Preimplantation embryonic development begins with fertilization and zygote formation. The division of zygote into a blastocyst involves a series of cell divisions accompanying changes in cell cycle characteristics and dynamics [1,2]. The cell cycle incorporates many aspects of embryonic development, which are essential for regulating cell proliferation, transcription, migration, and differentiation [3]. Histone acetylation is an important epigenetic modification that regulates gene expression by altering the chromatin structure and plays a key role in the cell cycle [4] and embryo development [5].

The E2 factor (E2F) family consists of 8 transcription factors (E2F1-E2F8) divided into activators (E2F1-E2F3) and repressors (E2F4-E2F8), which have been identified as major regulators of the cell cycle, differentiation, and apoptosis [[6], [7], [8]]. E2F4 has been classified as a repressor for a long time and is dependent on retinoblastoma (Rb) family proteins to enter the nucleus during the G0/G1 phase and suppress cell cycle regulators [9]. Phosphorylation of Rb by cyclin/cyclin-dependent kinases releases E2Fs, which activate their target genes and promote the G1 to S transition [10]. However, in mouse embryonic stem cells, E2F4 is required for proliferation and survival and as a transcription activator independent of the Rb family proteins [11]. E2F4 plays multiple roles during cell proliferation, such as in pRb−/− tumor cells, loss of E2F4 suppresses cell proliferation and inhibits tumor growth [12]. In contrast, E2F4 promotes the proliferation of bovine mammary epithelial cells and positively regulates the biosynthesis of milk components [6]. Therefore, E2F4 plays a dual role as a transcriptional factor.

In addition to its role in cell cycle regulation, E2F4 plays multiple roles in embryonic development including cell proliferation, growth, and protection against infections [13]. One of the studies has reported that E2F4−/− mice are smaller and more prone to bacterial infections than wild-type mice after birth [[14], [15], [16]]. In addition, E2F4 deficiency has been shown to reduce the size of the developing ventral telencephalon in mouse embryos [17]. Recently, a study suggested that the E2F4 mutant exhibited growth retardation in body length and weight, reduced intestinal epithelial cell proliferation, and increased apoptosis in zebrafish [10].

Based on the above-mentioned effects of E2F4, the objective of this study was to investigate the localization of E2F4 in the porcine embryo and its role in embryonic development. For this purpose, we knocked down E2F4 in porcine 1-cell embryos to deduce its influence on DNA damage and cell cycle progression in these embryos.