Transcription factor OTX2 silences the expression of cleavage embryo genes and transposable elements.

Upon mammalian fertilization, zygotic genome activation (ZGA) and activation of transposable elements (TEs) occur in early embryos to establish totipotency and support embryogenesis. However, the molecular mechanisms controlling the expression of these genes in mammals remain poorly understood. The 2-cell-like population of mouse embryonic stem cells (mESCs) mimics cleavage-stage embryos with transient Dux activation. In this study, we demonstrated that deficiency of the transcription factor OTX2 stimulates the expression of ZGA genes in mESCs. Further analysis revealed that OTX2 is incorporated at the Dux locus with corepressors for transcriptional inhibition. We also found that OTX2 associates with TEs and silences the subtypes of TEs. Therefore, OTX2 protein plays an important role in ZGA and TE expression in mESCs to orchestrate the transcriptional network.

Zygotic genome activation (ZGA) occurs robustly at the 2-cell (2C) stage of mouse embryos, which is essential for development beyond the 2C stage [1]. During this process, thousands of genes and transposable elements are activated to replace maternally stored RNAs, driving embryonic programs. Among these transcripts, Dux gene [2], zinc finger and SCAN domain containing 4 (Zscan4) clusters [1], and endogenous retroviruses (ERVs) [3] are hallmarks of zygotic genome activation because of their expression specificity [4] and potential functions in totipotency establishment [5, 6]. The Dux gene, which encodes a double-homeodomain protein, is activated at the onset of ZGA in early embryos [7,8,9]. Although Dux is not essential for pre-implantation development, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA [10,11,12]. Despite intensive studies for decades, the regulatory network of ZGA genes is still largely unknown. Embryonic stem cells (ESCs), which are isolated from the inner cell mass (ICM) of blastocysts, can generate any cell type of the fetus but have extremely low efficiency in producing cell types of extraembryonic tissues [13]. In recent years, isolated mouse ESC (mESC)-derived 2C-like cells have been a useful model for exploring totipotency and early embryonic development [14,15,16,17,18,19]. The 2C-like cells express high levels of ZGA transcripts, including the murine endogenous retrovirus (ERV)-L (MERVL) family of retroviruses and Zscan4 genes. In addition, these cells share some key epigenetic characteristics with 2C-stage embryos, such as high histone mobility [20] and chromatin accessibility [21]. Notably, Dux was transiently activated in 2C embryos and 2C-like mESC populations, and Dux knockout in mESCs prevents cells from cycling through the 2C-like state [7, 8].

OTX2 is a member of the OTX family expressed in mouse blastocysts and ESCs [22]. Otx2–/– mice exhibit embryonic lethality due to abnormal gastrulation, and Otx2+/– mice exhibit female-specific lethality and reduced male fertility [23,24,25]. Recently, the Otx2 gene has been suggested to regulate the stem cell state of the ICM and epiblast [22]. The OTX2 protein exhibits heterogeneous expression in ESCs and facilitates the transition into the early primed state [22, 26, 27]. Furthermore, Otx2 is required for mESC transition into epiblast stem cells (EpiSCs) and stabilizes the EpiSC [22]. However, the regulation of zygotic transcribed genes by OTX2 has not been reported.

mESCs are heterogeneous and contain subpopulations with distinct gene expression. A rare mESC subpopulation marked by the reactivation of endogenous retrovirus MERVL, called 2C-like ESCs, has been identified [28]. To examine the regulation of ZGA genes by OTX2 at the transcriptional level, we used mESCs and identified the upregulation of ZGA genes in the absence of the Otx2 gene. Further analysis showed that OTX2 acts as a transcriptional barrier for the zygotic transcriptional program to inhibit Dux expression through incorporation at the Dux locus.

Materials and Methods

Cell culture

mESCs were cultured on irradiated MEFs in N2B27 medium with 2i (PD0325901, 0.4 μM [Stemgent, San Diego, CA, USA] and CHIR99021, 3 μM [Stemgent]) and LIF (1000 u/ml) in tissue culture (TC) dishes pretreated with 7.5 µg/ml poly-L-ornithine (Sigma, St. Louis, MO, USA) and 5 µg/ml laminine (BD Biosciences, San Jose, CA, USA) [26].

RNA-seq dataset analysis

Raw reads [4, 26, 29] were processed with cutadapt v1.16 to remove adapters and perform quality trimming with default parameters, except for quality cutoff and minimum length, which were set at 20 each (GSE56138 and GSE22182). Cufflinks were used to calculate the FPKM values. The ZGA gene list was obtained from a previous report [21], and ZGA genes with abundance in MuERVL+ versus MuERVL– populations of mESCs [4] above 1.5 fold were used for further analysis. Dot and box plots, heatmaps, and bar plots were generated using R. Processed RNAseq datasets of mESC transcriptome before and after Dux overexpression were also used [30].

