Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 5, pp. 582-589 © Pleiades Publishing, Ltd., 2025.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 5, pp. 627-635.

582

FOS Promoter is Overactive Outside of Genome Context

and Weakly Regulated by Changes in the Na

Ratio

Andrey M. Gorbunov

, Dmitrii A. Fedorov

, Olga E.Kvitko

Olga D. Lopina

, and Elizaveta A. Klimanova

1,a

Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

e-mail: klimanova.ea@yandex.ru

Received February 10, 2025

Revised March 31, 2025

Accepted May 13, 2025

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.

DOI: 10.1134/S0006297925600371

Keywords: lithium, sodium, ouabain, FOS, transcription

* To whom correspondence should be addressed.

INTRODUCTION

The gradient of the Na

and K

ions between the

extracellular medium and cytoplasm of animal cells

is essential for the regulation of various cellular func-

tions and is strictly maintained mainly by the activity

of Na,K-ATPase [1]. However, some physiological and

pathological conditions might cause changes in the

intracellular concentrations of these cations, which

could affect expression of certain genes [2], in par-

ticular, the early response gene FOS coding for the

c-Fos transcription factor [3]. The baseline FOS expres-

sion is extremely low, but can increase dramatically

in response to external stimuli. Transcription of FOS

is controlled by multiple regulatory elements, includ-

ing serum-response element (SRE), cAMP-responsive

element (CRE), and activator protein 1 (AP-1) binding

site [4].

As demonstrated in various types of mammalian

cells, the effect of changes in the Na

ratio on the

gene expression is not mediated by the Ca

-depen-

dent signaling or alterations in the membrane po-

tential, intracellular pH, or cell volume [4-6]. Most of

these genes, including FOS, are early response genes

encoding transcription and translation factors [7].

The product of the FOS gene is the AP-1 family pio-

neer transcription factor c-FOS involved in the chro-

matin remodeling [8]. The study aimed at establishing

the nature of the Na

-sensitive regulation of FOS

expression have shown that the promoter regulatory

elements, such as SRE, CRE, and AP-1 binding site, are

not involved in the transcriptional activation of gene

expression driven by the Fos promoter in response to

ouabain (specific inhibitor of Na,K-ATPase) [5]. By us-

ing genetic constructs carrying the human FOS gene

with deletions in the promoter, Nakagawa et al. [9]

were able to identify two sequences responsible for

the ouabain-induced activation of transcription: the

SRE element and a region between −222 and −123 bp

upstream of the transcription start site (TSS) [9]. How-

ever, the authors did not investigate the mechanism

of such regulation. It is important to note that ac-

cording to the bioinformatic analysis, the identified

region (−222 to −123) contained a sequence potentially

capable of forming a G-quadruplex. G-quadruplexes

are non-canonical secondary DNA structures formed

FOS PROMOTER IS HYPERACTIVATED OUTSIDE OF GENOME CONTENT 583

BIOCHEMISTRY (Moscow) Vol. 90 No. 5 2025

by guanine-rich sequences [10] that can bind monova-

lent cations. The ability of monovalent metal ions to

stabilize G-quadruplexes decreases in the following or-

der: K

>Rb

>Na

>Cs

>Li

[11]. The presence of G-qua-

druplexes in the coding sequences and promoters of

some Na

-sensitive genes, including FOS, was pre-

dicted [12]. We hypothesize that these structures may

be involved in the Na

-dependent regulation of

transcription.

To further investigate the role of G-quadruplexes

in the regulation of FOS transcription, we obtained a

genetic construct encoding TurboGFP-dest1 (a desta-

bilized variant of TurboGFP) under control of the hu-

man FOS promoter and studied the effects of ouabain

and Li

ions on the TurboGFP-dest1 expression in

transfected HEK293T cells. Introduction of plasmid

DNA encoding TurboGFP-dest1 into the cells caused

accumulation, resulting in the hyperactivation of

the human FOS promoter (−549 to +155  bp relatively

to the TSS) and reduction of its sensitivity to monova-

lent metal cations.

