1 Génétique Moléculaire, UMR 8541 CNRS, Ecole Normale Supérieure, 46 rue dUlm, 75230 Paris Cedex 05, France
2 Dynamique de la Chromatine, UMR 144 CNRS, Institut Curie, 26 rue dUlm, 75231 Paris Cedex 05, France
* Present address: Service de Biochimie, Ecole Nationale Vétérinaire, 7 Av. du Général De Gaulle, 94704 Maisons-Alfort Cedex, France
Author for correspondence (e-mail: bensaude{at}biologie.ens.fr)
Accepted April 5, 2001
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SUMMARY |
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Key words: RNA polymerase, phosphorylation, mid-blastula transition, zygotic gene activation.
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INTRODUCTION |
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The determinism of zygotic transcriptional repression has been investigated during early development in different species including Xenopus. Three nonexclusive hypotheses have been formulated to account for the global repression before zygotic gene activation or MBT: (1) a high rate of mitosis and lack of G1/G2 phases are characteristics of the earlier embryonic cell cycles and might be incompatible with transcription (Edgar and Schubiger, 1986; Kimelman et al., 1987; Yasuda and Schubiger, 1992); (2) a large excess in maternal factors, such as histones stored during oogenesis, might repress the assembly of transcription complexes on promoters (Newport and Kirshner, 1982b; Prioleau et al., 1994; Prioleau et al., 1995); (3) transcriptional factors might be deficient (Almouzni and Wolffe, 1995) or absent (Bell and Scheer, 1999; Veenstra et al., 1999). According to these models, the transcriptional activation would be attributed, respectively, to lengthening of the cell cycle, progressive titration of histones by replication of the zygotic DNA, or maturation or neosynthesis of transcriptional factors.
In mammalian embryos, phosphorylation of RNA polymerase II (RNAPII) largest subunit (RPB1) has been shown to be abnormal prior to the burst of transcriptional activity (Bellier et al., 1997a). Such default might contribute to, or result from, the transcriptional repression because transcription involves a cycle of phosphorylation/dephosphorylation of the C-terminal domain (CTD) of the RPB1 subunit (reviewed by Dahmus, 1996). The CTD is composed of repetitions (up to 52 in mammals) of a consensus heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) with five potential acceptors of phosphate groups. In all species studied so far, RPB1 can be resolved into two extreme forms: a hyperphosphorylated form called IIo and a hypophosphorylated form called IIa. The equilibrium between the IIo and the IIa forms is dynamic and relies on a balance between the activities of CTD kinases and CTD phosphatases (Bensaude et al., 1999; Dahmus, 1996).
In Xenopus laevis, the phosphorylation of the CTD increases during meiotic maturation and relies on the activation of the Xp42 kinase, which is a CTD kinase (Bellier et al., 1997b). In this study, we show that the CTD is abruptly dephosphorylated after egg fertilisation. The period of transcriptional silence during Xenopus early development is characterised by low amounts in phosphorylated forms of RPB1. The major remaining phosphorylated form, designated IIe (embryonic), is not phosphorylated on serine-2 of the heptapeptide. The appearance of a fully hyperphosphorylated IIo form coincides with MBT. Thus, CTD phosphorylation is proposed as a new landmark of MBT in Xenopus embryos. We will discuss how changes in RNAPII phosphorylation may contribute to the global transcriptional regulation and RNA processing during Xenopus early development.
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MATERIALS AND METHODS |
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Cell culture
A6 cells derived from Xenopus kidney (Rafferty, 1969) were propagated in Leibovitz L-15 medium (SIGMA) supplemented with 10% heat-inactivated fetal calf serum (FCS; GIBCO-BRL), penicillin G (100 U/ml) and streptomycin (100 µg/ml) at 22°C. For serum stimulation, subconfluent A6 cells (24 hours after plating) were placed in serum-free medium for 16 hours. The resulting quiescent cells were serum-stimulated by the addition of FCS (20%) to the medium. U0126 (Promega) was dissolved in dimethylsulfoxide prior to use and added or not 30 minutes before serum stimulation.
