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, 2019, 2130973
eCollection

Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa

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Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa

Seung-Hun Kim et al. Stem Cells Int.

Abstract

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5' upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.

Figures

Figure 1
Figure 1
Schematic illustration of the constructed vector using the porcine OCT4 promoter. (a) EGFP reporter vectors carrying different elements of the pig Oct4 URS were constructed from a pEGFP-N1 vector. One RFP reporter vector carrying a 1.7 kb pig Oct4 URS was constructed from a pDsRed2 vector. (b) Four constructs containing the different elements of the OCT4 promoter in the pGL3-basic vector and the pGL3-basic vector alone as a control were transfected into cells as described in Materials and Methods. DE: distal enhancer; PE: proximal enhancer. The deleted regions are shown as dotted lines.
Figure 2
Figure 2
Mouse embryonic stem cells, embryonic carcinoma cells, and embryonic fibroblasts used in the experiments: (a) ES-E14TG2a mouse embryonic stem cells; (b) P19 mouse embryonic carcinoma cells; (c) mouse embryonic fibroblasts.
Figure 3
Figure 3
Alignment of porcine OCT4 transcriptional regulatory regions with those of five other mammalian orthologs and schematic representation of the porcine OCT4 transcriptional regulatory region. (a) Conserved region 4 (CR4) contains distal enhancer site 2A (DE2A) and the binding domain for Oct4 and Sox2 (Oct4/Sox2). Conserved region 3 (CR3). Conserved region 2 (CR2) contains proximal enhancer site 1B (PE1B). Conserved region 1 (CR1) contains the Sp1/Sp3 site, hormone responsive element (HRE), and three G/C-rich sites. The asterisks show the nucleotides that are identical among the six species. (b) The black line represents the 3.5 kb upstream region (located at positions -3500/-1) of OCT4 in pig. The four upper lines of CR1, CR2, CR3, and CR4 represent the four conserved regions, respectively. The circles show the locations of distal enhancer site elements 2A (DE2A) and 2B (DE2B) and the proximal enhancer site elements 1A (PE1A) and 1B (PE1B). The arrow indicates the translational initiation site of the OCT4 gene.
Figure 4
Figure 4
BlastN analysis and alignment of the putative transcription factor binding sites from mouse and pig using PROMO. (a) BlastN analysis between mouse and pig and human and pig. The color key for alignment scores: red: ≥200, purple: 80–200, and green: 50–80. (b) Putative transcription factor binding sites that exist in mouse sequences, but not in pig sequences. (c) Putative transcription factor binding sites that exist in pig sequences, but not in mouse sequences.
Figure 5
Figure 5
Luciferase reporter analysis of the porcine OCT4 regulatory regions in mouse E14 ES cells, mouse P19 cells, and mouse embryonic fibroblasts. (a) Four constructs containing the different elements of the OCT4 promoter in the pGL3-basic vector were transfected into mouse E14 ES cells, mouse P19 cells, and mouse embryonic fibroblasts. Luciferase activity was measured 48 h after transfection. Different letters indicate significant differences (P < 0.05). Error bars indicate the standard error of the means. (b) Reporter gene expression is tabulated on the right (compared to control): +++, high expression; ++, reduced expression; +, greatly reduced expression; and –, lack of expression in each of the different pluripotent-state cultured cell types, E14TG2a mouse embryonic stem cells, P19 mouse embryonic carcinoma cells, and mouse embryonic fibroblasts. OCT4-Luc: conserved regions 1, 2, 3, and 4 (CR1, CR2, CR3, and CR4); DE-Luc: CR1 and CR4; PE-Luc: CR1, CR2, and CR3; CP-Luc: CR1; pGL3-basic: control.
Figure 6
Figure 6
Comparison of naïve- or primed-state-related gene expression between low- and high-fluorescent cells sorted by fluorescence-activated cell sorting. Comparison of marker gene expression from naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, and Klf2) and primed (Gata6, Mixl1, Fgf5, and Otx2) cells using a dual-reporter assay consisting of DE-GFP (pOCT4-∆PE-eGFP) and PE-RFP (pOCT4-∆DE-DsRed2). (a) Embryonic stem cells sorted by the DE-GFP signal. Low GFP/high GFP means that the expression level of genes in GFP-low-expressing cells is divided by that in GFP-high-expressing cells. (b) Embryonic stem cells sorted by the PE-RFP signal. Low RFP/high RFP means that the expression level of genes in RFP-low-expressing cells is divided by that in RFP-high-expressing cells. (c) Embryonic carcinoma cells sorted by the DE-GFP signal. High GFP/low GFP means that the expression level of genes in GFP-high-expressing cells is divided by that in GFP-low-expressing cells. (d) Embryonic carcinoma cells sorted by the PE-RFP signal. High RFP/low RFP means that the expression level of genes in RFP-high-expressing cells is divided by that in RFP-low-expressing cells. The asterisk indicates that no values were observed.

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References

    1. Nichols J., Smith A. Naive and primed pluripotent states. Cell Stem Cell. 2009;4(6):487–492. doi: 10.1016/j.stem.2009.05.015. - DOI - PubMed
    1. Hanna J. H., Saha K., Jaenisch R. Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues. Cell. 2010;143(4):508–525. doi: 10.1016/j.cell.2010.10.008. - DOI - PMC - PubMed
    1. Hanna J., Cheng A. W., Saha K., et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(20):9222–9227. doi: 10.1073/pnas.1004584107. - DOI - PMC - PubMed
    1. Buecker C., Chen H. H., Polo J. M., et al. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells. Cell Stem Cell. 2010;6(6):535–546. doi: 10.1016/j.stem.2010.05.003. - DOI - PMC - PubMed
    1. Telugu B. P. V. L., Ezashi T., Sinha S., et al. Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos. The Journal of Biological Chemistry. 2011;286(33):28948–28953. doi: 10.1074/jbc.M111.229468. - DOI - PMC - PubMed

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