MATERIALS AND METHODS

Genetic constructs. pFOS-TurboGFP and pR-

PLP0-TurboGFP reporter vectors were constructed

that encoded TurboGFP-dest1 under control of the FOS

or RPLP0 promoters, respectively. The RPLP0 promot-

er was chosen as a control because RPLP0 belongs to

housekeeping genes and its promoter is not sensitive

to changes in the Na

ratio, as well as lacks pre-

dicted G-quadruplexes. The promoter regions of FOS

(−549 to +155 bp) and RPLP0 (−700 to +295 bp) were

amplified by PCR with an Encyclo Plus PCR kit (Euro-

gen, Russia) using genomic DNA from HeLa cells as a

template and primers carrying HindIII and AsiGI re-

striction sites (Online Resource 1). The FOS promoter

was amplified in a buffer proposed by Chashchina [13]

in which KCl was replaced by LiCl (60 mM Tris-HCl,

pH 9.5, 20 mM LiCl, 2.5 mM MgCl

, 5% DMSO). PCR

products were analyzed by sequencing. The FOS and

RPLP0 promoters were cloned into the promoterless

peTurboGFP-PRL-dest1 vector (Eurogen) using HindIII

and AsiGI restriction endonucleases (SibEnzyme, Rus-

sia). Full sequences and maps of the used plasmids

are presented in the Online Resource 1.

Culturing and transfection of HEK293T cells.

HEK293T cells were cultured in Dulbecco’s Modi-

fied Eagle Medium (DMEM; PanEco, Russia) con-

taining 4.5  g/L glucose, 0.58  g/L glutamine (PanEco,

Russia), 10% fetal bovine serum (FBS) (Cytiva, USA),

100 unit/mL streptomycin and penicillin (Life Tech-

nologies, USA) at 37°C in a humidified atmosphere

with 5%  CO

. Cell viability was determined using

alamarBlue reagent (Thermo Fisher Scientific, USA)

according to the manufacturer’s recommendations.

One day before the transfection, the cells were seeded

in 24-well plates (10

cells in 500μl of culture medium

per well) pre-coated with poly-D-lysine (MP Biomed-

icals, USA) and cultured for 24 h. The next day, 2 h

before the transfection, the cells were placed in the

culture medium of the same composition but without

antibiotics (500μl per well), and then transfected with

a mixture of 0.8 μg of plasmid DNA (pFOS-TurboGFP

or pRPLP0-TurboGFP) and 2 μl of Lipofectamine 2000

(Invitrogen, USA) in 50 μl of Opti-MEM (Gibco, USA)

per well and incubated for 24 h at 37°C in a hu-

midified atmosphere and 5%  CO

. The medium was

then replaced with low-serum DMEM (4.5  g/L glucose,

0.58  g/L glutamine, 0.1% FBS) and incubated for 24 h.

Cell treatment. For studying the effect of ouabain

on the Na

ratio, the cells were incubated in the

presence of 1μM ouabain for 3h. For the intracellular

accumulation of Li

ions, the cells were incubated for

5 h in the Li-medium containing 10 mM HEPES-LiOH

(pH  7.4), 130  mM  LiCl, 5.2  mM  KCl, 1  mM  CaCl

0.5  mM  MgCl

, 0.4  mM  MgSO

, 0.3  mM KH

, and

6 mM glucose; Na-medium of the same composition,

except containing 130 mM NaCl instead of 130 mM

LiCl, was used as a control (the composition of the

Na-medium mimicked the inorganic salt composition

of DMEM).

Assessment of the intracellular content of

, K

, and Li

by atomic absorption spectrome-

try. The intracellular ion content was determined ac-

cording to the previously described method [14] with

some modifications. Briefly, at the end of incubation,

the plates were placed on ice. The medium was dis-

carded, after which the cells were washed three times

with 3 ml of ice-cold 0.1  M  MgCl

. Next, 1.5 ml of 5%

trichloroacetic acid (TCA) was added to the wells and

incubated for several hours at 4°C. The contents of

the wells were scraped off, transferred into microcen-

trifuge tubes, and centrifuged for 10 min at 18,000g.

