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Tyr1 Phosphorylation Promotes Phosphorylation of Ser2 on the C-Terminal Domain

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Tyr1 Phosphorylation Promotes Phosphorylation of Ser2 on the C-Terminal Domain 1 Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain 2 of eukaryotic RNA polymerase II by P-TEFb 3 Joshua E. Mayfield1† *, Seema Irani2*, Edwin E. Escobar3, Zhao Zhang4, Nathanial T. 4 Burkholder1, Michelle R. Robinson3, M. Rachel Mehaffey3, Sarah N. Sipe3,Wanjie Yang1, 5 Nicholas A. Prescott1, Karan R. Kathuria1, Zhijie Liu4, Jennifer S. Brodbelt3, Yan Zhang1,5 6 1 Department of Molecular Biosciences, 2Department of Chemical Engineering, 7 3 Department of Chemistry and 5 Institute for Cellular and Molecular Biology, University of 8 Texas, Austin 9 4 Department of Molecular Medicine, Institute of Biotechnology, University of Texas 10 Health Science Center at San Antonio 11 †Current Address: Department of Pharmacology, University of California, San Diego, La 12 Jolla 13 * These authors contributed equally to this paper. 14 Correspondence: Yan Zhang ([email protected]) 15 16 1 17 Summary 18 The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates 19 Ser2 residues of C-terminal domain (CTD) of the largest subunit (RPB1) of RNA 20 polymerase II and is essential for the transition from transcription initiation to 21 elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in 22 vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and 23 hinders understanding of the transition from transcription initiation to elongation. 24 Through in vitro reconstruction of CTD phosphorylation, mass spectrometry 25 analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we 26 uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of 27 P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 28 phosphorylation causes an accumulation of RNA polymerase II in the promoter 29 region as detected by ChIP-seq. We demonstrate the ability of Tyr1 30 phosphorylation to generate a heterogeneous CTD modification landscape that 31 expands the CTD’s coding potential. These findings provide direct experimental 32 evidence for a combinatorial CTD phosphorylation code wherein previously 33 installed modifications direct the identity and abundance of subsequent coding 34 events by influencing the behavior of downstream enzymes. 35 The C-terminal domain of the RPB1 subunit of RNA polymerase II (CTD) is 36 composed of a species-specific number of repeats of the consensus amino acid heptad 37 YSPTSPS (arbitrarily numbered as Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, & Ser7) 38 (Jeronimo et al., 2016). The CTD undergoes extensive post-translational modification 39 (PTM) that recruits RNA processing and transcription factors that regulate progression 40 through the various stages of transcription. These modification events are dynamic, 41 highly regulated, and maintained through the complex interplay of CTD modification 2 42 enzymes. Collectively these PTMs and recruited protein factors constitute the “CTD 43 Code” for eukaryotic transcription (Buratowski, 2003). 44 Chromatin immunoprecipitation and next-generation sequencing technologies 45 have revealed how phosphorylation levels of CTD residues change temporally and 46 spatially during each transcription cycle (Eick and Geyer, 2013). The major sites of 47 phosphorylation are Ser5 and Ser2, directed by Transcription Factor II H (TFIIH (Feaver 48 et al., 1994) and P-TEFb in mammals (Marshall et al., 1996), respectively. The other 49 three phosphate-accepting residues (Tyr1, Thr4, and Ser7) are also subject to 50 modification, although their functions are less well-understood (Jeronimo et al., 2013). In 51 mammalian cells, the phosphorylations of Tyr1 and Ser7 rise and peak near the 52 promoter along with Ser5 and gradually decrease as transcription progresses towards 53 termination. The phosphorylation of Thr4 and Ser2, on the other hand, don’t appear until 54 later in the transcription cycle during elongation (Eick and Geyer, 2013). The molecular 55 underpinnings resulting in this orchestration are poorly defined. A particularly apparent 56 gap in current knowledge is if sequence divergence from the consensus heptad or 57 previously installed PTMs influence coding events. 58 The CTD code is generated through the interplay of CTD modifying enzymes 59 such as kinases, phosphatases, and prolyl isomerases (Bataille et al., 2012). Disruption 60 of this process is implicated in various disease states. P-TEFb is of particular interest 61 due to its overexpression in multiple tumor types and role in HIV infection (Franco et al., 62 2018). As a major CTD kinase, P-TEFb promotes transcription by contributing to the 63 release of RNA polymerase II from the promoter-proximal pause through its 64 phosphorylation of Negative Elongation Factor (NELF), DRB Sensitivity Inducing Factor 65 (DSIF), and Ser2 of the CTD (Wada et al., 1998). Interestingly, P-TEFb seems to 66 phosphorylate Ser5 of the CTD in vitro and mutation of Ser5 to alanine prevents the 67 phosphorylation of CTD substrates. However, mutation of Ser2 to alanine did not result 3 68 in this abolishment (Czudnochowski et al., 2012). These results are in contrast to in vivo 69 studies of P-TEFb specificity, where compromised P-TEFb kinase activity results in a 70 specific reduction in levels of Ser2 phosphorylation (Marshall et al., 1996). The 71 discrepancies between P-TEFb specificity in vitro and in vivo make it difficult to reconcile 72 P-TEFb’s function as a CTD Ser2 kinases (Bartkowiak et al., 2010; Czudnochowski et 73 al., 2012). 