Negative transcriptional modulation and silencing of the bi-exonic Rnf35 gene in the preimplantation embryo. Binding of the CCAAT-displacement protein/Cux to the untranslated exon 1 sequence.

Previous works have indicated promiscuous transcription from the zygotic genome immediately after fertilization. The mouse Rnf35 gene is bi-exonic in structure and is transcribed in the preimplantation embryo until it is permanently silenced at the blastocyst stage of development. We have previously shown that Rnf35 transcription is positively regulated by the nuclear factor Y. Using the uniquely permissive Chinese hamster ovary-K1 cell line in transient transfection assays, we demonstrate in this work that the Rnf35 promoter was negatively modulated by a cis-cognate repressor element, designated as the downstream exon 1 repressor, or DER, residing between +72 and +95 in the untranslated exon 1 of the Rnf35 gene. Simultaneous mutagenesis of the two half-sections, DER1 and DER2, of the DER sequence was required for derepression suggesting participation of multiple proteins in the DER-dependent transcriptional repression. Electrophoretic mobility shift assays demonstrated that the 3'-half of DER (DER2) was targeted by the repressor CCAAT-displacement protein (CDP)/Cux. Chromatin immunoprecipitation experiments further demonstrated in vivo CDP-DER association in the blastocyst and the 8.5 day embryo. Furthermore, the DER-dependent repression was partially relieved in vivo in co-transfection with an antisense CDP construct. Transcription of the Cdp gene was shown to first occur between the eight-cell and the blastocyst stages, correlating and possibly explaining the onset of Rnf35 silencing at the blastocyst stage. Taken together, our results suggest that the evolutionarily acquired exon 1 of Rnf35, and possibly exon 1 of other similarly structured bi-exonic early embryonic genes, contributes to transcriptional modulation and silencing in the developing mouse embryo.