Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase II’s C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes a reduction of phosphorylated Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.


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Chromatin immunoprecipitation and next-generation sequencing technologies 44 have revealed how phosphorylation levels of CTD residues change temporally and 45 spatially during each transcription cycle (Eick and Geyer, 2013). The major sites of 46 phosphorylation are Ser5, directed by TFIIH in mammals (Feaver et al., 1994), and Ser2, 47 installed by P-TEFb in mammals (Marshall et al., 1996). The other three phosphate-

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The CTD code is generated through the interplay of CTD modifying enzymes 58 such as kinases, phosphatases and prolyl isomerases (Bataille et al., 2012). Disruption To phosphorylate Ser2 and Ser5, CTD kinases must discriminate very similar SP 119 motifs in the CTD, Y 1 S 2 P 3 and T 4 S 5 P 6 , to maintain accuracy during transcription. Among 120 the flanking residues of these two motifs, the unique structure of the tyrosine side chain 121 likely contributes to the recognition of the serine residues subject to phosphorylation.

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Several factors suggest the chemical properties of residues at the Tyr1 position are 123 important for CTD modification. First, residues at this first position of the heptad are 124 highly conserved across species and substitution to non-aromatic residues is rare, 125 suggesting significance to function (Chapman et al., 2008). As evidence of this, even

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To determine the effect of the chemical characteristics of residues at the Tyr1 136 position on CTD modification, we searched for naturally occurring Tyr1 substitutions.

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To identify the position of phosphates added by P-TEFb when Tyr1 is 250 phosphorylated, we quantified pSer2 and pSer5 by immunoblotting with antibodies 251 recognizing Ser2 and Ser5 phosphorylations ( Figure 3B). Compared to a non-252 phosphorylated CTD, the pre-treatment of yCTD with c-Abl results in a significant 253 increase in Ser2 phosphorylation of nearly 300% as detected by pSer2 specific CTD 254 antibody 3E10, accompanied by little change in pSer5 by P-TEFb ( Figure 3B and 3C).

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The increase of pSer2 is unique for P-TEFb-mediated phosphorylation on CTD since 256 similar tandem treatment of c-Abl followed by either TFIIH or Erk2 showed no changes 257 on pSer2 levels ( Figure S3A).

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We propose two possible explanations for the apparent increase of pSer2 levels 259 upon c-Able/P-TEFb treatment: First, c-Abl interacts with and/or modifies P-TEFb and 260 alters its specificity from Ser5 to Ser2. Alternatively, c-Abl may phosphorylate substrate 261 CTD and these phosphorylations prime the P-TEFb specificity towards Ser2 residues of 262 the CTD. To differentiate these two models, we inactivated c-Abl after its reaction with 263 CTD but before the addition of P-TEFb. We used two independent methods to inactivate 264 c-Abl prior to P-TEFb addition: introduction of the potent Abl inhibitor dasatinib to 10 µM 265 or denaturation of c-Abl via heat-inactivation ( Figure S3B). In both experiments, P-TEFb 266 continues to install a greater amount of Ser2 phosphorylation relative to no c-Abl 267 treatment controls ( Figure S3B). Therefore, the increase in the apparent Ser2 268 phosphorylation is not due to P-TEFb's physical interaction with c-Abl but arises from c-

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Abl kinase activity against CTD substrates at Tyr1.

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To determine the phosphorylation pattern resulting from sequential kinase

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When treated with P-TEFb alone, we observed four single phosphorylated peptides: two 292 almost equally abundant peaks containing di-heptads with a single pSer5 ( Figure 3E and 293 S4B, peak 1 and 2) and two peaks about ~40 fold less in intensity with pSer2 or pSer7 294 ( Figure 3E and S4B, peak 3 and 4), consistent with our previously analysis that P-TEFb 295 strongly favors pSer5 in unmodified CTD substrates ( Figure 1A). Double phosphorylated 296 species are also detected for di-heptads with both Ser5 residues phosphorylated as the 297 predominant product ( Figure 3E and 3F and S4B, peak 5). Several very small peaks 298 (less than 100-fold lower in intensity) are identified as peptides containing both Ser5 and 299 pSer2 ( Figure 3E and S4B, peak 6). These results indicate that the existence of Lys 300 residue does not seem to bias kinase activity and is consistent with our previous results 301 that P-TEFb strongly prefers to phosphorylate Ser5.

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When treated in tandem with c-Abl followed by P-TEFb and digested with trypsin, 303 diheptads (YSPTSPSYSPTSPK) in a variety of phosphorylation states are generated.

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These species were separated in CL and revealed 9 di-heptad species of varying

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The high performance of LC chromotography also allowed us to confirm our 326 phosphomapping within in di-heptads in the yeast CTD that diverge from consensus 327 sequence ( Figure S1C). Three di-heptads of divergent sequence were generated 328 following trypsin digestion of the yCTD-Lys construct ( Figure

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To further corroborate the mass spectrometry results that the specificity of P-

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TEFb is altered from Ser5 to Ser2 upon Tyr1 phosphorylation, we generated a new 343 yeast CTD variant with 13 repeats (half of the full-length yeast CTD) with every single 344 Ser5 mutated to alanine (S5A construct, sequence in Figure S1H). Previously, it was 345 shown that replacing Ser5 in CTD prevents its phosphorylation by P-TEFb

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Kinetic data obtained from the two assays described above were analyzed in R 593 (Hamilton, 2015;Team, 2017) and fitted to the Michaelis-Menten kinetic equation to 594 obtain respective kinetic parameters k cat (s -1 ) and K m (µM).

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The proximal promoter region is defined as −50bp to +300bp around the transcription

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Supplementary Figure S4 -LC-UVPD-mass spectra yCTD-Lys treated with CTD kinases. Related to Figure 3. The tables show the fragments matched from UVPD spectra. The confirmed phosphorylated residues are highlighted with blue boxes in the sequence, and backbone cleavages that produce diagnostic fragment ions are designated by color-coded slash marks (a/x green, b/y blue, c/z red) in the sequence.