Báo cáo khoa học: Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation ˆ Benoıt Palancade and Olivier Bensaude pptx

Eur J Biochem 270, 3859–3870 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03794.x REVIEW ARTICLE Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation ˆ Benoıt Palancade and Olivier Bensaude Ge´ne´tique Mole´culaire, UMR 8541 CNRS, Ecole Normale Supe´rieure, Paris, France Phosphorylation of RNA polymerase II’s largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism Numerous enzymes, including cell cycledependent kinases and TFIIF-dependent phosphatases target the CTD However, the repetitive nature of the CTD prevents determination of phosphorylated sites by conventional biochemistry methods Fortunately, a panel of monoclonal antibodies is available that distinguishes between phosphorylated isoforms of RNA polymerase II’s (RNAP II) largest subunit Here, we review how successful these tools have been in monitoring RNAP II phosphorylation changes in vivo by immunofluorescence, chromatin immunoprecipitation and immunoblotting experiments The CTD phosphorylation pattern is precisely modified as RNAP II progresses along the genes and is involved in sequential recruitment of RNA processing factors One of the most popular anti-phosphoCTD Igs, H5, has been proposed in several studies as a landmark of RNAP II molecules engaged in transcription Finally, we discuss how global RNAP II phosphorylation changes are affected by the physiological context such as cell stress and embryonic development The C-terminal domain (CTD) of RNA polymerase II’s (RNAP II) largest subunit is essential for transcription [1–3], for its function as enhancer [4,5], for organization of transcription foci within the nucleus [6] and for pre-mRNA processing [7,8] The CTD consists of multiple repeats of a seven amino acid motif [9,10] (Fig 1) This motif has been conserved during evolution in eukaryotes, but the number of repeats varies depending on the species: 26 in yeast, 45 in flies and 52 in mammals Five out of seven amino acids in the consensus motif are phosphate acceptors and indeed phosphorylation is a major post-translational modification of the CTD in vivo [11] Serine O-glycosylation has also been reported as a minor modification with unknown functional significance [12] In contrast, CTD phosphorylation plays a major role in the transcriptional process [13–16] A large variety of kinases have been reported to phosphorylate the CTD of RNAP II in vitro [17,18] Of particular significance, CDK7, CDK8 and CDK9 are subunits of the TFIIH general transcription factor, of the mediator complex and of the positive transcription elongation factor (P-TEFb), respectively Interactions with the unphosphorylated CTD are involved in assembly of RNAP II with the mediator complex to form a holoenzyme or with general transcription factors to form a preinititiation complex of transcription Phosphorylation of the CTD is required to disrupt these interactions at elongation of transcription and to assist the recruitment of pre-mRNA modification enzymes [16,19] Initial studies generally considered CTD phosphorylation as an all or nothing process Here, we review recent evidence that multiple forms of CTD-phosphorylated RNAP II are present in cells These forms have been characterized using monoclonal antibodies that distinguish between phosphorylated amino acid residues within the CTD The phosphorylation site specificity of kinases and phosphatases that target the CTD is discussed next Variations in RNAP II phosphorylation are examined along the transcriptional process and as a function of physiological context including cell stress and early development ´ ´ ´ Correspondence to O Bensaude, Genetique Moleculaire, ´ UMR 8541 CNRS, Ecole Normale Superieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France Fax: + 33 44323941, Tel.: + 33 44323410, E-mail: bensaude@biologie.ens.fr Abbreviations: CDK, cyclin-dependent kinase; CTD, C-terminal