Portal de Periódicos da CAPES

The Rate of c-fos Transcription in Vivo Is Continuously Regulated at the Level of Elongation by Dynamic Stimulus-coupled Recruitment of Positive Transcription Elongation Factor b

2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês

10.1074/jbc.m607847200

1083-351X

Stephan Ryser, Toshitsugu Fujita, Silvia Tórtola, Isabelle Piuz, Werner Schlegel,

Retinoids in leukemia and cellular processes

In mammalian cells, multiple stimuli induce the expression of the immediate early gene c-fos. The specificity of c-fos transcriptional response depends on the activation of signaling protein kinases, transcription factors, and chromatin-modifying complexes but also on a regulated block to elongation in the first intron. Here we show by chromatin immunoprecipitation that finely tuned control of c-fos gene expression by distinct stimuli is associated with a dynamic regulation of transcription elongation and differential phosphorylation of the C-terminal domain of RNA polymerase II. Comparison of two stimuli of c-fos expression in the pituitary cell line GH4C1, namely the thyrotropin-releasing hormone versus depolarizing KCl, shows that both stimuli increase initiation, but only thyrotropin-releasing hormone is efficient to stimulate elongation and thus produce high transcription rates. To control elongation, the elongation factor P-TEFb is recruited to the 5′-end of the gene in a stimuli and time-dependent manner. Transition from initiation to elongation depends also on the dynamic recruitment of the initiation factors TFIIB and TFIIE but not TFIID, which remains constitutively bound on the promoter. It thus appears that tight coupling of signaling input to transcriptional output rate is achieved by c-fos gene-specific mechanisms, which control post-initiation steps rather than pre-initiation complex assembly. In mammalian cells, multiple stimuli induce the expression of the immediate early gene c-fos. The specificity of c-fos transcriptional response depends on the activation of signaling protein kinases, transcription factors, and chromatin-modifying complexes but also on a regulated block to elongation in the first intron. Here we show by chromatin immunoprecipitation that finely tuned control of c-fos gene expression by distinct stimuli is associated with a dynamic regulation of transcription elongation and differential phosphorylation of the C-terminal domain of RNA polymerase II. Comparison of two stimuli of c-fos expression in the pituitary cell line GH4C1, namely the thyrotropin-releasing hormone versus depolarizing KCl, shows that both stimuli increase initiation, but only thyrotropin-releasing hormone is efficient to stimulate elongation and thus produce high transcription rates. To control elongation, the elongation factor P-TEFb is recruited to the 5′-end of the gene in a stimuli and time-dependent manner. Transition from initiation to elongation depends also on the dynamic recruitment of the initiation factors TFIIB and TFIIE but not TFIID, which remains constitutively bound on the promoter. It thus appears that tight coupling of signaling input to transcriptional output rate is achieved by c-fos gene-specific mechanisms, which control post-initiation steps rather than pre-initiation complex assembly. Stimulus-transcription coupling mediates cellular responses, which require changes in gene expression such as proliferation, differentiation, or adaptive responses (e.g. neuronal plasticity) in higher eukaryotes. The induction of immediate early gene (IEG) 3The abbreviations used are: IEG, immediate early response gene; ChIP, chromatin immunoprecipitation; CTD, C-terminal domain; FAM, 6-carboxyfluorescein; pol II, RNA polymerase II; TRH, thyrotropin-releasing hormone; Ham's F10 serum-free medium, Ham's F10 serum-free medium; RT, reverse transcription; TAMRA, 6-carboxytetramethylrhodamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PIC, pre-initiation complex; cdk, cyclin-dependent kinase; RCFRD, reconstituted c-fos rat DNA; P-TEFb, positive transcription elongation factor b; PD, promoter distal; PP, promoter proximal; TBP, tata-binding protein; TF, transcription factor; GTF, general transcription factor. transcription via multiple signal transduction pathways (1.Morgan J.I. Curran T. Trends Neurosci. 