PGC7 maintains the pluripotency of F9 embryonic carcinoma cells by promoting Nanog translation

Abstract

Primordial germ cell 7 (PGC7) is prominently expressed in primordial germ cells (PGCs) and embryonic stem cells (ESCs), serving as a pivotal marker for discerning stem cell pluripotency. However, the role of PGC7 in regulating core pluripotency factors remains unclear. In this study, the expression dynamics of PGC7 and pluripotency-associated proteins are systematically evaluated by quantitative reverse transcription PCR (RT-qPCR) and western blot analysis. Complementary experimental approaches including confocal immunofluorescence and Co-immunoprecipitation (Co-IP) assays are subsequently employed to establish subcellular colocalization patterns and elucidate the molecular mechanisms associated with PGC7 function. The results show that PGC7 is closely associated with the pluripotency status of F9 embryonal carcinoma (EC) cells. Notably, PGC7 can counteract the decrease in pluripotency induced by retinoic acid (RA). Ectopic expression of PGC7 in F9 EC cells enhances the translation of Nanog. Mechanistic analysis reveal that PGC7 activates Y-box binding protein 1 (YBX1) phosphorylation by enhancing the interaction between YBX1 and AKT1. The subsequent phosphorylation of YBX1 reduces its binding to Nanog mRNA and promotes the translation of Nanog. These results shed light on a previously unknown role of PGC7 in supporting the translation of Nanog, offering valuable insights into the functions of PGC7 in F9 EC cells.

1. Introduction

With their potential for unlimited proliferation, pluripotency and low immunogenicity, embryonic stem cells (ESCs) have become crucial tools in the fields of regenerative medicine and disease treatment [1,2]. Primordial germ cell 7 (PGC7) is an essential maternal effect gene in early embryonic development and is specifically expressed in germ cells, oocytes, preimplantation embryos, and pluripotent cells [3]. The differential expression and functional diversity of PGC7 in these distinct cell types are crucial for comprehending its role in developmental biology [4,5]. PGC7 is a small, basic protein with a molecular weight of approximately 17 kDa that is characterized by a putative SAP-like domain and a splicing-like factor [6].

Current research on PGC7 has focused on its ability to regulate DNA methylation. In NIH-3T3 cells, the interaction between PGC7 and UHRF1 inhibits the recruitment of DNMT1, leading to genome-wide DNA hypomethylation [7]. In F9 embryonal carcinoma (EC) cells, PGC7 also regulates ERK-mediated DNMT1-Ser717 phosphorylation, affecting genome-wide methylation [8]. Furthermore, PGC7 promotes DNA demethylation by directly binding to the plant homeodomain of UHRF1, disrupting the association of UHRF1 with chromatin [9]. Additionally, PGC7 counteracts DNMT3A-mediated demethylation through its interaction with HP1BP3 [10]. During the maturation of oocytes, the absence of PGC7 results in abnormal accumulation of UHRF1 and DNMT1 in the nucleus. This ultimately causes excessive DNA methylation across the genome, including the promoters of inactive genes, and further impairs zygotic genome activation (ZGA) [11].

PGC7 must localize to the nucleus to protect the maternal genome from demethylation, a process initiated by the interaction between PGC7 and Ran-binding protein 5 (RBP5). Further studies revealed that PGC7 primarily binds to dimethylated histone H3 lysine 9 (H3K9me2), thereby inhibiting the activity of Tet3 methylcytosine oxidase in the maternal genome and at specific imprinted loci in the paternal genome [12–14]. Knocking out PGC7 results in widespread transcriptional dysregulation, which is characterized primarily by impaired activation of endogenous retroviruses (ERVs), ultimately leading to the failure of ZGA [15]. Although PGC7 is unique to mammals, it is also capable of inducing global DNA demethylation in non-mammalian species, such as Xenopus and medaka [16].