74 To resolve these inconsistencies, we utilize a multi-disciplinary approach to 75 investigate the specificity of P-TEFb. Identification of phosphorylation sites was carried 76 out using ultraviolet photodissociation (UVPD) mass spectrometry establishing the 77 specificity of P-TEFb in vitro in single residue resolution. We reveal the tyrosine kinase 78 c-Abl phosphorylates consensus and full-length CTD substrates in a conservative 79 fashion, with only half of the available sites phosphorylated. The unique phosphorylation 80 pattern of Tyr1 by tyrosine kinases like c-Abl directs the specificity of P-TEFb to Ser2. 81 The priming effect of pTyr1 on P-TEFb extends to human cells, where small-molecule 82 inhibition of c-Abl-like Tyr1 kinase activities leads to a reduction of Tyr1 phosphorylation. 83 Further ChIP-seq analysis shows that the loss of tyrosine phosphorylation increases 84 promoter-proximal pausing with an accumulation of RNA polymerase II. Overall, our 85 results reconcile the discrepancy of P-TEFb kinase activity in vitro and in cells, showing 86 that Tyr1 phosphorylation can prime P-TEFb and alter its specificity to Ser2. Importantly, 87 these findings provide direct experimental evidence for a combinatorial CTD 88 phosphorylation code wherein previously installed modifications direct the identity and 89 abundance of subsequent coding events, resulting in a varied PTM landscape along the 90 CTD allowing for diversified co-transcriptional signaling. 91 Results: 92 Determination of P-TEFb specificity in vitro using mass spectrometry 4 93 To define P-TEFb’s specificity directly on full-length RPB1 CTD substrates, we 94 applied matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and 95 liquid chromatography ultraviolet photodissociation tandem mass spectrometry (LC- 96 UVPD-MS/MS) to identify the substrate residues of this kinase. Ultraviolet 97 photodissociation (UVPD) using 193 nm photons is an alternative to existing collision- 98 and electron-based activation methods in proteomic mass spectrometry. This method 99 energizes peptide ions via a single absorption event of high-energy photons resulting in 100 a greater number of diagnostic fragment ions and the conservation of lower energy 101 bonds like those of some PTMs including phosphorylations (Brodbelt, 2014). This 102 method is applicable in both positive and negation ionization modes, results in a greater 103 degree of peptide fragmentation, better certainty in PTM localization, and conservation 104 of PTMs to ultimately ensure the detection of even low abundance or particularly labile 105 modifications. Because endogenous RNA polymerase II is heterogeneously modified, 106 we used recombinant yeast CTD GST fusion proteins, which contain mostly consensus 107 heptad repeats (20 of 26), as an unmodified substrate for PTM analysis (Figure 1-figure 108 supplement 1). The stability and consistency of GST yeast CTD (yCTD) make it ideal for 109 studying CTD modification along consensus heptads. With high kinase and ATP 110 concentration (2mM) and overnight incubation (~16 hours), P-TEFb generates two 111 phospho-peptides as detected by LC-UVPD-MS/MS: a major species phosphorylated on 112 Ser5 (Y1S2P3T4pS5P6S7) and a minor species phosphorylated on Ser2 113 (S5P6S7Y1pS2P3T4) (Figure 1A and Figure 1-figure supplement 2A-B). This is highly 114 similar to patterns observed previously for bona fide Ser5 CTD kinases Erk2 and TFIIH 115 (Mayfield et al., 2017). These experiments confirm P-TEFb’s inherent in vitro preference 116 for Ser5 when phosphorylating unmodified CTD (Czudnochowski et al., 2012; Portz et 117 al., 2017). 5 118 We next measured the total number of phosphates added to CTD by P-TEFb. 119 MALDI-MS analysis of yCTD treated with P-TEFb reveals a cluster of peaks with mass 120 shifts relative to no kinase control ranging from 1906.1 to 2318.4 Da, each interspaced 121 by 80 Da (Figure 1B and figure supplement 2C). This corresponds to the addition of 24 122 to 29 phosphates to yCTD’s 26 heptad repeats. This finding in combination with our LC- 123 UVPD-MS/MS analysis of P-TEFb treated yCTD indicates that P-TEFb phosphorylates 124 the CTD in an average one phosphorylation per heptad manner, and these heptads are 125 primarily phosphoryl-Ser5 (pSer5) in vitro. 126 Amino acid identity at the Tyr1 position is important for kinase activity 127 To phosphorylate Ser2 and Ser5, CTD kinases must discriminate very similar SP 128 motifs in the CTD, Y1S2P3 and T4S5P6, to maintain accuracy during transcription. Among 129 the flanking residues of these two motifs, the unique structure of the tyrosine side chain 130 likely contributes to the recognition of the serine residues subject to phosphorylation.
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