1989; 12: 459-462Abstract Full Text PDF PubMed Scopus (845) Google Scholar, 2.Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1984) Google Scholar, 3.Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar) is the first step in stimulus-transcription coupling. IEGs code mainly for transcription factors, which in turn will control the expression of further genes leading to the cellular responses. Distinct extracellular stimuli can induce in the same cell a similar panel of IEGs with the same kinetics but with clear differences in their levels of expression. Therefore, during any particular stimulation, specific signal transduction and transcription mechanisms must act in a concerted manner to obtain a distinct level of gene transcription on each specific IEG. In a recent review, Hazzalin and Mahadevan (4.Hazzalin C.A. Mahadevan L.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 30-40Crossref PubMed Scopus (342) Google Scholar) propose that the rate of IEG transcription varies continuously as a function of the strength of intracellular signaling events. Such dynamic control of IEG transcription implies an equally rapid reversal in addition to the well documented rapid induction of transcription. Transcription factors constitutively present on the promoter of IEGs may function as “rheostats,” sensing the degree of activation of several signal transduction pathways and driving continuously varying levels of gene transcription. In parallel, IEG transcription is also correlated with a dynamic regulation of phosphorylation and acetylation of histone (H3/H4) tails that modify the level of chromatin compaction within the gene (5.Thomson S. Clayton A.L. Mahadevan L.C. Mol. Cell. 2001; 8: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Thus, transcription factors and chromatin components function as sensors/integrators for signal transduction inputs. How is this information related to the basal transcription machinery, which has to modulate RNA polymerase II (pol II) to produce a continuously varying output of mRNA? We speculated that pol II features associated with either initiation or elongation could be differentially regulated by distinct signal transduction pathways. Specifically, we looked for changes in the phosphorylation levels of the C-terminal domain (CTD) of pol II during the expression of an IEG to understand how quantitative information can be converted from input intracellular signaling events to output rates of mRNA. During the transcription cycle, transition from initiation to processive elongation and termination is associated with changes in the phosphorylation state of the CTD (6.Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429Crossref PubMed Google Scholar, 7.Orphanides G. Reinberg D. Cell. 2002; 108: 439-451Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar). Different cyclin-dependent kinases (cdks) catalyze the differential phosphorylation of the serine residues in positions 2 and 5 in the YSPTSPS repeat of the CTD. These serine residues are phosphorylated in a dynamic fashion during the transcription of the entire gene (8.Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes & Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (302) Google Scholar, 9.Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar, 10.Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar, 11.Cheng C. Sharp P.A. Mol. Cell. Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar). Ser-5 is predominantly phosphorylated in pol II transcribing the 5′ part of the gene and progressively dephosphorylated during elongation while Ser-2 phosphorylation increases. Post-translational modifications of the CTD are also crucial for the maturation of the nascent RNA to mRNA in pol II genes (6.Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429Crossref PubMed Google Scholar, 7.Orphanides G. Reinberg D. Cell. 2002; 108: 439-451Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar): RNA capping enzyme and phospho-Ser-5 CTD are functionally associated (8.Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes & Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (302) Google Scholar, 9.Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar, 12.Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and so are phospho-Ser-2 CTD and 3′-end RNA-processing enzymes (13.Ni Z. Schwartz B.E. Werner J. Suarez J.R. Lis J.T. Mol. Cell. 2004; 13: 55-65Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 14.Ahn S.H. Kim M. Buratowski S. Mol. Cell. 2004; 13: 67-76Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). CTD phosphorylation combined with other CTD modifications such as proline cis-trans isomerization (15.Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (428) Google Scholar) generates different pol II structural conformations, with major consequences on the rate of gene transcription and mRNA synthesis (16.Buratowski S. Nat. Struct. Biol. 2003; 10: 679-680Crossref PubMed Scopus (260) Google Scholar). An important issue addressed with the present study is to understand how these different molecular mechanisms are regulated in vivo during IEG gene transcription by pol II and whether they are correlated in a quantitative manner with stimuli-dependent synthesis of mRNA. Cell Culture and Stimulation−GH4C1 pituitary cells were maintained in Ham’s F10 Gluta Max medium (Invitrogen) supplemented with 2.5% fetal bovine serum and 15% horse serum at 37 °C in a humidified atmosphere with 5% CO2. Confluent GH4C1 cells were incubated in Ham’s F10 Gluta Max serum free medium for 24 h and then stimulated for the indicated time with either 100 nm TRH, 10 nm epidermal growth factor, 10 ng/ml tumor necrosis factor, or 20 mm KCl. RNA Preparation and Quantitative RT-PCR−RNA preparation, RT-PCR, standard curves, and quantification procedures have been described previously (17.Ryser S. Tortola S. van Haasteren G. Muda M. Li S. Schlegel W. J. Biol. Chem. 2001; 276: 33319-33327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 18.Ryser S. Massiha A. Piuz I. Schlegel W. Biochem. J. 2004; 378: 473-484Crossref PubMed Google Scholar). mRNA levels of c-fos, GAPDH, H1, and of 18 S RNA were quantified by real-time RT-PCR (Applied Biosystems). c-fos gene expression was analyzed with 200 nm forward primer 5′-TGACAGATACGCTCCAAGCG-3′, 200 nm reverse primer 5′-TGGCAATCTCGGTCTGCA-3′, and 250 nm TaqMan probe 5′-FAM (6-carboxyfluorescein)-CGCAGACTTCTCGTCTTCAAGTTGATCTGTCT-TAMRA (6-carboxytetramethyrhodamine)-3′. GAPDH and H1 mRNA level were quantified similarly with primers pairs and probes described for the 3′-end (GAPDH) or the 5′-end (H1) of their coding sequences (see below). The TaqMan probe 18 S RNA Human was purchased from Applied Biosystems. Nascent and Free Nuclear RNA Preparation and Real-time RT-PCR Analysis−Nascent and free nuclear RNA fractions were prepared as described by (19.Wuarin J. Schibler U. Mol. Cell Biol. 1994; 14: 7219-7225Crossref PubMed Scopus (176) Google Scholar). c-fos nascent and free nuclear RNA at different times during the stimulation by TRH was quantified with the TaqMan probes for exons 1, 3, and 4; values were normalized to 18 S RNA in each individual fraction RT-PCR (17.Ryser S. Tortola S. van Haasteren G. Muda M. Li S. Schlegel W. J. Biol. Chem. 2001; 276: 33319-33327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 18.Ryser S. Massiha A. Piuz I. Schlegel W. Biochem. J. 2004; 378: 473-484Crossref PubMed Google Scholar). Chromatin Immunoprecipitation−GH4C1 cells were grown on 5 × 107 cells in 175-cm2 flasks. Prior to stimulation, the cells were incubated with serum-free Ham’s F10 Gluta Max medium for 24 h. The cells were stimulated by the addition directly into the 30 ml of Ham’s F10 serum-free medium of 100-fold concentrated stock solutions of TRH (100 nm final) and of KCl (20 mm final) and left for the indicated times at 37 °C in a humidified atmosphere with 5% CO2. At each time point of the stimulation, the dishes were rapidly placed on ice, and the culture medium was replaced with 30 ml of ice-cold fixing solution containing 1% formaldehyde, 9.08 mm NaCl, 4.5 mm HEPES, pH 8.0; incubation for cross-linking continued during 1 h at 4 °C, and then 127 mm glycine was added. The cells were washed 2× with phosphate-buffered saline, scrapped with