Transcriptional modulation of the pre-implantation embryo-specific Rnf35 gene by the Y-box protein NF-Y/CBF

Maternal-to-zygotic transition of a fertilized egg and the subsequent pre-implantation development of the embryo involve zygotic genome activation and reprogramming of gene expression. The goal of the present study is to establish a model suitable for the characterization of transcriptional modulation of mammalian pre-implantation development. Rnf35 is a mouse RING-finger protein gene that is temporally transcribed in the early embryo, but is permanently silenced before the blastocyst stage of development. We first show that the Chinese-hamster ovary-K1 cells are unique in supporting Rnf35 promoter activities in transient transfection assays. Using the permissive Chinese-hamster ovary-K1 cell line, we show that Rnf35 transcription is driven by an Inr (initiator) core promoter element in the absence of a TATA box; the Inr promoter function is confirmed by direct microinjection of mouse one-cell embryos. This is the first demonstration of the involvement of an Inr core promoter element in transcription in pre-implantation development. We show that the Rnf35 promoter is regulated by three obligatory Y-box (CCAAT-box) elements: two Y boxes (Y(I) and Y(II)) located at -81 are coupled in a palindrome and act synergistically in contributing to Rnf35 transcription; the third Y box (Y(III)) is situated at -13, just upstream of the Inr element, and may be an integral part of the Inr function. Electrophoretic mobility-shift assays and competition experiments further reveal that the Y(I) box is bound by the ubiquitous NF-Y (nuclear factor-Y)/CBF (CCAAT-binding factor) and that Y(II) is targeted by an unidentified protein(s) that acts synergistically with the NF-Y. We suggest that the NF-Y, targeting at a Y-box sequence, may function as an important activator in transcriptional regulation of the Rnf35 gene in the pre-implantation embryo.