domain (of Rpb1); DSIF, DRB sensitivity inducing factor; HIV-LTR, long-terminal repeats of the human immunodeficiency virus; FCP1, TFIIF-dependent CTD phosphatase; NELF, negative elongation factor; MAPK, mitogen-activated protein kinase; Rpb1, largest subunit of RNAP II; RNAP II, RNA polymerase II; SCP, small CTD phosphatase; TFIIx, x type general class II gene transcription factor (Received 12 June 2003, revised 13 August 2003, accepted 15 August 2003) Keywords: RNA polymerase II; CTD-phosphorylation; CTD-kinase; CTD-phosphatase; transcription; mRNA processing Multiple RNAP II phosphoisoforms Two distinct fractions of RNAP II were purified from mammalian cells in early studies and designated RNAP IIA and RNAP IIO [20] They differ in the phosphorylation status of their largest subunit (Rpb1), respectively, designated as IIa and IIo forms [13] The CTD is hypophosphorylated in the IIa form and multiphosphorylated in the IIo form It is noteworthy that some preparations of RNAP II contain a third fraction, RNAP IIB, which results from an Ó FEBS 2003 3860 B Palancade and O Bensaude (Eur J Biochem 270) Fig Sequence of the human CTD Selfalignment of human RNAP II largest subunit sequence from amino acids 1593–1970 (last residue before the stop codon) (GeneBank accession number NM_000937) Note that serine at position in the heptad motif is not conserved in the C-terminal section of the CTD Serines and 5, the major phosphorylated residues are in red Non-conserved amino acid residues are in blue in vitro proteolysis of the CTD thus generating the IIb form of Rpb1 The CTD is phosphorylated mainly on serine residues, although phosphothreonine and phosphotyrosine have been detected to a minor extent [21,22] CTD phosphorylation provokes a remarkable shift in electrophoretic migration equivalent to an apparent molecular weight increase from 210 to 240 kDa in vertebrates [23] The number of phosphates in the mammalian IIo form has been evaluated as close to 50 residues, corresponding to one phosphate per repeat on average [24] Phosphorylated forms that migrate between the IIa and IIo forms have also been described An IIi (intermediary) form develops during Herpes simplex virus infection [25,26] An IIm (MAP kinase-dependent) form is generated during serum-stimulation of quiescent cells or during osmotic and oxidative stress [27,28] An IIe (embryonic) form is found in nontranscribing early embryos from vertebrates [28,29] and insects [30] Hence SDS/PAGE analysis unravels the diversity in phosphorylated forms of RNAP II Phosphorylation-dependent epitopes within the CTD Due to the elevated number of phosphate acceptors and to the repetition of the consensus motif, it is a formidable task to determine precisely which serine residues are phosphorylated in a particular repeat Recently, a set of monoclonal antibodies became a powerful tool to investigate phosphorylated residues within the CTD (Table 1) These antibodies have been characterized by Western blot using yeast CTD fusion proteins in which serines at positions or in all 26 heptads had been replaced either by alanine or by glutamate [31] Some of these antibodies react preferentially with the unphosphorylated CTD whereas recognition by others requires phosphorylated serines at specific positions It is worth mentioning that monoclonal antibodies H5 and H14 are often referred to as specific for phosphoserines and 5, respectively Table Phosphorylation dependence of anti-CTD monoclonal antibodies Characterization of CTD phosphoepitopes: (1) using a set of yeast CTD fusion proteins mutated or not [31]; (2) using a set of synthetic peptides phosphorylated or not ([34]; A Albert, S Lavoie and M Vincent, personal communication); (3) using purified RNAP II phosphorylated or not by MAP kinase [27] Name Epitope Method Reference 8WG16 V15 V6 MARA3 B3 H14 H5 CC3 MPM2 Unphosphorylated Ser2 Unphosphorylated Unphosphorylated Ser2 Ser2-P or Ser5-P Ser2-P or Ser5-P Ser5-P Ser2-P Ser2-P Ser2-P and