a rubber policeman in phosphate-buffered saline with 1 mm phenylmethylsulfonyl fluoride (PMSF), and collected by centrifugation. Then the cell were lysed in cell lysis buffer (10 mm Tris, pH 8.1, 1 mm EDTA, pH 8.0, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 50 mm NaF, 1 mm ortho-vanadate, 1 μg/ml aprotinin, 1 μg/ml leupeptin), and nuclei were collected by centrifugation. The nuclei were further lysed in nuclear lysis buffer (10 mm Tris, pH 8.1, 1 mm EDTA, pH 8.0, 0.5 m NaCl, 1% Triton, 0.5% Sod-deoxy, 0.5% Sarkosyl, 1 mm phenylmethylsulfonyl fluoride, 50 mm NaF, 1 mm ortho-vanadate, 1 μg/ml aprotinin, 1 μg/ml leupeptin) and spun down, and the pelleted chromatin was resuspended in sonication buffer (10 mm Tris, pH 8.1, 1 mm EDTA, pH 8.0, 100 mm NaCl, pH 8.0). Formaldehyde-cross-linked chromatin extract was fragmented with a Branson 250 sonifier with an output control at 50% and 20-s hold/cycle; 10 cycles of sonication for each sample were supplemented with glass beads (Fluka). DNA fragments were <1 kb and averaged 450 bp as verified by agarose gel electrophoresis (data not shown). The amount of chromatin was estimated based on the DNA concentration measured by optical density at 260 nm. Chromatin extract was stored in aliquots at -80 °C until use. Immunoprecipitation was performed with 100 μg (DNA content) of chromatin extracts, diluted in radioimmune precipitation assay buffer (1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 140 mm NaCl, 1 mm EDTA, 10 mm Tris-HCl, pH 8.0, 1 mm phenylmethylsulfonyl fluoride) were pre-cleared with Protein A-Sepharose beads (Amersham Biosciences) for 1 h and then incubated overnight (16 h) with 20 μg of antibody at 4 °C. 8WG16, H14, and H5 antibodies were purchase from Covance, anti-pol II (N20), -cyclin T1 (H-245), -TBP (SI-1), -TFIIH p62 (Q-19), -TFIIB, and -TFIIE-α (C17) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-cdk8 was from Neomarkers. Protein A-Sepharose beads or (for H5 and H14 antibodies) anti-mouse IgM antibody-coupled agarose beads (Sigma) were then added to the mixture for 3 h at 4 °C. Beads were washed as described previously (11.Cheng C. Sharp P.A. Mol. Cell. Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar). Reversal of the cross-links in chromatin was achieved by overnight incubation at 65 °C with proteinase K and SDS 1%. DNA was extracted with phenol-chloroform and precipitated with EtOH supplemented with 0.3 m sodium acetate, pH 5.2, and 100 μg of glycogen/sample. DNA was resuspended in 240 μl of NaOH (8 mm pH 8.0) and stored at -20 °C. Real-time PCR was performed with 5 μl of DNA solution obtained by ChIP. Except for the 5′-end of GAPDH, c-fos proximal promoter, and intron 1 (analyzed with the SYBR Green PCR Master Mix, Applied Biosystems), all other PCR reactions were carried out with 200 nm primers pairs, 250 nm TaqMan probes (see supplemental material) and the Universal PCR master mix (Applied Biosystems), in a final volume of 25 μl. Real-time PCR was performed in triplicates in 96-well plates with the ABPrism 7700 Sequence Detection System. Initially each data point was calculated as the percentage of input DNA. To this end, a standard curve generated by serial dilution of genomic DNA purified from GH4C1 cells was included in each quantification. The percent input for mock ChIP (no primary antibody) was maximally 0.01%; thus for pol II the lowest ChIP values (obtained for the GAPDH 3′-end probe with pol II immunoprecipitated with the H5 antibody) represent twice the background thus measured. Immunoprecipitation was repeated at least three times from separate cross-linked chromatin for each time point. For the ChIP criteria used in Fig. 6, absolute values could not be directly compared between ChIP experiments with different antibodies because of the uncertainties of the efficiencies of the antibody to precipitate their targets (10.Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar). The ChIP data after each PCR were considered significant when the lowest signal observed for one antibody was minimally 8 times higher than the mock signal. Nonspecific chromatin-antibody interaction for each antibody (background level) can be estimated in basal condition with the distal promoter probes (-350) or the exon 3 probe (data not shown). List of Primer Pairs and TaqMan Probes−GenBank™ accession number and position of the 5′ sequence is indicated for each forward (F), reverse (R), and TaqMan (T) probe oligonucleotides. The reconstituted c-fos rat DNA (RCFRD) was designed based on several rat genomic sequences present in DNA databases (data not shown). Rat H1f0 (H1 histone family, member 0; GenBank™ accession number X72624) and GAPDH (GenBank™ accession number X00972) gene sequences were selected for the analysis their gene transcription. In Fig. 2A, the 5′ and 3′ GAPDH primer pairs and probe are located, respectively, in the exons 1 and 8 of GAPDH, 4kb apart. The primer and probes are as follows. For c-fos PD: RCFRD-926F, 5′-TGCGCTGCACCCTCAGA-3′; RCFRD-972R, 5′-TGTGTAAAGGAGGGAGGGATTG-3′; and RCFRD-970TR, 5′-FAM-AGCCGGCGAGCTGTTCCCG-TAMRA-3′. For c-fos PP: RCFRD-1239F, 5′-CTCATGACGTAGTAAGCCATTCAAG-3′; RCFRD-1295R, 5′-GCAATCGCGGTTGGAGTAGT-3′. For c-fos exon 1: RCFRD-1423F, 5′-CCCTCGCCGAGCTTTGC-3′; RCFRD-1487R, 5′-GCCTCGTAGTCCGCGTTGA-3′; RCFRD-1441T, 5′-FAM-CAAACCACGACCATGATGTTCTCGGGT-TAMRA-3′. For c-fos intron 1: RCFRD-1802F, 5′-CGCGGCAGGTTTACTCTGA-3′; RCFRD-1871R, 5′-AGCGAGTCTTTGCTAGAGACTTGTT-3′. For c-fos exon 2: RCFRD-2444F, 5′-CAGCCCACTCTGGTCTCCTC-3′; RCFRD-2531R, 5′-CGTAAGCCCCGGTCGAC-3′; RCFRD-2482T, 5′-FAM-CAGAGCGCCCCATCCTTACGGACT-TAMRA-3′. For c-fos exon 3; RCFRD-3046F, 5′-AAGGGAAAGGAATAAGATGGCTG-3′; RCFRD-3118R, 5′-CGCTTGGAGCGTATCTGTCA-3′; RCFRD-3091TR, 5′-FAM-CCTCCGATTCCGGCACTTGGCT-TAMRA-3′. For c-fos exon 4: RCFRD-3723F, 5′-TCCCAGCTGCACTACCTATACG-3′; RCFRD-3795R, 5′-TGCGCAGCTAGGGAAGGA-3′; RCFRD-3746T, 5′-FAM-CTTCCTTTGTCTTCACCTACCCCGAGGC-TAMRA-3′. For GAPDH 5′ region: X00972-1F, 5′-CTCTCTGCTCCTCCCTGTTCTA-3′; X00972-35R, 5′-CTGGCACTGCACAAGAAGA-3′. For GAPDH 3′ region: X00972-667F, 5′-GGGCAGCCCAGAACATCA-3′; X00972-723R, 5′-CCGTTCAGCTCTGGGATGAC-3′; X00972-687T, 5′-FAM-CCCTGCATCCACTGGTGCTGCC-TAMRA-3′. For histone H1 5′ region: X72624-87F, 5′-CGGACCACCCCAAGTATTCA-3′; X72624-151R, 5′-GCCGGCGCGGTTCT-3′; X72624-115T, 5′-FAM-CGTGGCTGCCATCCAGGCAGATAMRA-3′. For histone H1 3′ region: X72624-1308F, 5′-TAGGAGGACGTTGTTCGTTTCC-3′; X72624-1380R, 5′-GAACTGAAGTGGCACCAAGCA-3′, X72624-1332T: 5′-FAMTCCCCTCTTCCTGTGTAAGATGTGGCA-TAMRA-3′. Various Stimuli Induce c-fos Transcription to Distinct Levels−We undertook to study the expression of the c-fos oncogene, a highly inducible IEG for which transcription control at the level of initiation and a block to elongation situated in the first intron are well documented (17.Ryser S. Tortola S. van Haasteren G. Muda M. Li S. Schlegel W. J. Biol. Chem. 2001; 276: 33319-33327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20.Collart M.A. Tourkine N. Belin D. Vassalli P. Jeanteur P. Blanchard J.M. Mol. Cell Biol. 1991; 11: 2826-2831Crossref PubMed Scopus (82) Google Scholar, 21.Werlen G. Belin D. Conne B. Roche E. Lew D.P. Prentki M. J. Biol. Chem. 1993; 268: 16596-16601Abstract Full Text PDF PubMed Google Scholar, 22.Coulon V. Veyrune J.L. Tourkine N. Vie A. Hipskind R.A. Blanchard J.M. J. Biol. Chem. 1999; 274: 30439-30446Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 23.Yamada T. Yamaguchi Y. Inukai N. Okamoto S. Mura T. Handa H. Mol. Cell. 2006; 21: 227-237Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). c-fos expression is a ubiquitous sign of cellular activation, particularly striking in brain (24.Curran T. Morgan J.I. J. Neurobiol. 1995; 26: 403-412Crossref PubMed Scopus (303) Google Scholar). In the rat neuroendocrine cell line GH4C1, c-fos is induced to different levels following stimulation with diverse extracellular stimuli (Fig. 1A). TRH and epidermal growth factor both strongly induced c-fos gene transcription compared with the moderate stimulation obtained with tumor necrosis factor-α or by membrane depolarization with 20 mm KCl. The two latter stimuli however still increased significantly the level of c-fos mRNA, whereas two housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Histone H1, were as expected unaffected by any stimuli (Fig. 1A and data not shown). Dynamic Control of Elongation Determines the Rate of c-fos