Biochemical and Biophysical Research Communications
Copper chaperone ATOX1 regulates pluripotency factor OCT4 in preimplantation mouse embryos
Introduction
Copper (Cu) is an essential nutrient in living organisms acting as a cofactor in proteins facilitating for example respiration, iron transport, oxidative stress protection, peptide hormone production, pigmentation, and blood clotting [1], [2]. In order to avoid toxicity of free Cu ions, intracellular Cu is regulated by devoted Cu transport proteins that assist uptake, efflux, and distribution of the metal ion to target Cu-dependent proteins [2], [3]. In human cells, the Cu chaperone ATOX1 transports Cu to ATP7A and ATP7B in the trans-Golgi network [1]. These P1B-type ATPases use ATP hydrolysis to transfer Cu to the lumen for loading of target Cu-dependent enzymes [2].
It was recently reported that ATOX1 also could act as a Cu-dependent transcription factor [4] that promoted expression of cyclin D1 [5], [6] and SOD3 [7], [8]. ATOX1 was also shown to promote inflammatory neovascularization by acting as a transcription factor for NADPH oxidase [9] and it was essential for platelet-derived growth factor-induced cell migration [10]. Like the other studies, we detected ATOX1 in the nucleus of mammalian HeLa cells, but we found no binding to the proposed DNA promotor sequence in vitro [11]. Nonetheless, from a yeast two hybrid screen, using ATOX1 as ‘bait’, several nuclear and signaling protein partners were detected [12]. Moreover, when we investigated RNA transcript levels of all known Cu-binding proteins in humans, we found upregulation of several Cu-dependent proteins (including ATOX1) in various cancers [13]. Fascinatingly, in aggressive breast cancer cell lines, we discovered that ATOX1 localized to membrane protrusions and aided in cell migration [14].
Maintaining proper Cu levels is essential during pregnancy for normal child development [15] and even temporal nutritional Cu deficiency during pregnancy can result in long-lasting neurological effects on the offspring [5], [15]. Mice in which Atox1 has been knocked out often died after birth, and those that survived had severe malfunctions and malformations, suggesting that ATOX1 is important during embryo development [5]. Before embryo implantation, during the first week after fertilization, the very first developmental steps take place and involve strictly regulated networks of expressed genes.
The first cell division of a pre-implantation embryo depends on maternal transcripts in the oocytes allowing for the maternal-to-embryo transition at the 2-cell stage where reprogramming of gene expression occurs [16]. When reaching the 8-cell stage, the embryo undergoes cell compaction to form the morula in which individual blastomeres increase their cell-cell contacts and localization of cellular proteins become polarized [17]. Subsequent asymmetric cell divisions result in the late morula stage at which the inner cell mass (ICM) and the outer layer of cells, the trophectoderm, TE, are separated. When reaching the late blastocyst stage, the embryo is ready to implant into the wall of the uterus.
There are a number of transcription factors (TF) known to regulate embryo differentiation during early development. If expression of any of these TF genes is perturbed during oogenesis, or in the zygote, most embryos arrest development [18]. The pluripotency TF OCT4, encoded by the Pou5f1 or Oct4 gene, regulates maternal gene networks that are important during the maternal-to-embryo transition [19]. The expression of embryonic OCT4 begins around 8-cell stage and is essential for maintaining the pluripotency of the ICM [20], [21], [22], [23], [24]. The first distinct cell-fate decision involves the spatial separation of TE and ICM cells at the 8- to 16-cell stages. While Oct4 expression is connected to ICM, expression of Cdx2 [25], Tead4 [26] and Eomes [27] genes are connected to TE. The next cell-fate decision occurs at the 16- to 32-cell stages and involves cell sorting from within the ICM to form the epiblast (EPI), a pluripotent population of cells that will later shape the foetus, and the primitive endoderm (PrE), which is a monolayer of blastocoel-facing cells that will contribute to form the yolk sac [28]. Nanog [29] is a TF gene involved in EPI regulation whereas Gata6/4 and Sox2 genes are linked to PrE [30], [31], [32].
Unexpectedly, the Atox1 gene was recently shown to be one of 80 genes directly regulated by OCT4 during the maternal-to-embryo transition [19]. OCT4 was shown to downregulate Atox1 in the oocyte, whereas it upregulated Atox1 expression at the 2-cell stage. To expand on that finding, we here explored the expression of Atox1 in mouse embryos from zygote to late blastocyst stages and, using reported single-cell RNA transcript data, we correlated Atox1 RNA transcript levels with those corresponding to known early embryo TFs. Based on the results, we propose that ATOX1 is a (co)regulator of OCT4 in preimplantation embryos.
Section snippets
Animals handling
All B6D2F1 mice were purchased from a certified supplier (Janvier Labs, France) and kept in the local laboratory animals' facility (EBM, Sweden). Five-week-old females were super-ovulated using previously described [33] protocols, with intra-peritoneal injection of 5 I.U. of the two hormones PMSG (pregnant mare's serum gonadotropin) and hCG (human chorionic gonadotropin, both from Sigma Aldrich, Sweden). The super-ovulating females were immediately mated with male B6D2F1 mice. The presence of a
Presence of Atox1 during first week of embryo development
Zuccoti et al. showed that OCT4 upregulated transcription of Atox1 at the 2-cell stage [19]. Here, we analyzed single-cell RNA transcript data for subsequent stages of pre-implantation mouse embryos taken from Deng at al [35]. In Fig. 1A, the whiskers box-plot data show that Atox1 expression increases dramatically at the early blastocyst stage (8 cells) and then remains high. In Fig. 1B we display the Atox1 expression levels for individual cells and it is clear that the Atox1 transcript levels
Discussion
Recent unprecedented findings have suggested that the cytoplasmic Cu chaperone ATOX1, in addition to its normal Cu transport activity, also acts as direct or indirect TF for several genes [4], [6], [7], [8], [10] as well as facilitates cancer cell migration by accumulating at cell edges [13], [14]. Work in mice have demonstrated that absence of Atox1 results in death or dysfunction of the fetus and, recently, using transcriptomics profiles [19], Atox1 was shown to be among 80 genes directly
Acknowledgement
The Swedish Natural Research Council, the Knut and Alice Wallenberg Foundation, and Chalmers Foundation (PWS) and the Swedish Society for Medical Research provided financial support. The Centre for Cellular Imaging and the Laboratory for Experimental Biomedicine at the Sahlgrenska Academy and the National Bioinformatics Infrastructure Sweden are acknowledged for support.
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