Ser5-P (1) (3) (3) (1) (1) (1) (1) (1) (2) [123] [27] [27] [31] [124] [110] [110] [125] [37] (2) (2) (2) (2) Limitations in epitope data interpretation The specificity of monoclonal antibodies should be handled cautiously For instance, depending on investigators, the 8WG16 antibody binds to the unphosphorylated CTD exclusively, or to both phosphorylated and unphosphorylated CTD indistinctly [31–33] In addition, although ELISA assays confirmed that H5 and H14 antibodies distinguished peptides phosphorylated on serines or at low antigen concentration, this specificity was lost at high antigen concentrations [34] Moreover, a weaker binding with peptides phosphorylated simultaneously on serines and suggests that phosphorylation of adjacent sites might impair recognition Furthermore, little is known about the consequences of amino acid substitution occurring at nonphosphorylated positions on degenerated repeats as found in the C-terminal half of the mammalian CTD Finally, some of these antibodies such as H5, CC3 and MPM2 have been shown to cross-react with proteins other than the RNAP II largest subunit [35–37] Ó FEBS 2003 RNA polymerase II CTD phosphorylation (Eur J Biochem 270) 3861 Despite these restrictions in establishing a correspondence between serine or serine phosphorylation and H5 or H14 recognition, these antibodies provide useful tools with which to characterize the phosphorylation of the CTD As will be seen below, H5 and H14 reactivities unravel variations in the phosphorylation sites and may reflect differential action of CTD-kinases and CTD-phosphatases Moreover, H5 reactivity might be used as a marker for transcribing RNAP II molecules Amino acid residues targeted by CTD-kinases in vitro CTD-kinases belong to three subclasses: cyclin-dependent kinases, mitogen-activated protein kinases and others (see Table and [15,17,18]) Two methods have been used to determine phosphorylation site preference for a given CTDkinase The first method uses synthetic peptides consisting in one or more CTD heptads with the phosphorylable serine, threonine or tyrosine replaced by alanine [38,39] Serine phosphorylation is preferred for CDK7 [39–41], CDK8 [42,43], CDK9 [38] and MAPK [39] Kinases such as CDK1 and CDK2 phosphorylate both serine and serine [41,44] Efficiencies of the different kinases are influenced by the position of the targeted repeats within the CTD [38,45] and by the neighboring residue’s identity [38,39,43] Slightly different conclusions were reached when determining phosphoepitopes that were generated on purified RNAP II For example, CDK7 (TFIIH) generates H14 epitope (serine 5) as predicted [46,47] but also the H5 one [48] and the CC3 one [45], which are specific for phosphoserine [31] Furthermore, CDK8 generates both H14 and H5 reactivity upon long phosphorylation times [46] In discrepancy with peptide-driven conclusions, CDK9 generates H5 reactivity [47,49,50] One might consider that genuine RNAP II CTD is a complex array of degenerated repeats differing from the peptides used in biochemical studies Transcription factors influence phosphorylation site preference CTD kinases phosphorylate RNAP II within large supramolecular complexes that may influence the phosphorylation reaction course Recruitment of CDK7 in a DNA-bound transcription preinitiation complex increases its efficiency towards RNAP II CTD [51] and TFIIE enforces its preference for serine [48] Transcriptional regulators contribute to recruit CTD kinases Activation of HIV-LTR transcription by the viral transactivator Tat provides a paradigm of such a phenomenon [52,53] In the presence of Tat, CDK9 phosphorylates serine (H14) in addition to serine (H5) [50] Several cellular activators also recruit CDK9 to enhance genespecific transcription [54–57] In contrast, the glucocorticoid receptor bound to its hormone represses nuclear factorkappa B activation and this effect correlates with a decreased phosphorylation of serine (H5) on target genes [58] Amino