FOS promoter is overactive outside of genome context and weakly regulated by changes in the Na+i/K+i ratio

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Abstract

Changes in the Na+ and K+ intracellular concentrations affect expression of the FOS gene. Here, we obtained a genetic construct coding for the TurboGFP-dest1 protein under control of the human FOS promoter (−549 to +155) and studied its expression in HEK293T cells exposed to monovalent metal cations. Amplification of the FOS promoter sequence from genomic DNA was efficient only in the presence of Li+ ions. Incubation of cells with ouabain or in a medium containing Li+ ions instead of Na+ ions caused intracellular accumulation of Na+ and Li+ ions, respectively. In addition, both stimuli increased the levels of endogenous FOS mRNA and the average fluorescence intensity of TurboGFP-dest1 in transfected cells. The mRNA levels of TurboGFP-dest1 were significantly higher than the FOS mRNA levels and were little affected by the stimuli.

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About the authors

A. M. Gorbunov

Lomonosov Moscow State University

Email: klimanova.ea@yandex.ru

Department of Biochemistry, Faculty of Biology

Russian Federation, 119234, Moscow

D. A. Fedorov

Lomonosov Moscow State University

Email: klimanova.ea@yandex.ru

Department of Biochemistry, Faculty of Biology

Russian Federation, 119234, Moscow

O. E. Kvitko

Lomonosov Moscow State University

Email: klimanova.ea@yandex.ru

Department of Biochemistry, Faculty of Biology

Russian Federation, 119234, Moscow

O. D. Lopina

Lomonosov Moscow State University

Email: klimanova.ea@yandex.ru

Department of Biochemistry, Faculty of Biology

Russian Federation, 119234, Moscow

E. A. Klimanova

Lomonosov Moscow State University

Author for correspondence.
Email: klimanova.ea@yandex.ru

Department of Biochemistry, Faculty of Biology

Russian Federation, 119234, Moscow

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Supplementary files

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2. Fig. 1. Reporter vectors used to study the activity of the FOS gene promoter region. a – Schematic representation of the pFOS-TurboGFP and pRPLP0-TurboGFP vectors encoding TurboGFP-dest1 under the control of the FOS promoter (from −549 to +155 bp) or the RPLP0 promoter (from −700 to +295 bp). TSS – transcription start site; ATG – start codon. b – Electrophoresis in 1.5% agarose gel stained with ethidium bromide of PCR products of the FOS gene promoter (from −549 to +155 bp) obtained using different buffer solutions: 1 – commercial buffer solution for Encyclo polymerase; 2 – commercial buffer solution for GC-rich sequences; 3 – modified buffer solution in which K+ ions were replaced by Li+ ions. The amplified DNA had introduced restriction sites for subsequent cloning, the expected length of the PCR product was 728 bp.

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3. Fig. 2. Effect of different experimental treatments on intracellular levels of monovalent metal cations in HEK 293T cells. a – Transfection of HEK 293T cells with pFOS-TurboGFP and pRPLP0-TurboGFP vectors increased Na+i levels by 60% and 40%, respectively, and had no significant effect on K+i. The transfection reagent Lipofectamine 2000 alone had no effect on Na+i and K+i levels. b – Incubation of HEK 293T cells in the presence of 1 μM ouabain for 3 h increased Na+i levels by approximately 12-fold and had the opposite effect on K+i. c – HEK 293T cells incubated in Li medium (containing 135 mM Li+ and 5.5 mM K+) for 5 h accumulated 740 nmol/mg Li+ protein and decreased Na+i and K+i levels relative to Na medium (135 mM Na+ and 5.5 mM K+), a control medium simulating the composition of inorganic salts in DMEM. d – Photographs of HEK 293T cells incubated in the presence of 1 μM ouabain for 3 h, in Na medium, or in Li medium for 5 h. Data are presented as mean values ​​(n = 6–8) and box plots with whisker boundaries equal to 1.5 interquartile ranges. Statistically significant differences were identified using the t test or ANOVA followed by Tukey's test (* p < 0.05)

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4. Fig. 3. Expression of the TurboGFP-dest1 reporter protein under the control of the FOS or RPLP0 promoter in HEK 293T cells. a – Confocal micrographs of HEK 293T cells transfected with pFOS-TurboGFP or pRPLP0-TurboGFP. Forty-eight hours after transfection, the cells were incubated in the presence of 1 μM ouabain for 3 h, in Na or Li medium for 5 h. Scale bar: 100 μm. b – Distributions of the mean fluorescence intensity of TurboGFP-dest1 in individual cells (n = 500–1000). Statistical analysis of the data revealed a small but significant increase in reporter protein fluorescence in response to ouabain compared to the control (DMEM). A similar effect was observed in cells incubated in Li-medium compared to Na-medium. Statistically significant differences were identified using the Mann–Whitney U-test (* p < 0.05)

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5. Fig. 4. Amount of endogenous FOS or TurboGFP-dest1 mRNA under the control of the FOS or RPLP0 promoter in HEK 293T cells. HEK 293T cells were transfected with pFOS-TurboGFP or pRPLP0-TurboGFP. Forty-eight hours after transfection, the cells were incubated in the presence of 1 μM ouabain for 3 h, in Na-medium or in Li-medium for 5 h. The level of endogenous FOS or TurboGFP-dest1 mRNA was measured in the cell lysates using quantitative real-time PCR. Data are presented as geometric mean (GM) with whisker ranges from GM × SD (standard deviation) to GM/SD with experimental values ​​superimposed (n = 4). Statistical analysis revealed a significant increase in endogenous FOS mRNA levels in response to ouabain or Li-medium compared to control or Na-medium, respectively. Statistically significant differences were determined using the t-test (* p < 0.05)

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6. Appendix
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