ChIP-seq and ATAC-seq dataset analyses

Raw reads were processed with cutadapt v1.8.1 to remove adapters and perform quality trimming. Trimmed reads were mapped to the UCSC mm10 assembly using Bowtie with default parameters, and only uniquely aligned sequences were retained. The reported ChIP-seq and ATAC-seq results were obtained from GSE56138 [26], GSE17642 [31], GSE76823 [32], and GSE99746 [33].

Statistical analysis

The Wilcoxon rank sum test with continuity correction was used to calculate P-values and statistical significance was set at P < 0.05.

Results and Discussion

The transcription factor Otx2 has been shown to act as a negative switch in the regulation of the transition from naive to primed pluripotency in mouse pluripotent stem cells [22, 26, 27]. Thus, we carefully examined the transcriptome of Otx2 knockout mESCs [26]. Many typical ZGA genes were significantly upregulated upon Otx2 depletion, including Zscan4d, Sp110, Zfp560, and Zfp352 (Fig. 1A). In early mouse development, the Zscan4 gene family is restrictedly enriched in the 2C stage, and these genes are typical ZGA genes that play important roles in driving the expression of cleavage embryo genes [34]. Here, we show that Zscan4 gene family members are significantly activated when Otx2 is deficient in mESCs (Fig. 1B). We then showed the enrichment of the genes in our ZGA gene list in the MuERVL+ ESC population (Fig. 1C), and we found that the overall expression of ZGA genes was activated after Otx2 depletion (Fig. 1D).

Fig. 1.

Otx2 depletion leads to upregulation of cleavage embryo genes in mouse embryonic stem cells (mESCs). A: Heatmap showing the abundance of representative ZGA genes in control and Otx2 knockout (Otx2-KO) mESCs. B: Heatmap of abundance showing the upregulation of Zscan4 family genes after Otx2 knockout (Otx2-KO) in mESCs. C: Dot-and-box plot depicting the abundance of ZGA genes in MuERVL– and MuERVL+ populations of mESCs. D: Dot-and-box plot depicting the abundance of ZGA genes in control and Otx2 KO mESCs. E: Barplot indicating the log-ratio RNA abundance of reported ZGA genes in Otx2 knockout (Otx2-KO) versus control mESCs. Orange indicates ZGA activators and aqua indicates ZGA repressors. Note that Dux is significantly upregulated after Otx2 knockout in mESCs. F: Barplot indicating the log-ratio RNA abundance of representative maternal factors in Otx2 knockout (Otx2-KO) versus control mESCs. G: Barplot indicating the log-ratio RNA abundance of representative pluripotent factors in Otx2 knockout (Otx2-KO) versus control mESCs.

To identify how ZGA genes are activated in the absence of the Otx2 gene in mESCs, multiple reported ZGA gene regulators were examined, including ZGA activators and repressors [4, 16, 30, 35, 36] (Fig. 1E). For example, a recent study using overexpression screening showed that the maternal factors Dppa2 and Dppa4 activate Dux to drive the expression of downstream ZGA genes [36]. Notably, only the expression of Dux and Zscan4 increased significantly in Otx2 knockout mESCs, while Zscan4 family genes are downstream targets of Dux. Therefore, Otx2 removal in mESCs leads to specific upregulation of Dux, the master activator of ZGA genes.

We next examined the expression of representative maternal factors that were highly enriched in oocytes (Fig. 1F), and we did not find significantly different gene expression after Otx2 depletion. Otx2 has been reported to be an early factor in the differentiation of mESCs, and its activation was correlated with the downregulation of Nanog and Oct4 during ESC differentiation [22]. Otx2, which is required for ESC transition into EpiSCs, stabilizes the EpiSC state by suppressing the mesendoderm-to-neural fate switch together with BMP4 and FGF2 in pluripotent cells [22]. Specifically, OTX2 binding was reported to be significantly enriched in the promoter/enhancer regions of Oct4, Sox2, and Nanog in EpiSCs [37]. Therefore, we also tested the expression of representative pluripotent regulators in mESCs (Fig. 1G) and found limited expression changes in these pluripotent regulators when Otx2 was depleted. Taken together, our analysis showed that Otx2 depletion did not significantly affect the maternal or pluripotency network.

To examine whether Otx2 regulates ZGA regulators and ZGA genes directly, we analyzed the OTX2 ChIP-seq results in mESCs (Fig. 2A). We noticed significant enrichment of OTX2 occupancy at the Dux locus and no signal at Zscan4 or other ZGA gene loci. We also identified similar binding patterns of two corepressors, SETDB1 [18] and RYBP [38], which were reported to inhibit ZGA genes (Fig. 2B). ATAC-seq results at the Dux locus in mESCs (Fig. 2B) also showed a peak at the Dux locus in mESCs. Taken together, our analysis indicates that the Dux locus is bound by OTX2-containing repressing complexes for transcriptional silencing, thereby blocking the activation of Dux-targeted ZGA genes.