Non-denaturing cell lysis and fractionation
Cells were washed twice with cold PBS and lysed on ice in a low salt buffer (20 mM sodium glycerophosphate, 1 mM EGTA, 5 mM MgCl2, 1% Nonidet P-40, 10% glycerol and 0.5 mM DTT adjusted to pH 7.5). The cell lysate was fractionated into a low-salt supernatant and a pellet by centrifugation at 15,000 g for 10 minutes at 4°C. The resulting low-salt supernatant was supplemented with Laemmli sample buffer. Whole-cell lysates were obtained by directly adding Laemmli sample buffer on the cells.
Denaturing electrophoresis and western blots
The volumes of lysate per lane correspond to 0.25 egg, 0.25 embryo or 30,000 A6 cells. All samples were heated at 95°C before loading on sodium dodecyl sulfate 6% polyacrylamide gels. Proteins were transferred on nitrocellulose membranes (Schleicher & Schull) and the blots were probed with primary antibodies. The immunoreactive bands were visualised using anti-mouse IgG horseradish peroxidase conjugates (Promega) and enhanced chemiluminescence (Pierce).
Antibodies
The monoclonal antibody POL3/3 recognises the RPB1 subunit at an evolutionary conserved epitope located outside the CTD and was kindly provided by E. K. Bautz (Kontermann et al., 1995). Various phosphoepitopes in the CTD (Bonnet et al., 1999; Patturajan et al., 1998) were recognised by the following monoclonal antibodies, CC-3 (gift from Michel Vincent), MARA3 (gift from Bart Sefton), V6 (gift from Marc Vigneron), H5 and H14 (gift from Stephen Warren). The anti-ERK2 MAP kinase antibody was purchased from Santa Cruz Biotechnology (reference no. sc1647).
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RESULTS |
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A new CTD phosphorylation pattern is established at MBT
To test whether the appearance of the IIo form could be inhibited when the MBT is impaired, embryos were treated or not at the two-cell stage with aphidicolin. This potent inhibitor of DNA replication slows down cell-cycle progression, which arrests at the 11th to 13th cleavage, and embryos do not undergo MBT (Clute and Masui, 1997). The IIo form was recovered in control untreated embryos before 6.5 hours p.f. (Fig. 2, lane 3). By contrast, in aphidicolin-treated embryos from the same animal, the IIo form was not detected even at 11.5 hours p.f. (lane 10). Thus, owing to DNA replication arrest, the hyperphosphorylated IIo form of RPB1 does not appear when MBT is prevented.
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When the POL3/3 antibody was used, the CTD was again seen to dephosphorylate shortly after fertilisation (Fig. 3A, lane 2) and the IIo began to increase as early as 5.75 hours p.f., which is the expected time for MBT (lane 7). It had been reported that, in nematodes and flies, the onset of transcription correlates with immunostaining with the monoclonal antibody H5, which binds to a specific phosphoepitope of the CTD (Seydoux and Dunn, 1997). The H5 antibody detected a single band comigrating with the IIo band and present in the unfertilised egg (Fig. 3A, lane 1). However, the H5 reactivity disappeared abruptly after fertilisation (lane 2) and increased after 5.75 hours p.f. (lane 7).
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This set of experiments suggests that the hyperphosphorylation of RNAPII during the blastula stage coincides with MBT and therefore, with zygotic gene activation.
Incomplete phosphorylation of the CTD in pre-MBT embryos
Several monoclonal antibodies directed against distinct phosphoepitopes on the CTD have been characterised and unravel the heterogeneity of the phosphorylated forms of the largest RNAPII subunit (Bonnet et al., 1999; Dubois et al., 1997; Patturajan et al., 1998). For example, H5 antibodies recognise serine-2 phosphorylation on the consensus CTD heptapeptide, whereas H14 recognise serine-5 phosphorylation. All four anti-phospho-CTD antibodies, H5, H14, MARA3 and CC-3, strongly reacted with a band comigrating with the IIo form in the lanes loaded with either A6 cell (Fig. 4, lane 1) or unfertilised egg lysates (lane 2). By contrast, the V6 monoclonal antibody reacted with the IIa form and a smear below the IIo form. These antibodies were used to characterise further the pattern of CTD phosphorylation during Xenopus development. In pre-MBT embryos, H14 and MARA3 antibodies clearly stained a band (Fig. 4, lane 3). By contrast, the V6, the CC-3 and H5 antibodies did not detect any IIe band in pre-MBT embryos (lane 3). At MBT, the MARA3 and H14 signals increased (lane 4) and, more significantly, the levels of a phosphorylated form recognised by H5 and CC-3 increased abruptly (lane 4). The post-MBT, MARA3, H14, H5 and CC-3 bands comigrated with the IIo form found in A6 cells.