acid residues targeted by CTD-phosphatases In contrast to the large number of characterized CTDkinases, few CTD-phosphatases have been identified (Table and references therein); most studies concern the TFIIF-dependent CTD phosphatase (FCP1) [59] Recently, three small FCP1 related proteins, SCP1/NLI-IF, SCP2/ OS4 and SCP3/HYA22, have been identified as also being CTD phosphatases [60] In addition, the contribution of type protein phosphatase (PP1) has been discussed in two reports [61,62] and the SSU72 transcription/processing factor has been hypothesized as a phosphotyrosine CTD phosphatase [63,64] In contrast to CTD-kinases that accept a wide range of substrates, the substrate specificity of FCP1 phosphatase is strictly restricted to the CTD within RNA polymerase II core enzyme [59] The site preference of CTD phosphatases was determined using various RNAP IIO isozymes: FCP1 dephosphorylates RNAP IIO purified from mammalian cells [65] or RNAP IIO generated in vitro by phosphorylation of RNAP IIA with purified kinases such as CDK7, CDK9, CDK1 or MAPK [66] Consistently, FCP1 phosphatase removes both H5 and H14 phosphoepitopes from mammalian RNAP IIO [66] Therefore, in higher eukaryotes, phosphates on serine (H5) and serine (H14) appear equally removed by FCP1 phosphatase In contrast, it has been proposed that fission yeast FCP1 specifically target serine [34] However, the Schizosaccharomyces pombe FCP1 assays were performed using peptides and highly acidic nonphysiological conditions Saccharomyces cerevisiae experiments involving FCP1 inactivation followed by chromatin immunoprecipitation (see below) suggested the occurrence of a distinct serine 5-specific phosphatase [34] Interestingly, in contrast to FCP1, the small FCP1-related CTD phosphatase, SCP1, might preferentially dephosphorylate phosphoserine as CDK7-phosphorylated RNAP IIO is its preferred substrate [60] Changes in RNAP II phosphorylation along gene transcription Initial studies established that RNAP II is hypophosphorylated when recruited to a promoter and then hyperphosphorylated during messenger RNA synthesis [67–70] Serine phosphorylation by the mediator complex subunit Srb10 (CDK8 ortholog) precludes this recruitment [42] An elegant study using step by step in vitro transcription in mammalian nuclear extracts has indicated that serine (H14) is phosphorylated first in the initiation complex (likely by CDK7) and that serine (H5) is phosphorylated by CDK9 upon entry into elongation [49] When engaged in transcription elongation complexes, RNAP II becomes a poor substrate for FCP1, suggesting that conformational changes or protecting factors affect the efficiency of the phosphatase [71,72] In support of the latter hypothesis, proteins that bind the phosphorylated CTD inhibit FCP1 activity (B Palancade, N F Marshall, O Bensaude, M Dahmus & M.-F Dubois, unpublished observation) Cellular DNA can be cross-linked to proteins in its vicinity and then immunoprecipitated with antibodies directed against these proteins The presence of specific DNA sequences in the immunoprecipitated material is probed by polymerase chain reaction (PCR) amplification Such chromatin immunoprecipitation (CHIP) assays have helped to map the evolution of CTD phosphorylation along a transcribed gene Antibodies directed to the hypo- and the Cyclin Cyclin H Ccl1 Cyclin C Srb11 CDK7 (MO15) Kin28 CDK8 Srb10 MAT1 Tfb3? Other partners Ctk2 Bur2 c-abl Others DNA-PK Mitogen-activated protein kinases ERK 1–2 Mpk1 Ctk1 Bur1 (Sgv1) Ku70 Ku86 Ctk3 Ser ND ND Tyr DNA-PK Ser Ser CTDK-I Ser 5* Mediator Ser 5 5 ND ND H14 H5 H14 H5 (H14 ?) H14 (H5 ?) H14 (H5 ?) H5 H14 ND Function in transcription Repression of initiation [21] [151] [39] [39,149] [27] ? ? ? [38,49,50, Entry into processive elongation 139,140,141] Tat-dependent HIV-1 transcription [145–147] Elongation? [148] Elongation? [42,43,46, 134–136] Repression of transcription during mitosis ? [44] Tat-dependent HIV-1 transcription ? [39–41,46–48, Initiation, recruitment of 130] capping enzymes [41,127] Peptides Phosphoepitopes Reference Site preference Ser Ser Ser Ser TFIIH or Ser CAK MPF Complex CDK9 (PITALRE) Cyclin T1, T2, K 7SK snRNA P-TEFb MAQ1 (HEXIM1) Cyclin E CDK2 Cyclin-dependent kinases CDK1 (cdc2) Cyclin A, B Kinase Associated factor DNA-PK, PI3-kinase, several nuclear proteins [153] p53, chromatin components [152] c-fos, c-jun, transcription factors [150] ND ND Spt5 [36,142] Rb [143], MyoD [144] CDK1, CDK2 [131] p53 [132], RARa [156], TBP, TFIIE, TFIIF [133] Cyclin H [107] Gcn4, Msn2 [137], Gal4 [138] Structural proteins, cell cycle regulators [128] Cell cycle regulators [129] Other substrates Table CTD-kinases and their features Usual mammalian protein names are indicated in bold characters, other nomenclatures are in parenthesis and S cerevisiae orthologues are in italic Ctk1 and Bur1 have both been proposed as budding yeast CDK9 counterparts Site preferences have been deduced from in vitro phosphorylation of synthetic peptides, or from phosphoepitopes generated on the CTD ´ ` ´ ` MPF, maturation-promoting factor; CAK, CDK-activating kinase; RARa, retinoic acid receptor; MAT1, Ômenage-a-troisÕ; MAQ1, Ômenage-a-quatreÕ [126] *, Although CDK9 preferentially phosphorylates serine on peptides, it generates phosphoserine (H5 epitope) in vitro and in vivo ND, not determined; ?, unknown 3862 B Palancade and O Bensaude (Eur J Biochem 270) Ó FEBS 2003 Ó FEBS 2003 RNA polymerase II CTD phosphorylation (Eur J Biochem 270) 3863 Table The CTD-phosphatases and their features The site preference has been determined by CTD-phosphatase assays in vitro Previous nomenclature of SCPs is given in parenthesis NLI-IF, nuclear LIM interactor–interacting factor; OS4, osteosarcoma gene 4; HYA22, human orthologue of S pombe YA22 locus; ND, not determined; ?, unknown Phosphatase Regulatory protein TFIIF-dependent phosphatases FCP1 RAP74 (TFIIF) Site preference References Function in transcription Other substrates Ser (H5) [59,66] Recycling of RNAP II after termination Regulation of elongation RNAP II mobilization CTD dephosphorylation after initiation? ? Developmentally regulated transcription? ? ? TFIIB Ser (H14) SCP1 (NLI-IF) RAP74 (TFIIF) Ser [60] SCP2 (OS4) RAP74 (TFIIF) ND [60,154] SCP3 (HYA22) ? ND [60] Phosphoserine/phosphothreonine protein phospshatases (PPP) PP1 At least seven different Ser (H5) [62] regulatory subunits Ser (H14) [155] hyperphosphorylated CTD of Drosophila RNAP II demonstrated that RNAP II molecules pause at the 5¢ ends of heat shock genes in an unphosphorylated state [73] In mammalian cells, phosphoserine (H14) is concentrated near the promoter while serine phosphorylation (H5) is observed throughout the gene [74] In yeast, serine is phosphorylated at initiation in a CDK7/Kin28-dependent manner [75,76] The Ctk1-dependent serine (H5) phosphorylation develops next at the beginning of elongation and decrease in immunoprecipitation suggested that serine was dephosphorylated at this stage [34,76] Alternatively, the latter finding might be interpreted as a loss of phosphoserine recognition when both serines and are phosphorylated Taken together, the chromatin immunoprecipitation experiments demonstrate that distinct residues are phosphorylated on the CTD during the progression of RNAP II along the genes in vivo (Fig 2) Interestingly, an alanine scanning analysis of the CTD in yeast RNAP II had suggested that phosphorylation on both serine and serine position in the repeats was required for viability but a search for suppressor mutations indicated that these positions differed functionally [77,78] Changes in CTD phosphorylation facilitate pre-mRNA processing The CTD mediates the coupling between transcription and pre-mRNA processing The phosphorylated CTD recruits the pre-mRNA processing machinery [79] Serine phosphorylation of the CTD by CDK7/Kin28 is sufficient to recruit and stimulate capping enzymes upon initiation [75,76,80] The capping enzyme may