Evidence that transcription factor AP-2γ is not required for Oct4 repression in mouse blastocysts

Abstract

In mouse blastocysts segregation of the inner cell mass (ICM) and the trophectoderm (TE) is regulated by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2 expressed in the ICM and TE, respectively. In contrast, in other species such as bovine and human, Oct4 is not restricted to the ICM and continues to be expressed in the Cdx2-positive TE. A recent comparative study of the bovine and mouse Oct4 promoters revealed that additional mechanisms might act in conjunction with Cdx2 to downregulate Oct4 expression in the mouse TE lineage. For instance, the mouse Oct4 distal enhancer contains an AP-2γ (Tcfap2c) binding motif that is absent in the bovine and human Oct4 distal enhancer. Nonetheless, the functional relevance of Tcfap2c in Oct4 repression during mouse preimplantation development was not tested. To elucidate the role of Tcfap2c in Oct4 expression an RNA interference approach was utilized. Depletion of Tcfap2c triggered a decrease in Oct4 expression at the 8-cell and morula stage. Remarkably, at the blastocyst stage depletion of Tcfap2c and/or its family member Tcfap2a had no effect on Oct4 repression. To test whether Tcfap2c interacts with Oct4 to positively regulate Oct4 expression, chromatin immunoprecipitation and in situ co-immunoprecipitation analyses were performed. These experiments revealed Tcfap2c and Oct4 binding were enriched at the Oct4 distal enhancer in embryonic stem (ES) cells, but were rapidly lost during differentiation into trophoblast-like cells when Oct4 became repressed. Moreover, Tcfap2c and Oct4 interactions were detected at the morula stage, but were lost during blastocyst formation. In summary, these data demonstrate that Tcfap2c is not required for Oct4 silencing in mouse blastocysts, but may be necessary for the maintenance of Oct4 expression during the 8 cell-to-morula transition. These findings support the notion Cdx2 is the predominant negative regulator of Oct4 expression during blastocyst formation in mice.

Figure 1. Depletion of Tcfap2c downregulates Oct4 expression during preimplantation development.

(A) Validation of siRNA-mediated knockdown (KD) of Tcfap2c transcripts and relative quantification of Oct4, Nanog, and Tead4 transcripts in 8-cell embryos and morulae by qRT-PCR; RQ (Relative Quantification). Three to four biological replicates with 10–20 embryos per replicate were used. (B) Average levels of pixel intensities of Oct4-fluorescent signal in the nuclei of Tcfap2c KD 8-cell embryos and morulae were determined with the ImageJ software and compared to those of control (RI; relative intensities). Three biological replicates with five embryos per replicate were used to measure fluorescence intensities of nuclei in control and Tcfap2c KD embryos (8-cell stage = 120 nuclei/group; morula stage = 400 nuclei/group). (C) Protein expression and localization of Tcfap2c and Oct4 in Tcfap2c KD and control embryos. Error bars represent mean ± s.e.m. Asterisk symbol indicates P<0.05(*) compared with the control group.

Figure 2. Tcfap2c is not required for restriction of Oct4 expression in the blastocyst ICM.

(A) Validation of siRNA-mediated KD of Tcfap2c transcripts and relative quantification of Tcfap2a, Oct4, and Nanog transcripts in blastocysts by qRT-PCR; RQ (Relative Quantification). Three to four biological replicates with at least 10 embryos per replicate were used. (B) Average levels of pixel intensities of Oct4 and Tcfap2c fluorescent signals in Tcfap2c KD blastocysts were determined with the ImageJ software and compared to those of control (RI; relative intensities). Three biological replicates with at least five embryos per replicate were used to measure the intensities of Oct4 and Tcfap2c in nuclei of control and Tcfap2c KD blastocysts. (C) Two representative images of Oct4 expression in Tcfap2c KD blastocysts. Error bars represent mean ± s.e.m. Asterisk symbol indicates P<0.05(*) compared with the control group.

Figure 3. Depletion of Cdx2 inhibits Oct4 repression and restriction to the ICM.

(A) Expression and localization of Tcfap2c and Oct4 in Cdx2 KD (100 or 10 µM Cdx2 siRNA) and control blastocysts. (B) Validation of siRNA-mediated KD of Cdx2 transcripts (100 or 10 µM Cdx2 siRNA), and expression of Oct4 and Tcfap2c in blastocysts by qRT-PCR; RQ (Relative Quantification). Three biological replicates with at least 10 embryos per replicate were used. Error bars represent mean ± s.e.m. Asterisk symbol indicates P<0.05(*) compared with the control group.

Figure 4. Tcfap2c and Oct4 interactions at the CR4 region of Oct4 during early development.

(A) Schematic diagram of the Oct4 genomic region; DE (Distal Enhancer), PE (Proximal Enhancer), CR (Conserved Region), TSS (Transcription start site), Oct/Sox(Oct4/Sox2 binding site), AP2(Tcfap2 binding site); black line and numbers (amplified regions for ChIP-qPCR and sequences from TSS). (B) ChIP analysis of Tcfap2c, Oct4, Tcfap2a binding to CR4 in uninduced ES cells (0 h) and Cdx2 induced TS-like cells (96 h). (C) ChIP analysis of Tcfap2c, Oct4, Tcfap2a binding to CR1/TSS region. (D) A proximity ligation assay (Duolink) in morulae and blastocysts. A subset of embryos were injected with Oct4 siRNA to deplete Oct4. Red dotted fluorescence indicates sites of interaction of Oct4 and Tcfap2c. Two to three biological replicates were used. Error bars represent mean ± s.e.m.