In mammalian embryos, zygotic gene transcription initiates after a limited number of cell divisions through a two-step process termed the zygotic gene activation (ZGA). Here we report that RNA polymerase II undergoes major changes in mouse and rabbit preimplantation embryos during the ZGA. In transcriptionally inactive unfertilized oocytes, the RNA polymerase II largest subunit is predominantly hyperphosphorylated on its carboxy-terminal domain (CTD). The CTD is markedly dephosphorylated several hours after fertilization, before the onset of a period characterized by a weak transcriptional activity. The largest subunit of RNA polymerase II then lacks immunological and drug-sensitivity characteristics related to its phosphorylation by the TFIIH-associated kinase and gradually translocates into the nuclei independently of DNA replication and mitosis. A phosphorylation pattern of the largest subunit, close to that observed in somatic cells, is established in both mouse and rabbit embryos at the stage when transcription becomes a requirement for further development (respectively at the 2-and 8/16-cell stage). As these events occurred in the presence of actinomycin D, the nuclear translocation of RNA polymerase II and the phosphorylation of the CTD might be major determinants of ZGA.

Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34 cdc2 kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.In Xenopus laevis, as in many species, transcription is arrested in the matured gametes. During oogenesis, proteins and RNAs accumulate to support early embryogenesis. Although the fully grown oocyte contains as much RNA polymerase II (RNAPII) activity as 10 5 individual somatic cells (43), little is known about its properties at maturation, after the germinal vesicle breakdown. From this perspective, we decided to investigate the phosphorylation of the RNAPII largest subunit (RPB1) in the developing and maturing oocytes. The RNAPII core enzyme is an assembly of 12 subunits (29, 45). Extensive studies have assigned an important role to the phosphorylation of the C-terminal domain (CTD) of RPB1 in regulating the initiation of transcription (11, 18). The hypophosphorylated RPB1 (IIa subunit) binds to the TATA box binding protein (TBP) within a preinitiation complex of transcription, this interaction being abolished by phosphorylation of the CTD (28, 52). The steady-state distribution between the hypophosphorylated IIa and the hyperphosphorylated IIo forms of RPB1 results from the antagonistic activity of CTD kinases and CTD phosphatases. A CTD phosphatase has recently been purified and characterized, but few data are available concerning...

The phosphorylation of the C-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 microgram/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5,6-dichlorobenzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), and actinomycin D (over 5 micrograms/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced CTD-phosphorylation process that might contribute to the regulation of transcription during stress.