Incomplete RNA polymerase II phosphorylation in Xenopus laevis early embryos

In many metazoans such as vertebrates and insects, transcription is arrested during oocyte maturation (Davidson, 1986). This transcriptional silence is maintained after fertilisation during the first cell cycles of development, which do not require RNA synthesis. The onset of transcription or zygotic gene activation occurs abruptly at a stage of development that is species specific: for example, it can range from the two-cell stage in mice to cell cycle 12-14 in flies. Although a very weak transcriptional activity has been detected in Xenopus laevis embryos after six cleavage divisions, mRNA synthesis bursts out after the twelfth cleavage division, at the mid-blastula transition (MBT) (Kimelman et al., 1987; Newport and Kirshner, 1982b; Shiokawa et al., 1994).

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.

Metaphase II-arrested eggs and embryos were prepared as previously described (Almouzni and Wolffe, 1995). Briefly, female Xenopus laevis were primed by injection of 50 U of Pregnant Mare Serum Gonadotrophin (InterVet) 3 days before the experiment and 500 U of Human Chorionic Gonadotrophin (InterVet) the day before. Eggs were laid in High Salt Barth’s medium and monospermic fertilisation was performed in vitro with freshly dilacerated testes. Eggs and embryos were dejellied in 2% cysteine 0.1× Barth’s medium (pH 7.8) for 10 minutes and washed extensively with 0.1× Barth. The embryos were then kept in 0.1× Barth supplemented with 100 U of penicillin G and 100 μg of streptomycin per ml. When indicated, aphidicolin (100 μg/ml) was added to the medium at the two-cell stage. To obtain polyspermic embryos, freshly laid eggs were incubated in 30 mM NaCl, 40 mM NaI, 0.4 mM CaCl₂ and 0.4 mM KCl for 5 minutes before fertilisation (Grey et al., 1982). After fertilisation, the procedure was the same as for monospermic embryos (see above). Batches of ten embryos were lysed in Laemmli sample buffer and lysates were centrifuged 15 minutes at 15,000 g.

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.

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.

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).

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).

The phosphorylation pattern of RPB1 in Xenopus laevis embryos was investigated by western blot using the POL3/3 monoclonal antibody (mAb), which maps outside the CTD (Kontermann et al., 1995). This antibody detected two bands in Xenopus laevis kidney A6 cells (Fig. 1, lane 1). The 210 kDa band and the 240 kDa band corresponded to the hypophosphorylated (IIa) and the hyperphosphorylated (IIo) forms of the largest subunit, respectively. Both forms gave signals of similar intensities as previously observed in cells from other species (Venetianer et al., 1995). In metaphase II-arrested eggs, phosphorylated and unphosphorylated forms were also found in similar amounts (Fig. 1, lane 2). The eggs were fertilised synchronously and the embryos maintained at 16°C were sampled at timely intervals until gastrulation was clearly visible upon visual inspection at 10.5 hours post fertilisation (p.f.). The signal intensity corresponding to the phosphorylated IIo form decreased abruptly 1 hour p.f. (lane 3). A form migrating slightly faster than the IIo form remained visible during the cleavage stages (Fig. 1, lanes 3-5). This form was designated as IIe (for embryonic form). At 7.5 hours p.f., a new band appeared just above the IIe (lane 6). This new band comigrated with the IIo form found in A6 cells. From 8 to 10.5 hours p.f., the intensity of this new band increased gradually (lanes 7-10).

Thus, two major transitions were detected in CTD phosphorylation during the first hours of Xenopus development. Fertilisation was followed by a massive dephosphorylation of RNAPII largest subunit. As signs of gastrulation were seen at 10.5 hours p.f., the appearance of the IIo form might coincide in time with the 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.

The MBT can be experimentally advanced either by shifting the temperature or by increasing the number of nuclei in the developing embryo by polyspermy (Newport and Kirshner, 1982a). To strengthen the connection between MBT and the IIo appearance, another fertilisation was performed. This time, the embryos were maintained at 23°C and seen to cleave into two cells, four cells and eight cells at 77±5, 99±5 and 124±5 minutes, respectively. A small number of embryos that fell outside this timing were discarded to improve the synchrony. The above cleavage division timing was consistent with the reported occurrence of MBT at 6 hours p.f. at this temperature (Kimelman et al., 1987).

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).

In parallel with the above experiment, metaphase II-arrested oocytes from the same female were fertilised in conditions leading to polyspermy and maintained at 23°C (Grey et al., 1982). The sampled polyspermic embryos were analysed by western blot with the H5 antibody, which is characteristic of the IIo form (see below). Polyspermic fertilisation also led to the loss of the H5 epitope (Fig. 3B, lane 2). However, in this case, the H5 immunoreactivity was already recovered at 5 hours p.f. (lane 4), 45 minutes earlier than with the monospermic embryos from the same animal. As polyspermic embryos have been shown to activate mRNA synthesis two divisions before monospermic (Newport and Kirshner, 1982a), once again the appearance of the IIo form might be linked to MBT.

This set of experiments suggests that the hyperphosphorylation of RNAPII during the blastula stage coincides with MBT and therefore, with zygotic gene activation.

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.

Taken together, these results demonstrate that the phosphorylated form of RNA polymerase largest subunit present in low amounts in pre-MBT embryos, the IIe form, is distinct from the IIo form found in somatic cells as well as unfertilized eggs. Indeed, this form does not react with several anti phospho-CTD antibodies.

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).

The antigenicity of the IIm form was investigated next with the same set of anti-CTD antibodies used for the analysis of the IIe form. The H14 and the V6 mAb recognised the IIm form (Fig. 5C, lane 3). By contrast, neither the CC-3, H5 or MARA3 mAbs recognised the IIm form. Such antigenic pattern is similar to that previously described in mammalian cells (Bonnet et al., 1999). Both the IIm and IIe forms were recognised by H14; however, they differed in their V6 and MARA3 recognition. Hence, they correspond to distinct forms of RNAPII largest subunit.

In this study, we show that the CTD of RNA polymerase II largest subunit undergoes two major developmental transitions following fertilisation of Xenopus oocytes. Fertilisation of Xenopus eggs is followed by massive dephosphorylation of RNAPII, leaving the hypophosphorylated IIa form predominant along with a minor form exhibiting incomplete phosphorylation. A marked increase of the hyperphosphorylated IIo form occurs at the mid-blastula transition and leads to the phosphorylation profile usually observed in somatic cells.

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.

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.

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.

This work was supported by grants from the Association pour la Recherche sur le Cancer, ARC 5216 (to O. B.). We are much indebted to Drs Jacek Kubiak, Michel Charbonneau, Marie Françoise Dubois and colleagues from our team for critical discussion and to Dr Olivier Hyrien and his team for help in Xenopus handling.