35 Characterization of the promoter region of ZNFO, an oocyte-specific gene in cattle

Abstract

Maternal factors are essential aspects of oocyte competence, which orchestrate early embryonic development. ZNFO is a Krüppel-associated box domain (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocyte. Previous studies have demonstrated that ZNFO is essential for early embryonic development. However, the mechanisms regulating ZNFO transcription remain elusive. The objective of present study is to elucidate regulatory mechanisms of ZNFO transcription in vitro, and specifically to identify putative core promoter and transcription factor(s) regulating ZNFO expression. 5′ Random amplification of cDNA ends (RACE) was performed using RNA isolated from 100 germinal vesicle (GV) stage oocytes to identify the transcription start site (TSS) of ZNFO. To elucidate the molecular mechanisms of ZNFO transcription, a 1.7-kb fragment of the 5′ regulatory region was obtained and cloned into pGL4.14 promoterless vector. The luciferase reporter assay was performed to confirm the promoter activity of the regulatory region. To further identify the core promoter region of the putative ZNFO promoter, a series of 5′ deletions in the ZNFO promoter followed by luciferase reporter assay was performed. The luciferase results indicated that the core promoter region of ZNFO was located within a region 57 to 31 bp upstream of the transcription start site. Bioinformatics analysis indicated that a putative USF1/USF2 binding site (GGTCTCGTGACC) is located within the core promoter region. USF1 is a basic helix–loop–helix leucine zipper transcription factor, which regulates the expression of various maternal genes, which are essential for oocyte maturation and early embryonic development in cattle. To confirm that USF1 regulated ZNFO expression, bovine USF1 open reading frame (ORF) was cloned into pcDNA3.1A-HA vector, generating a USF1 overexpression construct. Overexpression of USF1 by transfecting USF1 plasmid enhanced ZNFO promoter activity within HEK293 cells, confirming that ZNFO expression is regulated by USF1. From these results, we conclude that USF1 activates the ZNFO promoter by binding to its target site, GGTCTCGTGACC.