The supernatant was collected into separate tubes,

the residual TCA was aspirated, and the precipitate

was dissolved in 0.1 M NaOH containing 0.1% sodi-

um deoxycholate. The amount of protein was deter-

mined by the Lowry method [15]. The intracellular

content of Na

, Li

, and K

ions was measured by

atomic absorption spectrometry using a Kvant-2m1

spectrometer (Kortek, Russia) in a propane-air mix-

ture at 589.6, 670.8, and 766.5 nm, respectively. NaCl

(0.05-2  mg/L  Na

), LiCl  (0.05-2  mg/L  Li

), and KCl

(0.5-2  mg/L  K

) solutions were used as standards.

The contents of Na

, Li

, and K

in each sample were

normalized to the protein content.

Laser scanning confocal microscopy and im-

age analysis. HEK293T cells were transfected with

pFOS-TurboGFP or pRPLP0-TurboGFP and subject-

ed to experimental treatments as described above.

GORBUNOV et al.584

BIOCHEMISTRY (Moscow) Vol. 90 No. 5 2025

Cell microscopy was performed with an Olympus

IX83P2ZF microscope (Japan) in the confocal imag-

ing mode (diameter of confocal membrane aperture,

130 μm) at 37°C in 5% CO

in a live-cell incubation

chamber (Tokai Hit, Japan) using a 10× objective with

a numerical aperture of 0.4. Fluorescence emission

was excited with a 488-nm laser (absorption max-

imum of turboGFP-dest1, 482 nm) and registered at

490-540nm. The images acquired were processed with

the Fiji/ImageJ 1.54f program. Individual cells were se-

lected using “masks” by setting a critical intensity lev-

el to visually distinguish them from the background,

and the average fluorescence intensity within the se-

lected cells was calculated.

Analysis of pFOS-TurboGFP and pRPLP0-

TurboGFP transcription by qPCR. The plates with

the cells were placed on ice, the cells were washed

with 1 ml of ice-cold Ca

- and Mg

-free Hank’s salt

solution, and lysed by adding 400μl of Trizol reagent

(Invitrogen) to each well. The lysates were treated

with a chloroform-mixture; the aqueous phase was

collected and used for RNA isolation with a Qiagen

RNeasy kit (Netherlands). Reverse transcription was

performed with an ImProm-II™ Reverse Transcription

System kit (Promega, USA) according to the manufac-

turer’s recommendations (0.5 μg of isolated RNA per

reaction). qPCR was performed with a Bio-Rad Real-

Time PCR System (Bio-Rad, USA) in the following re-

gime: denaturation at 95°C for 5 min followed by 40

cycles of denaturation at 95°C for 10s, primer anneal-

ing at 58°C for 10 s, and elongation at 72°C for 20 s.

The primers were selected using the BLAST NCBI da-

tabase and synthesized by Syntol (Russia). The primer

sequences are given in the Online Resource 1. Gene

expression levels were calculated by the ∆∆Ct method

using the RPLP0 gene as a reference [16]. The average

level of FOS expression in control samples was taken

as 100%. PCR products were analyzed by sequencing.

Statistical analysis. The Origin software pack-

age (OriginLab Corporation, USA) was used for statis-

tical data processing. The samples were tested for the

normality of distribution using the Shapiro–Wilk test.

To compare two independent groups, the Student’s

t-test for normally distributed samples and the Mann–

Whitney U-test for non-normally distributed samples

were used. Multiple comparisons for normally distrib-

uted data were performed using the one-factor analy-

sis of variance (ANOVA) followed by the Tukey’s test.

The differences were considered statistically signifi-

cant at p < 0.05.

RESULTS AND DISSCUSSION

To investigate the mechanism of Na

-depen-

dent regulation of transcription, we constructed the

pFOS-TurboGFP and pRPLP0-TurboGFP reporter vec-

tors encoding TurboGFP-dest1 under control of the

human FOS promoter (−549 and +155) and RPLP0 pro-

moter (−700 and +295bp, no predicted G-quadruplex-

es), respectively (Fig. 1a). The sequences of the FOS

and RPLP0 promoters were amplified using genom-

ic DNA from HeLa cells as a template. Importantly,

we were able to amplify the RPLP0 promoter using

commercial buffer solution for Encyclo polymerase,

while amplification of the FOS promoter under these

conditions was unsuccessful. For this reason, we used

a buffer for GC-rich sequences for the FOS promoter

amplification; however, the length of the resulting PCR

product was shorter than the expected one by 78 bp

(Fig.1b), presumably, due to the presence of G-quadru-

plexes in the FOS promoter sequence. G-quadruplexes

are known to disrupt PCR in the presence of K

ions,

and most commercial buffers for DNA polymerases

contain K

ions. Hence, we used a modified buffer

solution suggested by Chashchina et al. [13] contain-

ing Li

ions instead of K

ions, to successfully amplify

the FOS promoter sequence.

