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Estrogen inducibility of VTG-CpGL <t>luciferase</t> reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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Estrogen inducibility of VTG-CpGL <t>luciferase</t> reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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Estrogen inducibility of VTG-CpGL <t>luciferase</t> reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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Estrogen inducibility of VTG-CpGL <t>luciferase</t> reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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Estrogen inducibility of VTG-CpGL luciferase reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Journal: Molecular and Cellular Biology

Article Title: Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding

doi: 10.1128/MCB.00436-19

Figure Lengend Snippet: Estrogen inducibility of VTG-CpGL luciferase reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: The enhancer/promoter region of the chicken vitellogenin II gene, spanning nucleotides −638 to −1, was cloned into a CpG-free firefly luciferase plasmid (pCpGL-basic; InvivoGen) using HindIII and NcoI.

Techniques: Luciferase, Plasmid Preparation, Transfection, Expressing, In Vitro, Methylation, Sequencing, Purification, Quantitative RT-PCR, Isolation, Comparison