Transcription−Quantitative analysis of c-fos nascent transcripts showed a very rapid rise in transcription rates that reached maximal levels at 12 min of stimulation (Fig. 1B). Beyond this point, transcription was attenuated but remained significantly elevated over starting levels for up to 96 min after addition of TRH. Consequently, a peak for c-fos mature transcripts in the free nuclear RNA fraction was observed for ∼24 min; thereafter, an elevated steady-state level of c-fos mRNA was maintained (Fig. 1C). To understand how transcriptional rates are dynamically controlled we quantified at various time points the distribution of pol II on the c-fos gene and assessed the phosphorylation of its CTD tail. Combining ChIP with quantitative real-time PCR made this possible in intact cells. In particular we could specifically address whether continuous dynamic control is exerted upon the transcription elongation steps. Indeed previous work, including our own, had suggested that Ca2+ signals could relieve a block to elongation in the first intron of the c-fos gene (17.Ryser S. Tortola S. van Haasteren G. Muda M. Li S. Schlegel W. J. Biol. Chem. 2001; 276: 33319-33327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20.Collart M.A. Tourkine N. Belin D. Vassalli P. Jeanteur P. Blanchard J.M. Mol. Cell Biol. 1991; 11: 2826-2831Crossref PubMed Scopus (82) Google Scholar, 21.Werlen G. Belin D. Conne B. Roche E. Lew D.P. Prentki M. J. Biol. Chem. 1993; 268: 16596-16601Abstract Full Text PDF PubMed Google Scholar, 22.Coulon V. Veyrune J.L. Tourkine N. Vie A. Hipskind R.A. Blanchard J.M. J. Biol. Chem. 1999; 274: 30439-30446Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, none of those earlier studies have addressed the dynamic control of this block with different stimuli in vivo. The ChIP experiments shown in Figs. 2 and 3 used three different anti-pol II antibodies (8WG16, H14, and H5), which recognize different epitopes of the CTD. 8WG16, which binds non-phosphorylated CTD, was used to follow total pol II, assuming that among the 52 heptad repeats of the mammalian CTD there will always be some that are not phosphorylated. ChIP data obtained with 8WG16 and with a different anti-pol II antibody were indeed indistinguishable (data not shown). H14, recognizing phospho-Ser-5 CTD and H5, directed against phospho-Ser-2 CTD distinguished between phospho-Ser-5 CTD and doubly (Ser-2/Ser-5) phosphorylated CTD plus phospho-Ser-2 CTD. A comment on the specificities of these commonly used antibodies 8WG16, H14, and H5 may be found in the supplemental material. The pol II distribution on exons 1, 3, or 4 at different times during the activation and attenuation phases of TRH-induced c-fos gene transcription was quantified by real-time PCR with the corresponding TaqMan primers and probes (Fig. 2A). Prior to stimulation, total pol II and phospho-Ser-5 CTD forms of pol II were present on exon 1 indicating a certain level of constitutive transcription initiation (Fig. 2, B and C). However, pol II was not detected on exons 3 and 4, consistent with a block at the level of elongation in resting cells (17.Ryser S. Tortola S. van Haasteren G. Muda M. Li S. Schlegel W. J. Biol. Chem. 2001; 276: 33319-33327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20.Collart M.A. Tourkine N. Belin D. Vassalli P. Jeanteur P. Blanchard J.M. Mol. Cell Biol. 1991; 11: 2826-2831Crossref PubMed Scopus (82) Google Scholar, 21.Werlen G. Belin D. Conne B. Roche E. Lew D.P. Prentki M. J. Biol. Chem. 1993; 268: 16596-16601Abstract Full Text PDF PubMed Google Scholar, 22.Coulon V. Veyrune J.L. Tourkine N. Vie A. Hipskind R.A. Blanchard J.M. J. Biol. Chem. 1999; 274: 30439-30446Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 23.Yamada T. Yamaguchi Y. Inukai N. Okamoto S. Mura T. Handa H. Mol. Cell. 2006; 21: 227-237Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Stimulation with TRH led to a rapid increase of total pol II distribution all along the gene until 9-12 min of stimulation (Fig. 2B). Strong transcription activation occurring during this period is correlated with a high level of Ser-5 and Ser-2 phosphorylation of the CTD of pol II (Fig. 2, C and D). Both initiation