DETERMINATION OF THE CONSENSUS TARGET SEQUENCES RECOGNIZED BY ZNFO, A NOVEL OOCYTE-SPECIFIC ZINC FINGER TRANSCRIPTION FACTOR IN CATTLE

Most of the early embryonic loss in livestock species occurs at or close to maternal to embryonic transition (MET), a process in which embryos switch from maternal factors to the factors encoded by their own genome. Maternal factors stored at oocytes during the process of folliculogenesis as well as oocyte maturation have essential role in regulation of early development including MET. Zinc finger proteins are one of the largest protein families in eukaryotes. The classic two cysteine and two histidine residue (C2H2) zinc finger proteins compose the largest transcription factor repertoire in mammals. Around one-third of them have an N-terminal KRAB domain. The zinc finger domain can make contact with DNA to locate zinc finger proteins in certain positions of the genome while the KRAB domain can interact with KAP1, which serves as a scaffold for various factors. ZNFO is a novel oocyte-specific transcription factor identified from a bovine oocyte library. It is a classic C2H2 zinc finger protein, having an N-terminal KRAB domain and a Cterminal zinc finger domain which is composed of nine zinc finger motifs. Our previous work revealed an essential function of ZNFO during early embryonic development in cattle. Depletion of ZNFO blocks the development of embryos to blastocyst stage. As a maternal transcription factor, ZNFO may repress certain target gene(s) to orchestrate post-fertilization events in cattle. Defect of ZNFO in vivo may cause early embryonic loss in cattle. To investigate the molecular function of ZNFO, we characterized the binding property of ZNFO to DNA. Using a cyclic amplification and selection of targets (CASTing) assay, we identified a potential ZNFO binding element (ZBE), ATATCCTGN5ANCCC. To confirm the binding specificity of ZNFO to the identified element, an electrophoretic mobility shift assay (EMSA) was performed using IDye 700 labeled probes containing the target sequence and purified Halo-tagged ZNFO fusion protein. A competitive binding assay was also performed using 10-fold and 100-fold molar excess of unlabeled cold competitors containing the target element and two mutated elements. The results confirmed the interaction between ZNFO and ZBE, and showed that both ATATCCTG and CCC are critical for the binding of ZNFO to ZBE. Further analysis of promoter regions of candidate bovine genes that contain ZBE may lead to the discovery of specific genes regulated by ZNFO.