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An incompletely phosphorylated form of RPB1 found in somatic cells is distinct from the IIe form
An incompletely phosphorylated form of RPB1, designated IIm, is generated in somatic cells upon various stimulations that activate MAP kinases (Bonnet et al., 1999). It was therefore questioned whether the IIm and IIe forms might be distinct from each other. When serum-starved Xenopus A6 cells were stimulated by addition of serum to the medium, a new band, which migrated slightly faster than the IIo form, was detected by the POL3/3 antibody (Fig. 5A). The intensity of this Xenopus IIm form increased rapidly after 10 minutes of treatment (lane 3) and remained for at least 60 minutes (lane 6). When quiescent A6 cells were lysed in a low-salt buffer, the POL3/3 antibody only detected the IIa form in the cytosolic fraction (Fig. 5A, lane 7). In contrast to the IIo form, the IIm form was readily extracted in a low-salt buffer (lanes 8 and 10). In mammalian cells, the appearance of the IIm form has been attributed to the ERK-type MAP kinases (Bonnet et al., 1999). MAP kinase activation occurs upon serum stimulation; in addition, it requires a phosphorylation, which decreases its electrophoretic mobility (Fig. 5B). MAP kinase activation, as well as the appearance of the Xenopus IIm form, was inhibited in the presence of the MAP kinase kinase inhibitor U0126 (Favata et al., 1998) (lane 5). Identical results were obtained with the MAP kinase kinase inhibitor PD098059 (Alessi et al., 1995) (data not shown).
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DISCUSSION |
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The embryo-specific IIe form of RNA polymerase II largest subunit
Between fertilisation and MBT, a minor phosphorylated form of RNA polymerase II largest subunit remains: the IIe form. This form is recognised by monoclonal antibodies directed against the phosphorylated CTD. However, there are anti-phospho CTD antibodies such as H5 and CC-3 that do not react with the IIe form. This observation, as well as the faster electrophoretic migration of this form, suggests that it is less phosphorylated than the IIo form. Determining the precise mapping of the in vivo phosphorylation sites on the CTD is a formidable task. In a recent study, the phosphoepitopes recognised by several anti-phospho-CTD monoclonal antibodies were characterised (Patturajan et al., 1998). The clearest results were obtained for mAbs H5 and H14, which recognise phosphoserine in position 2 and phosphoserine in position 5, respectively, in the consensus heptapeptide (see Introduction). Recognition by H14 indicates that the IIe form is phosphorylated on serine-5. The deficient H5 and CC-3 recognition suggests that most serine-2 residues are not phosphorylated on the IIe form.
The ERK-type MAP kinases phosphorylate in vitro the CTD on genuine mammalian RNA polymerase II and generate a phosphorylated form indistinguishable from the mammalian IIm form; the H14 immunoreactivity is generated without suppression of the V6 immunoreactivity (Bonnet et al., 1999). We now show that MAP kinase activation in Xenopus cells correlates with the appearance of a IIm form very similar to the mammalian one. The IIe and IIm forms of RNAPII largest subunit are distinct: the IIe migrates slower than the IIm (not shown) and it is slightly more phosphorylated as it reacts with a larger number of anti phospho-CTD antibodies but not with the V6 antibody. However, both forms lack serine-2 phosphorylation.
The kinase generating the IIe form remains unknown, but if there is a contribution from the transiently activated Xp42 (Guadagno and Ferrell, Jr, 1998), the involvement of an additional kinase might be required to generate the MARA3 phosphoepitope. Indeed, MAP kinase does not generate this epitope on the CTD in vitro (Bonnet et al., 1999). Alternatively, the low amounts of phosphorylated forms may reflect a general defect of CTD kinases in the pre-MBT embryos.