in turn, assist the recruitment of CDK9 as in S pombe, the CDK9 ortholog binds Pct1, a subunit of the capping apparatus [81] Consistently, inactivation of capping enzyme in Caenorhabditis elegans results in a loss of serine (H5) phosphorylation [82] Capping might be a prerequisite to elongate transcription and contribute to a transcriptional checkpoint: Tat-dependent HIV-1 transcription? ? ? ? Transcription factors, splicing factors, cell cycle regulators [155] only capped mRNAs would be transcribed efficiently The phosphorylated CTD next recruits SR proteins involved in splicing [83–85] followed by the cleavage-polyadenylation factors [86] The serine 2-kinase Ctk1 is required for 3¢-end processing in S cerevisiae [87] Consistently, the Pcf11p cleavage-polyadenylation factor preferentially associates to the CTD phosphorylated on serine rather than serine [88] Hence, transcription-dependent changes in CTD phosphorylation sites might be required to coordinate sequential recruitment of the processing machinery In addition to phosphorylation, degeneracy of the CTD adds a further level of complexity (Fig 1) Indeed, the N-terminal half of the CTD only stimulates capping whereas the C-terminal half supports capping as well as splicing and 3¢-end processing [89,90] Different repeats in the CTD may serve distinct functions Changes in CTD phosphorylation determine the recruitment of chromatin modification enzymes Recently, CTD phosphorylation has been shown to be involved in histone methylation control in budding yeast Indeed, phosphorylation of RNAP II mediates the recruitment of histone H3 methyltransferases from the SET family [79,91] Serine 5-phosphorylated RNAP II contributes to the association of the Set1/COMPASS methyltransferase complex onto the 5¢-portion of actively transcribed genes shortly after initiation, thereby enhancing histone H3 K4 trimethylation and providing a molecular mark of recent transcriptional activity [92,93] Subsequently, the Set2 histone H3 K39 methyltransferase is recruited to the elongation complex through direct binding to the CTD phosphorylated by Ctk1 on serines at position [94,95] Thus, distinct phosphorylated RNAP II isoforms contribute to chromatin imprinting Specific CTD phosphorylation patterns therefore determine and coordinate the interactions with protein partners such as transcription factors, histone-modifying complexes or mRNA-processing enzymes 3864 B Palancade and O Bensaude (Eur J Biochem 270) Ó FEBS 2003 Fig CTD phosphorylation and the transcription cycle (A) Recycling The ÔfreeÕ RNAP II core enzyme is not phosphorylated on the CTD It may assemble with coactivators such as the mediator complex (Med) thus forming a holoenzyme Premature CTD phosphorylation by CDK8 prevents the assembly of RNAP II on the promotor (B) Preinitiation The unphosphorylated RNAP II core or holoenzyme assemble onto the promoter sequences with general transcription factors such as TFIID, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH thus forming a preinititiation complex of transcription The CTD is phosphorylated on serines at position (5P) by the CDK7 subunit of TFIIH (C) Initiation Transcription begins The phosphorylated CTD recruits the capping enzymes and the nascent transcript is capped at its 5¢ end (D) Elongation Phosphorylation of the CTD on serines at position (2P) by the CDK9 subunit of the positive transcription elongation factor (P-TEFb) is required to remove to block opposed by the DSIF/NELF factors and elongate transcription (E) RNA processing and transcription termination The phosphorylated CTD recruits the splicing machinery to remove introns and, finally recruits the cleavage and polyadenylation factors that cleave the transcript and add a polyadenylic tail at its 3¢ end This step signals transcription to terminate and RNAP II falls off its DNA template The resulting mRNA is then exported to the cytoplasm (F) To be recycled for another transcription round, RNAP II is dephosphorylated