Fig 2: Two Cdk9-regulated phosphatases help order the transcription cycle.a Schematic of the MYC gene, indicating positions of primer pairs used in ChIP-qPCR analysis. b ChIP-qPCR analysis of PP4C, PP4R2, and PP1γ on the MYC gene in unperturbed HCT116 cells (n = 2 biological replicates). c ChIP-qPCR analysis of PP4R2 and PP4R2-pThr173 after inhibition of Cdk9 with NVP-2 (250 nM) or mock treatment (DMSO) for 1 h (n = 2 biological replicates). d Cells transfected with siRNA targeting PP4R2 or nontargeted negative control siRNA were subjected to formaldehyde cross-linking, chromatin isolation, and reversal of cross-linking before analysis by immunoblotting with indicated antibodies to measure the depletion of PP4R2 and phosphorylation of Spt5 at Ser666 and Thr806. e ChIP-qPCR analysis on MYC shows Pol II distribution with or without PP4R2 depletion. Error bars (+s.d.) and p values were calculated by two-sided Student’s t test from four biological replicates (n = 4); individual data points from biological replicates are shown in the plots (b, c, and e). f Two distinct Cdk9-phosphatase switches govern transitions in Spt5-phosphorylation state—a model. At the 5′ pause prior to P-TEFb activation, the PP4 complex is active and both CTR1 and the KOW4–KOW5 loop are unphosphorylated, At the 3′ pause, PP1 becomes active to dephosphorylate CTR1 but not Ser666, distinguishing the two paused complexes. During elongation, Cdk9 phosphorylates Spt5, and inhibits PP4 and PP1. Source data are provided as a Source data file.

Fig 3: PP4 is active toward pSer666 and pThr806 and negatively regulated by Cdk9.a PP4R2 was immunoprecipitated from HCT116 cell extracts, incubated in vitro with purified, recombinant Cdk9/cyclin T1 and ATP, and immunoblotted with antibodies to PP4R2-pThr173, total PP4R2, or PP4C, as indicated at right. b HCT116 cells were mock treated (DMSO) or treated with inhibitors of Cdk9 (NVP-2) or Cdk1 (RO-3306), as indicated, for 1 h before lysis and extract preparation. Anti-PP4R2, anti-PP4C, or control IgG immunoprecipitates, as indicated, were incubated with Spt5-derived phosphopeptides containing pSer666 or pThr806, as indicated, and phosphate release was measured colorimetrically. Error bars indicate ±s.d. of six biological replicates (n = 6). c Anti-PP4R2 immunoprecipitates were incubated with 5 ng purified, recombinant Cdk9/cyclin T1 and ATP or mock treated (as indicated), washed and tested for phosphatase activity toward an Spt5-derived phosphopeptide containing pSer666 or pThr806, as in b. Activity of Cdk9-treated samples is plotted as a fraction of activity of the corresponding, mock-treated immunoprecipitate (defined as 1.0). Error bars indicate ±s.d. from three biological replicates (n = 3). Individual data points from biological replicates are shown in the plots (b and c). d HCT116 cells were infected with lentivirus expressing shRNA targeting PP4C (three different hairpins, sh1-3) or a nontargeted control vector (V), and chromatin fractions were immunoblotted for Spt5-pSer666, Spt5-pThr806, total Spt5 (to ensure equal loading),and total PP4C (to assess efficiency of depletion). Source data are provided as a Source data file.

Fig 4: ZWC complex-mediated SPT5 phosphorylation regulates divergent antisense transcription. (A) Establishment of a cell line system expressing WT or S6Y2→A mutant FLAG-SPT5 with endogenous SPT5 depletion. Schematic representation of SPT5 mRNA from endogenous genes and FLAG-SPT5 mRNA from a chromosome-integrated plasmid indicating the SPT5 siRNAs targeting the 3′ UTR used to selectively deplete endogenous SPT5 (blue lines) and the RT-qPCR primers used to measure the levels of total and endogenous SPT5 mRNA (black lines). Note that the primers for endogenous SPT5 were designed to amplify the region between the 5′ UTR and gene body of SPT5 (Endo-SPT5), and are thus specific for endogenous SPT5 mRNA but not FLAG-SPT5 mRNA. Note also that siSPT5 #1 and #2 were used for this figure and Supplementary Figure S6, respectively. (B and C) WT and S6Y2→A mutant FLAG-SPT5 HEK293T cell lines were treated with either control or SPT5 3′ UTR-targeted (#1) siRNAs as indicated. mRNA levels of total and endogenous SPT5 were measured by RT-qPCR analyses. Error bars denote standard deviations from three independent experiments (B). Total and FLAG-tagged SPT5 protein levels were measured by immunoblot analyses with the indicated antibodies (C). (D) Schematic representation of GAPDH, EIF3K and HDAC6 loci and amplicons used for qPCR measurement of the levels of sense and divergent antisense transcripts. Numbers next to the amplicons for divergent antisense transcript indicate base pairs from the TSS of the gene (top). Sense and divergent antisense transcript levels in either control or SPT5 3′ UTR-targeted (#1) siRNA-treated cell lines were measured by RT-qPCR analysis. All transcript levels were normalized to those of β-actin mRNA. Error bars denote the standard deviations from three independent experiments (*P <0.05, **P <0.01; Student's t-test) (bottom).

Fig 5: A Cdk9-PP1 switch governing Spt5 phosphorylation is conserved in human cells.a Purified, recombinant Cdk9/cyclin T1 phosphorylates wild-type (WT) GFP-PP1γ, expressed in human cells and recovered by anti-GFP immunoprecipitation, but not a Thr311→Ala (TA) mutant variant. Phosphorylation was detected with antibody specific for the carboxy-terminal phosphorylation site (Thr320) in PP1α isoform, analogous to Thr311 of PP1γ. b Inhibition of Cdk9 or Cdk1 diminishes PP1γ-inhibitory phosphorylation in human cells. HCT116 cells were treated with DMSO, a Cdk1 inhibitor (RO-3306), a Cdk9 inhibitor (NVP-2), or both, as indicated. Extracts were analyzed by direct immunoblotting (lanes 1–4), or anti-PP1γ immunoprecipitation (IP) followed by immunoblotting (lanes 5–8), with the indicated antibodies. c Cdk9 inhibition diminishes phosphorylation of Spt5-Thr806 but not Ser2 of the Pol II CTD. HCT116 cells were treated with the indicated concentrations of NVP-2 for 1 h and extracts were immunoblotted with the indicated antibodies. d HCT116 cells were treated with 250 nM NVP-2 for indicated times and extracts were immunoblotted with antibodies specific for Spt5, Spt5-pThr806, and Spt5-pSer666, as indicated. Immunoblot (n = 1) signals were quantified with ImageJ software. e Spt5-derived phosphopeptides containing pSer666 or pThr806 or a control histone H3-derived phosphopeptide containing pSer10, as indicated, were incubated with purified PP1 or lambda phosphatase, as indicated, and phosphate release was measured colorimetrically. Individual data points are shown from three biological replicates (n = 3); error bars indicate ±standard deviation (±s.d.) from mean. f HCT116 cells were transfected with an siRNA cocktail targeting all three PP1 catalytic-subunit isoforms or a scrambled control (C) siRNA, as indicated, and extracts were immunoblotted for the indicated proteins or protein modifications. Experiments were performed twice with similar results (a–c, f). Source data are provided as a Source data file. Uncropped blots can be found in the Supplementary information.