Characterization of Two KRAB-Containing Zinc Finger Transcription Factors In Bovine Preimplantation Embryonic Development

Abstract

Oocyte developmental competence or oocyte intrinsic quality describes the capability of oocytes to resume meiosis, cleave and develop to blastocyst stage after fertilization, implant and develop to term in a good health. A growing number of evidences indicate that the majority of embryonic mortality occurs during early embryonic development in different species, including human, horse and cattle primarily due to poor oocyte quality. Maternal effect genes are key aspects of oocyte quality, which are transcribed during the process of oogenesis and folliculogenesis. The maternal factors are accumulated in oocytes, orchestrating various early developmental events including fertilization, epigenetic reprogramming and zygotic genome activation (ZGA). The key step to acquire development competence is oocyte maturation. The fully matured oocytes, which obtain required maternal factors, are determining factor for fertility. During the process of in vitro maturation, manipulation of synchronization of meiotic maturation and cytoplasmic maturation, which determines the acquisition of maternal factors, increases the oocyte competence. C2H2 (Cys2-His2) zinc finger domain represents one of most common domains of transcription factor in mammals, which dominate around 53% of mammalian transcription factor repertoire. Approximately 2/3 of C2H2 zinc finger transcription factors contain a Küppel associated box (KRAB) domain, which is known to interact with KRAB-associated protein-1 (KAP1) corepressor. KAP1 serves as a scaffold to recruit repressive complexes. Interestingly, even though KRAB domain is present in some C2H2-zinc finger proteins, the interaction with KAP1 is not guaranteed, especially for those that have a SCAN domain. Despite being highly abundant in mammalian genome, the KRAB containing zinc finger proteins are still poorly understood. Our laboratory previously identified a novel member of KRAB-ZFPs family, ZNFO. As a maternal effect gene, ZNFO transcript is highly abundant in germinal vesicle (GV), MII-stage oocytes, and early-stage embryos but barely detectable in morula and blastocyst stage embryos. RNAi experiments demonstrated that ZNFO is indispensable during early embryonic development in cattle. However, the molecular mechanism regulating ZNFO transcription and regulatory mechanism downstream of ZNFO remain elusive. In the present study, we identified the core promoter that controls the ZNFO basal transcription. Using 5’RACE followed by Sanger sequencing, the 5’ untranslated region (UTR) and the transcription start site (TSS) of ZNFO transcript were identified. A 1.7 kb of putative promoter region of ZNFO spanning from -1665 to +36 was cloned into pGL4.14 luciferase reporter vector. A series of 5′ deletion in the ZNFO promoter followed by luciferase reporter assays indicated that the core