Identification of the DNA binding element of ZNFO, an oocyte-specific zinc finger transcription factor in cattle

The maternal effect genes are essential components of oocyte competence, which orchestrate the early developmental events before zygotic genome activation (ZGA). The Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KRAB-ZFPs) constitute the largest transcription factor family in mammals. As a novel maternal effect gene, ZNFO was identified previously in our laboratory. The gene codes for a KRAB-ZFP specifically expressed in bovine oocytes and early embryos and gene silencing experiments have demonstrated that ZNFO is required for early embryonic development in cattle. In the present study, we identified a consensus sequence, ATATCCTGTTTAAACCCC, as the DNA binding element of ZNFO (ZNFOBE) using a library of random oligonucleotides by cyclic amplification of sequence target (CAST) analysis. Sequence-specific binding of ZNFO to the DNA binding element was confirmed by an electrophoretic mobility shift assay (EMSA), and the key nucleotides in the ZNFOBE that are required for specific binding by ZNFO were further determined by a competitive EMSA using mutant competitors. Through a luciferase-based reporter assay, it was confirmed that the interaction between ZNFO and ZNFOBE is required for the repressive function of ZNFO. These results provide an essential step towards the identification of ZNFO regulated genes that play important roles during early embryonic development.