by the FCP1 CTD phosphatase Global changes in RNAP II phosphorylation The IIa and IIo RNAP II phosphoisoforms coexist in a dynamic equilibrium in yeast [33], insect [96], amphibian [28] and mammalian cells [97] Importantly, this dynamic exchange does not require transcription as it is also observed in a nontranscribing cell system such as the amphibian egg [98] Addition of purified phosphorylated RNAP IIO to a crude egg extract demonstrated a half turnover of CTD phosphates of 2–4 In this system, the antagonistic action of FCP1 and of ERK type MAP kinase accounts for the distribution between phosphorylated and unphosphorylated RNAP II forms [98,99] In transcribing cells, the CTD phosphorylation dynamic is unraveled upon CTD kinase/phosphatase inactivation In S cerevisiae, inactivation of either Kin28 (the CDK7 orthologue) or Ccl1, its cyclin partner, results in a rapid decrease in the amount of CTD-phosphorylated RNAP II [33,100] Conversely, Fcp1 inactivation leads to an increase in the amount of phosphorylated RNAP II [32] Selective gene inactivation in mammalian cells is less straightforward but, fortunately, numerous chemical inhibitors are available instead Kinase inhibitors have generally limited specificity but this problem might be circumvented by using a panel of compounds belonging to distinct chemical families and by comparing efficiencies in vitro and in vivo This procedure has been used to suggest that CDK9 is the major CTD kinase generating the bulk IIo form in interphasic mammalian cells [36,97,101] RNAP II hyperphosphorylation increases following treatments such as exposure to a-amanitin [102] or actinomycin D [101], or upon UV irradiation [103] As this increase is suppressed by CDK9 inhibitors [101], it is suggested to result from an enhanced activity of CDK9 provoked by the transcriptional arrest [104,105] Similarly, Ctk1 is required for serine phosphorylation during the diauxic shift or following DNA damage in S cerevisiae [106] Ó FEBS 2003 RNA polymerase II CTD phosphorylation (Eur J Biochem 270) 3865 However, the effect of kinase inhibition on CTD phosphorylation may be indirect For instance, CDK8 phosphorylates cyclin H thereby inactivating CDK7 [107] In turn, TFIIH inhibits the autophosphorylation of CDK9, which enhances its kinase activity [47] Therefore, a body of arguments is required to strengthen the interpretation For instance, the identification of ERK1/2 MAP kinase as the major CTD kinase generating the IIm forms relies on several arguments [27,98,99]: (a) the appearance of the IIm form correlates with MAP kinase activation; (b) it is insensitive to CDK9 inhibitors but suppressed in the presence of inhibitors of MAP kinase activation; (c) the immunogenicity of the IIm form matches that of RNAP II phosphorylated in vitro by MAP kinase Serine phosphorylation (H5) as a landmark of transcribing RNAP II Immunofluorescence analysis of polytene chromosomes revealed that hyperphosphorylated RNAP II is associated with actively transcribed sequences such as Drosophila ecdysone-induced puffs [108] Interestingly, this study also suggested that transcribing RNAP II is less phosphorylated under heat-shock conditions Although the H14 monoclonal antibody stains diffusely nuclei of interphasic mammalian cells, H5 reveals punctuated nuclear structures that match transcription foci [109] During mitosis, at metaphase, both H14 and H5 epitopes are excluded from chromatin [110] However, H14 staining is diffuse whereas H5 decorates mitotic granules (MIGs) which are speckle-like structures containing pre-mRNA processing material [111] At telophase, H14 immunostaining develops in daughter nuclei simultaneously with recruitment of the general transcription machinery Nuclear H5 staining develops at early G1 phase, in coincidence with the onset of transcription In many metazoans, early embryonic development following fertilization is characterized by global repression of zygotic transcription Gene expression