and elongation are stimulated during this phase of c-fos gene transcription. After 12 min, transcription was attenuated, and pol II density was decreased on exons 3 and 4 with a reduction in the number of pol II phosphorylated on the two serine residues. Interestingly at 48 min, the level of total pol II (8WG16) and Ser-5-phosphorylated pol II (H14) in the exon 1 was still high compared with 9-12 min of stimulation (Fig. 2, B and C). This result suggests that the recruitment and initiation of pol II at the promoter continues over a long period even though the amount of elongating pol II has decreased. Hence, following stimulation by TRH, temporal control of transcription initiation may be distinct from the control of transcription elongation. Elongation can be best illustrated by the ratio between pol II ChIP signals from exon 3 versus exon 1 (Fig. 2E). For total pol II (8WG16), this ratio increased during the activation phase demonstrating that elongation becomes more efficient to drive pol II than new recruitment. Following the peak of transcriptional rates, the ratio decreased progressively to a new steady state, which was maintained. The ratio curve was strikingly parallel to the kinetics of transcriptional rates (Fig. 2E versus Fig. 1C). This illustrates that the kinetics of transcriptional rates result from the dynamic control of elongation. In summary, c-fos like many other immediate early genes displays biphasic kinetics of gene expression after extracellular stimulation. Induction of c-fos by TRH is characterized by a short and transient phase of very active gene transcription (9-12 min) followed by a longer attenuation phase. CTD phosphorylation of initiated and of elongating pol II changes during the different phases of gene transcription. At the peak of active gene transcription CTD is highly phosphorylated both on Ser-2 and Ser-5; thereafter, for pol II on exon 1, phospho-Ser-2 and doubly phosphorylated pol II (H5) decreased, whereas phospho-Ser-5 (H14) was stable (Fig. 2, C and D). Like for CTD kinases (9.Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar, 10.Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar) this suggests that the control of elongation could be also regulated by specific CTD phosphatases in a position- and time-dependent manner. Such phosphatases able to distinguish between the Ser-2 and Ser-5 positions have already been described (25.Meinhart A. Kamenski T. Hoeppner S. Baumli S. Cramer P. Genes Dev. 2005; 19: 1401-1415Crossref PubMed Scopus (261) Google Scholar). RNA Polymerase Transcribing Constitutively Expressed Genes Is Unaffected during Stimulation of c-fos Transcription−Changes in CTD phosphorylation upon transcription activation were observed so far for inducible genes known to harbor a promoter proximally paused pol II (10.Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar, 11.Cheng C. Sharp P.A. Mol. Cell. Biol. 2003; 23: 1961-1967Crossref PubMed Scopus (69) Google Scholar). We wondered whether pol II on unregulated genes would show an altered kinetic pattern of CTD phosphorylation after stimulation by TRH. We choose two housekeeping genes known to be regulated differentially at the level of elongation: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which presents an elongation block in the 5′-end, and Histone H1 which does not (10.Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar, 26.Uptain S.M. Kane C.M. Chamberlin M.J. Annu. Rev. Biochem. 1997; 66: 117-172Crossref PubMed Scopus (400) Google Scholar). Nascent transcript RT-PCR and ChIP assays were performed as in Figs. 1 and 2 with TaqMan probes corresponding to the 5′ and 3′ transcribed sequences of both housekeeping genes (Fig. 3A). No further induction of gene transcription was observed for GAPDH and H1 after stimulation by TRH; nascent or free nuclear transcripts of both genes were constant (Fig. 3B). TRH modified neither pol II density nor CTD phosphorylation on either gene (Fig. 3, C-E). However, a clear difference in spatial pol II distribution was observed between GAPDH and H1: for GAPDH, pol II, both total (8WG16) and phospho-Ser-5 CTD (H14), was more abundant in the 5′ region, consistent with paused