Transitions in CTD phosphorylation allow the definition of a new landmark of MBT during Xenopus development
The results obtained in this study show that RNAPII phosphorylation is modified during the development of amphibian embryos at MBT. The reappearance of the IIo form is correlated with MBT. It does not occur when MBT is prevented. By contrast, increasing the temperature of development or increasing the number of nuclei by allowing polyspermy - treatments which both advance MBT - also accelerate IIo phosphorylation. The IIo form can be monitored using the CC-3 and the H5 monoclonal antibodies. Thus, we propose the appearance of the hyperphosphorylated form of RNAPII as a landmark of MBT during Xenopus development.
In rabbit embryos, a fully phosphorylated IIo form also replaces a phosphorylation-deficient IIe form at the onset of zygotic transcription (Bellier et al., 1997a). In nonvertebrate metazoans such as Caenorhabditis elegans and Drosophila melanogaster, the phosphorylation of RNAPII is also correlated to zygotic genome activation during early development (Dantonel et al., 2000; Leclerc et al., 2000; Seydoux and Dunn, 1997). The H5 phosphoepitope appears in differentiated cell nuclei in coincidence with zygotic gene activation but remains absent from germ cells until gastrulation, when the latter cells become transcriptionally active. By contrast, the H14 epitope is continuously present in both somatic and germ cells. Thus, in these species, the phosphoepitopes evolution is very similar to the one observed in vertebrates. The control of phosphorylation of RNAPII might be an essential feature of early embryogenesis in many metazoans and the CTD phosphorylation characteristics may therefore be used as an evolutionary conserved landmark of transcriptional activation.
Changes in CTD phosphorylation as a potential mRNA processing regulator
CTD phosphorylation is required for transcription (Dahmus, 1996). The H5 mAb stains transcriptional foci detected by immunofluorescence (Zeng et al., 1997). Furthermore, chromatin immunoprecipitation assays with this antibody indicated that serine-2 is phosphorylated in elongating polymerase molecules but not in initiation complexes (Komarnitsky et al., 2000). As the IIe form is not phosphorylated on serine-2, it is unlikely to be engaged in transcription. Indeed, transcription does not occur during the period characterised by the presence of the IIe form. Alternatively, phosphorylation of RNAPII may contribute to regulate mRNA processing. RNAPII can directly stimulate mRNA processing reactions such as capping and cleavage-polyadenylation (Cho et al., 1997; Hirose and Manley, 1998; Ho and Shuman, 1999; McCracken et al., 1997). Phosphorylation of the CTD enhances its binding to cleavage-polyadenylation factors (Barillà et al., 2001; Rodriguez et al., 2000) and the phosphorylated polymerase is more efficient than the unphosphorylated form in a cleavage-polyadenylation assay (Hirose and Manley, 1998). Furthermore, serine-5 phosphorylated CTD is more efficient in stimulating capping (Ho and Shuman, 1999). Thus, the IIe form phosphorylated on serine-5, might be competent in regulating maternal mRNA capping and polyadenylation.
There are evidences that modifications of the 5' cap of mRNA may regulate gene expression during early development (Caldwell and Emerson Jr, 1985). During Xenopus oocyte maturation, the activity of a cytoplasmic guanine-7-methyltransferase increases markedly (Gillian-Daniel et al., 1998) as well as phosphorylation of the CTD (Bellier et al., 1997b). Furthermore, developmentally regulated cytoplasmic polyadenylation influences the polysomal recruitment of specific mRNAs during Xenopus early development (Paris and Philippe, 1990). It is worth noting that both mos mRNA polyadenylation (Howard et al., 1999) and RNAPII phosphorylation (Bellier et al., 1997b) are mitogen-activated protein kinase-dependent at this stage. In oocytes and early embryos, capping and polyadenylation are cytoplasmic events, whereas RNAPII is cytosolic (data not shown). Thus, a parallel can be drawn between RNAPII properties (phosphorylation and localisation) and mRNA processing. Unfortunately, assaying capping and polyadenylation in embryos is technically difficult and has been unsuccessful in our hands. However, it might be speculated that the phosphorylated forms of RNAPII are involved in maternal mRNA processing and subsequent translatability during meiosis and the first hours of amphibian development.
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ACKNOWLEDGMENTS |
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