat monoclonal | Chapman et al. (2007) | 1.2 × 108 (2.5 × 107*) | 24 µg/240 µl (5 µg/50 µl*) | 8x/1x | 1 | NA | 13.98 | 1 | 146 | NR | NR |
| 2 | NA | 3.57 | 1 | 216 | |||||||||
| Ser7P | Ser7P (4E12) | Rat monoclonal | Chapman et al. (2007) | 1 × 108 | 10 µg/100 µl | 5x/1x | 1 | NA | 16.46 | 1 | 156 | NR | NR |
| 2 | NA | 1.92 | 1 | 226 | |||||||||
| Short-RNA-seq | NR | NR | NR | 1 × 107 | NR | NR | 1 | NA | 9.87 | 1 | NR | NR | NR |
| MNase-seq | NR | NR | NR | 2 × 107 | NR | NR | 1 | 90.00 | 289.60 | 1 | 152/NR midpoints** | NR | NR |
| Input | NR | NR | NR | NR | NR | NR | 1 | 20.10 | 18.18 | 1 | 126 | NR | NR |
| 2 | NA | 29.74 | 1 | 146 | |||||||||
| 3 | 15.41 | 24.93 | 1 | 118 | |||||||||
| 4 | 11.20 | 28.32 | 1 | 196 | |||||||||
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**
For MNase-seq, the experiment was performed and processed in pair-end. For nucleosome density, tags were not elongated but connected and the indicated sequence average length is withdrawn by our analysis pipeline using the pair-end information. For midpoints analyses, elongation does not apply and data treatment is indicated earlier in ‘Materials and methods–Processing of sequenced tags’.
Additional files
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Source code 1
Folder containing the different scripts used in this study.
- https://doi.org/10.7554/eLife.02105.022