is initiated abruptly at the so-called mid-blastula transition (MBT) in amphibians, or zygotic genome activation (ZGA) in mammals This period is marked by important CTD phosphorylation changes that have been documented in nematodes, flies [30,112], fishes [113], amphibians [28] and mammals [29] Before ZGA or MBT, the RNAP II CTD does not react with H5, which is consistent with the absence of transcription at this period H5 reactivity has therefore been proposed as a landmark of zygotic gene activation in early embryos [28] In nematode embryos, a mixed diffuse/punctuated H14 staining develops in nuclei of somatic cells throughout embryogenesis whereas transcription-inactive germline cells show only two discrete foci [112] In contrast, H5 staining appears in conjunction with ZGA at the four-cell stage and remains restricted to the transcriptionally active somatic cells, except for mutants that disrupt the asymmetric divisions of germline cells [114] In contrast to H14, the H5 phosphoepitope is absent in embryos when transcription is globally impaired following RNA interference inactivation of essential components of the transcription machinery such as CDK9 [115] or TBP-like factor (TLF) [116,117] Both H14 and H5 staining are decreased upon CDK7 [118], RGR-1 mediator subunit [119], TFIIB or TAFIIs [120] inactivation Similar data are available for vertebrate embryos H5-reactive RNAP II is absent from zebrafish embryos inactivated for TBP [121], and both H5 and H14 staining are attenuated in mouse embryos following inactivation of MAT1, the CDK7 partner [122] These findings are consistent with an H14 staining attributed in part to initiating polymerases (productive or abortive) and an H5 staining of elongating polymerases Conclusion Determining the sites phosphorylated in vivo within RNAP II CTD is a formidable task due to the repetitive nature of this domain However, phosphospecific antibodies such as H5 and H14 have been successfully used in a wide range of experiments (Table 1) Using these tools, qualitative changes in the CTD phosphorylation pattern have been found to occur during the course of transcription or in response to physiological stimuli These changes might contribute to synchronize the successive steps of nuclear mRNA metabolism (Fig 2) Many data point out that recognition of the CTD by the H5 antibody might be specific for RNAP II molecules engaged in mRNA synthesis It is expected that an expanded use of other anti-CTD antibodies will contribute to improve our knowledge of CTD phosphorylation Deciphering this ÔCTD codeÕ may help our global understanding of gene expression mechanisms [58] Acknowledgements We are much indebted to Keith Blackwell and Marie-Francoise Dubois ¸ for critical reading of the manuscript We thank Michael E Dahmus, ă Michel Vincent and members of their lab for sharing unpublished information This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC 6250), the Ligue Nationale ´ Contre le Cancer (Comite de Paris) and the Agence Nationale de Recherche sur le SIDA References Corden, J.L (1993) RNA polymerase II transcription cycles Curr Opin Genet Dev 3, 213–218 Meininghaus, M., Chapman, R.D & Eick, D (2000) Conditional expression of RNA polylmerase II in mammalian cells Deletion of the carboxy-terminal domain of the large subunit affects early steps in 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Yeast carboxyl-terminal domain kinase I positively and Ó FEBS 2003 3870 B Palancade and O Bensaude (Eur J Biochem 270) 148 149 150 151 negatively regulates RNA polymerase II carboxyl-terminal domain. .. recycled for another transcription round, RNAP II is dephosphorylated by the FCP1 CTD phosphatase Global changes in RNAP II phosphorylation The IIa and IIo RNAP II phosphoisoforms coexist in a dynamic... (DRB) and isoquinoline sulfonamide derivatives (H-8 and H-7*), promote the dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II largest subunit J Biol Chem 269, 13331–13336 Palancade,