Semester

Spring

Date of Graduation

2022

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Division of Animal and Nutritional Sciences

Committee Chair

Jianbo Yao

Committee Co-Chair

Vagner A Benedito

Committee Member

Vagner A Benedito

Committee Member

Ida Holásková

Committee Member

I-Chung Chen

Committee Member

Daniel J Mathew

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 promoter region has to include the sequence located between 57 bp to 31 bp upstream of the TSS. Sequence analysis revealed that a putative upstream stimulatory factor 1/2 (USF1/USF2) binding site (GGTCACGTGACC) containing an enhancer box (E-box) motif (CACGTG) is located within the essential region. Depletion of USF1/USF2 by RNAi and E-box mutation analysis demonstrated that the USF1/USF2 binding site is required for the ZNFO basal transcription. Furthermore, EMSA and super-shift assays indicated that the observed effects are dependent on the specific interactions between USF proteins and the ZNFO core promoter. From these results, it is concluded that USF1 and USF2 are essential for the basal transcription of the ZNFO gene. Regarding the regulatory mechanism downstream ZNFO, the previous study identified an 18-nucleotide ZNFO binding element (ZNFOBE), ATATCCTGTTTAAACCCC. The present study confirmed the sequence-specific binding of ZNFO to its target element using EMSA in combination with competition assays. Furthermore, it was confirmed that the interaction between ZNFO and ZNFOBE is required for the repressive effect of ZNFO via a luciferase reporter assay.

Zinc Finger Imprinted 2 (ZIM2) is isolated from a highly conserved Paternally Expressed 3 (PEG3) imprinted domain. Compared to mouse Zim2, the human and bovine ZIM2 maintain the protein coding ability. Both human and bovine ZIM2 encode a KRAB-containing zinc finger protein. In addition, SCAN domain, a protein interaction domain is also present in the N-terminal of human and bovine ZIM2 protein. It has been reported that ZIM2 is highly abundant in testis. Consistent with human microarray data, analysis of RNA-seq data from our laboratory revealed that ZIM2 is highly abundant in bovine oocytes as well. In the present study, characterization of ZIM2 transcript expression revealed that ZIM2 mRNA is expressed in testis, oocytes, and early embryos. Interestingly, ZIM2 mRNA is not detectable in morula but re-transcribed in blastocyst. In addition, western blot analysis using a customized ZIM2 antibody indicated that ZIM2 protein is present in oocytes and 2-cell, 4-cell, 8-cell, 16-cell embryos, morula, and blastocyst. The RNAi-mediated knockdown indicated that deletion of ZIM2 by microinjecting siRNA targeting ZIM2 reduced the blastocyst rate. In addition, using a GAL4-luciferase reporter assay, ZIM2 was demonstrated to contain an intrinsic repressive effect. Furthermore, ZIM2 interacted with a highly conserved co-repressor KAP-1. Present studies demonstrated that maternally derived ZIM2 is indispensable for early embryonic development, presumably serving as a transcription repressor.

Overall, the present projects elucidate the molecular mechanism regulating basal transcription of ZNFO, as well as the downstream regulatory mechanism of ZNFO. In addition, ZIM2 was confirmed to be a transcription repressor, which might be indispensable for bovine early development.

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