Volume 21, Issue 2, Pages 227-237 (January 2006) P-TEFb-Mediated Phosphorylation of hSpt5 C-Terminal Repeats Is Critical for Processive Transcription Elongation  Tomoko Yamada, Yuki Yamaguchi, Naoto Inukai, Sachiko Okamoto, Takashi Mura, Hiroshi Handa  Molecular Cell  Volume 21, Issue 2, Pages 227-237 (January 2006) DOI: 10.1016/j.molcel.2005.11.024 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Structure of the hSpt5 CTR and Its Mutants (A) Evolutionary conservation of the Spt5 CTR pentapeptide motif. The consensus sequence is shown at the top. The heptapeptide motif of the RNA Pol II CTD is also shown for comparison. (B) Partial sequences of wild-type and mutant hSpt5 proteins used in this study. Schematic structure of hSpt5 is shown at the top. Boxes in amino acid sequence indicate the pentapeptide motifs. (C) hSpt5 and its mutants that were expressed in bacteria and purified extensively. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 CTR Thr-4 Is Required for Activation Activity, but Not for Repression Activity, of DSIF (A) Reactions containing either normal NE or NEΔDSIF, recombinant proteins, and pTF3-6C2AT were preincubated. Transcription was allowed to proceed for 10 min in the presence or absence of DRB. “DSIF” represents an equimolar mixture of hSpt4 and one of the hSpt5 proteins. An arrow indicates a full-length 380 nt transcript. (B and D) pSLG402 was used as a template in place of pTF3-6C2AT. Arrows indicate promoter-proximal (40–124) and -distal (1512–1888) fragments of transcripts. In (D), P-TEFb and DSIF were incubated in the presence of 60 μM ATP at 30°C for 10 min in separate reactions, which were then combined with mixtures containing NEΔDSIF and the template prior to addition of nucleotides, as shown in the diagram. (C) Transcription reactions were carried out essentially as in (B), using [γ-32P] ATP instead of [α-32P] UTP. hSpt5 was immunoprecipitated, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and autoradiographed. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 CTR Thr-4 Is Phosphorylated by P-TEFb In Vitro (A) P-TEFb and one of the hSpt5 proteins were incubated in the presence of [γ-32P] ATP for the indicated times, resolved by SDS-PAGE, and visualized by silver staining and autoradiography. (B) To quantify the radioactivity incorporated into hSpt5, protein bands were excised and subjected to liquid scintillation counting. Reaction rates were calculated and plotted against time as described in the Experimental Procedures. (C) Phosphoamino acid analysis using GST-CTR as a substrate. A 32P labeled CTR peptide was acid hydrolyzed, electrophoresed on a TLC plate, and autoradiographed (lane 2). Lane 1 shows ninhydrin-stained phosphoamino acid standards. An asterisk indicates phosphopeptides generated by partial hydrolysis. Abbreviations: pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine; and ori, origin. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 DSIF and CTR Thr-4 Are Important for EGF-Inducible c-fos Transcription (A) Experimental design. In F-WT cells, endogenous hSpt5 is constitutively knocked down, and shRNA-resistant Flag-hSpt5 is conditionally expressed from a tetracycline-repressible promoter (TRE). (B) Immunoblot analysis for hSpt5. Knockdown (KD) cells are F-WT cells that have been cultured for 4 days in the presence of 2 μg/ml tetracycline. Anti-hSpt5 and anti-Flag antibodies detect total and exogenous hSpt5, respectively. Topoisomerase I (topo I) serves as a loading control. (C and E) Real-time RT-PCR analysis for c-fos and GAPDH mRNAs. Total RNA was prepared from cells that were stimulated for the indicated times with 0.1 μg/ml EGF. The results shown are means ± SEM from three independent experiments. Values derived from unstimulated F-WT cells are set to 1. (D) Real-time RT-PCR analysis for hSpt5 mRNA. hSpt5 expression was quantified by using primer sets that distinguish between endogenous and exogenous hSpt5 sequences. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 DSIF and CTR Thr-4 Are Important for Progression of RNA Pol II along c-fos Gene after Induction (A) Indicated cell types were stimulated with EGF or not and subjected to ChIP with anti-RNA Pol II or anti-Flag antibody. Promoter, coding, and 3′ regions of c-fos and a control, intergenic region on chromosome 2 were amplified by real-time PCR. Each bar represents a mean ± SEM from three independent experiments. In the diagram of the c-fos gene, open and closed boxes represent introns and exons, respectively. (B) Cell lysates were immunoblotted with antibodies against Thr-202- and Tyr-204-phosphorylated ERK, ERK, Ser-383-phosphorylated Elk-1, and topoisomerase I (topo I). (C) ChIP was performed as in (A), using anti-TBP antibody (Santa Cruz, sc-273). Each bar represents a mean ± SEM from three independent experiments. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 CTR Thr-4 Is Phosphorylated by P-TEFb In Vivo (A) HeLa cells were treated with various concentrations of Flavopiridol for 1 hr, and total cell lysates were immunoblotted with anti-hSpt5 antibody. (B) In vivo phosphopeptide mapping. hSpt5 was immunoprecipitated from cells that were metabolically labeled with 32P, resolved by SDS-PAGE, isolated, digested with trypsin, and analyzed on TLC plates. Where indicated, Flavopiridol was added to a final concentration of 0.5 μM 1 hr prior to harvest. The dot and the circle of each panel mark the origin and the signals corresponding to the CTR phosphopeptides, respectively. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Models Showing the Role of DSIF in EGF-Inducible c-fos Transcription (A) The distribution of RNA Pol II (filled circle) over the c-fos gene. In the induced state, the DSIF knockdown results in only a weak reduction in RNA Pol II associated with the promoter region, but it has a more pronounced effect on RNA Pol II associated with downstream regions. (B) Upon EGF stimulation, P-TEFb-mediated phosphorylation of the RNA Pol II CTD and the hSpt5 CTR occurs and converts DSIF from a repressor to an activator. The phosphorylated CTR may function in a manner analogous to that of the phosphorylated CTD by recruiting other factors capable of activating transcription elongation. Molecular Cell 2006 21, 227-237DOI: (10.1016/j.molcel.2005.11.024) Copyright © 2006 Elsevier Inc. Terms and Conditions