TRANSIENT EXPRESSION OF TRANSLATION INITIATION-FACTOR EIF-4C DURING THE 2-CELL STAGE OF THE PREIMPLANTATION MOUSE EMBRYO - IDENTIFICATION BY MESSENGER-RNA DIFFERENTIAL DISPLAY AND THE ROLE OF DNA-REPLICATION IN ZYGOTIC GENE ACTIVATION

Zygotic gene activation (ZGA) definitely occurs by the 2-cell stage in the mouse embryo. Analysis of protein synthesis by two-dimensional ge l electrophoresis reveals a class of genes whose expression transientl y increases in the 2-cell embryo. Although the paucity of biological m aterial has prevented a systematic identification of these genes, the mRNA differential display method circumvents this problem. Using this approach we find a transient increase in the mRNA abundance of the tra nslation initiation factor eIF-4C that is inhibited by alpha-amanitin and correlated with a transient increase in the relative rate of prote in synthesis for eIF-4C. We confirm the transient increase in eIF-4C m RNA abundance by a reverse transcription-PCR-based assay using eIF-4C- specific primers. The first round of DNA replication seems critical fo r eIF-4C expression, since addition of aphidicolin prior to S phase in the 1-cell embryo inhibits the magnitude of the increase in eIF-4C ex pression. Aphidicolin treatment also inhibits the synthesis of an acce pted marker for ZGA, the transcription-requiring complex (TRC), which is also transiently expressed during the 2-cell stage. Incubating late 1-cell/early 2-cell embryos in medium containing aphidicolin reveals that the second round of DNA replication is not required for the incre ase in eIF-4C expression but DNA replication is required for the decre ase in both eIF-4C expression and TRC synthesis. The decrease in eIF-4 C expression, however, does not require cytokinesis or mitosis, since it occurs when 2-cell embryos are cultured in the presence of cytochal asin D or nocodazole, respectively. Changes in chromatin structure may be involved in the decrease in both eIF-4C and TRC expression, since neither decrease occurs when 2-cell embryos are cultured in trapoxin, which is a specific and irreversible inhibitor of histone deacetylase. Results of these experiments suggest that the first round of DNA repl ication is permissive with respect to ZGA and that the second round is repressive. (C) 1996 Academic Press, Inc.