Fig. 2.

OTX2 associates with Dux locus. A: Genome browser screenshot of OTX2 ChIP-seq signals at Dux, Zscan4, Zfp352, Zscan4d, and histone loci in mESCs. B: Genome browser screenshot of ChIP-seq and ATAC-seq signals of corepressors at Dux locus in mESCs. C: Scatterplot depicting the co-relationship of Dux overexpression (OE) and Otx2 knockout (Otx2-KO)-mediated changes in ZGA gene expression.

To determine how Dux activity contributes to Otx2-mediated repression of ZGA, we compared the changes in the expression of ZGA genes in Otx2 knockout and Dux-overexpressing mESCs (Fig. 2C). Generally, higher upregulation of ZGA genes upon Otx2 knockout correlates with higher upregulation upon Dux overexpression. Therefore, we propose that Dux overexpression is the leading reason for the activation of ZGA genes in the absence of Otx2.

Transposable elements (TEs) help establish gene regulatory networks both in ESCs and embryonic development, including the long interspersed nuclear elements (LINEs), long terminal repeat (LTR) elements, and short interspersed nuclear elements (SINEs) [4, 39]. Our results showed that the number of reads of some TE subtypes was increased in Otx2 KO mESCs compared to the control (Figs. 3A and B). According to OTX2 ChIP-seq data in mESCs, OTX2 was enriched in TE elements (Fig. 3C). Remarkably, reads at the ERV-K family and LTR retrotransposon (MaLR) in LTR elements accounted for most positions at LTR loci, and L1 accounted for most positions at LINE loci (Fig. 3C). Interestingly, the transition from the 2-cell stage and the development progression to the blastocyst stage depend on LINE-1 expression [14, 40]. In addition, we found that the changes in the L1 family in Otx2 knockout mESCs are not associated with the evolutionary year of L1 (Fig. 3D). These results indicate that OTX2 is also associated with TEs and regulates TE expression.

Fig. 3.

OTX2 regulates the expression of transposable elements (TEs). A: Total reads of TE expression in control and Otx2 knockout (Otx2-KO) mouse embryonic stem cells (mESCs). B: Heatmap of abundance depicting the differentially expressed genes of TE subtypes after Otx2 knockout (Otx2-KO) in mESCs. C: Pie charts depicting the distribution of OTX2 at TEs, including the long interspersed nuclear elements (LINEs), long terminal repeat (LTR) elements, and short interspersed nuclear elements (SINEs) in mESCs. D: Relative expression ratio of Otx2 knockout (Otx2-KO) to control mESCs versus evolutionary ages of L1 subfamilies (left). Enrichment of OTX2 at L1 subfamilies versus evolutionary ages of L1 subfamilies (right).

OTX2 is regarded as a factor that facilitates ESC differentiation. Our results suggest that OTX2 may participate in ESC differentiation by increasing the transient totipotency state of stem cells, which were reported to have higher chromatin opening. During the transition of stem cell conditions, the appearance of transient totipotency status with activated ZGA genes and overall enhanced chromatin accessibility across the genome [21] may be important regulatory factors.

The OTX family of homeobox genes is a vertebrate ortholog of the Drosophila orthodenticle homeobox gene [41]. It is expressed in both early embryos and ESCs in mice (Supplementary Fig. 1: online only) [22, 29, 37]. Otx2 is first detected in the murine blastula [42] and is then restricted to ICM with Nanog expression [37]. Otx2 expression is gradually downregulated in the epiblast, where it is virtually absent from E3.7 to E4.5 [37]. The RNA-seq data showed that Otx2 was undetectable in mouse 2-cell embryos, whereas Otx1 could be detected at this stage. However, Otx2 was detected in ESCs instead of Otx1. This indicates that other OTX family proteins may be essential during development. At late embryonic stages, the expression of Otx1, although more restricted, is detected in the same areas as those of Otx2 [42]. Based on the different expression patterns of OTX family genes, we propose that orthologous genes Otx1 and Otx2 regulate ZGA and TE in early mouse embryos and mESCs, respectively.

Until now, the roles of OTX-family genes in early mouse embryos have been poorly understood. Our finding that the transcription factor Otx2 regulates Dux and TE expression in mESCs provides new clues for the developmental functions of OTX family genes.

Collectively, our analysis shows that Otx2 represses the activation of ZGA genes and subfamilies of TE expression by directly binding to genomic loci of the Dux gene and TE elements (Fig. 4).

Fig. 4.

Scheme of how Otx2 regulates cleavage embryo genes and TE in mESC.

Conflict of interests

The authors declare no conflict of interest.