Next, we evaluated the effect of transfection itself

on the cell intracellular ion composition. Figure 2a

shows that lipofection with pFOS-TurboGFP or

pRPLP0-TurboGFP (but not the treatment with Lipo-

fectamine 200 alone) was accompanied by a 40-60%

increase in the Na

concentration even 48 h after

transfection. There are at least two factors that may be

responsible for this effect. First, ion transport could be

affected by the induction of interferon expression by

foreign AT-rich DNA via an RNA polymerase III-depen-

dent pathway [17]. On the other hand, expression of

TurboGFP-dest1 could influence various intracellular

functions. For example, TurboGFP-dest1 was found to

affect the NF-κB signaling pathway in T cells, HEK293

cells, and HeLa cells [18]. It is possible that TurboG-

FP-dest1 expression can have an impact on the ion

transport as well. However, the precise mechanisms

of this phenomenon remain unclear.

To compare the effects of Li

and Na

ions on

the FOS promoter activity, the cells were incubated in

DMEM containing 0.1% FBS in the presence of 1 μM

ouabain or in the Li-medium containing 135 mM Li

Figure 2b shows that incubation in the presence of

1 μM ouabain for 3 h caused an 11.5-fold decrease

in the K

concentration and an 11.8-fold increase in

the Na

concentration in the cells. Incubation in the

Li-medium for 5  h led to the intracellular accumula-

tion of Li

(to 740  nmol/mg protein), while the con-

centration of Na

and K

decreased by 85 and 90%,

respectively, compared to the cells incubated in the

Na-medium (Fig.2c). Exposure to ouabain or Li-medi-

um did not affect the cell viability (data not shown),

although the morphology of cells in the Li-medium

was slightly altered (Fig. 2d).

FOS PROMOTER IS HYPERACTIVATED OUTSIDE OF GENOME CONTENT 585

BIOCHEMISTRY (Moscow) Vol. 90 No. 5 2025

Fig. 1. Reporter vectors used to study the FOS promoter activity. a)pFOS-TurboGFP and pRPLP0-TurboGFP vectors en-

coding TurboGFP-dest1 under control of the FOS promoter (−549 to +155bp) and RPLP0 promoter (−700 to +295bp),

respectively. b) Electrophoresis of the FOS promoter amplification products obtained by PCR with different buffer

solutions in 1.5% agarose gel stained with ethidium bromide: 1,commercial buffer solution for Encyclo polymerase;

2,commercial buffer solution for GC-rich sequences; 3,modified buffer solution with K

ions substituted by Li

ions.

The amplified DNA fragments contained HindIII and AsiGI restriction nuclease sites for subsequent cloning; expected

length of PCR product, 728 bp; TSS, transcription start site.

To assess the activity of the FOS promoter in trans-

fected cells, we measured the fluorescence intensity

of TurboGFP-dest1 using confocal microscopy, as well

as determined the levels of TurboGFP-dest1 mRNA by

qPCR. Figure 3 shows that the median fluorescence

intensity in the cells transfected with pFOS-TurboGFP

and incubated in the presence of 1 μM ouabain or

(Li-medium) increased by 21 and 11%, respective-

ly, while no significant changes in the fluorescence

intensity were observed for the cells transfected with

pRPLP0-TurboGFP. However, contrary to our expecta-

tions, the fluorescence intensity of cells transfected

with pFOS-TurboGFP and pRPLP0-TurboGFP was very

similar, despite the fact that the levels of endoge-

nous RPLP0 mRNA (Ct  =  18.4  ±  0.12) were orders of

magnitude higher compared to the levels of endog-

enous FOS mRNA (Ct  =  28.3  ±  0.07). No significant

differences were found between the TurboGFP-dest1

gene transcription levels (control vs. ouabain-treated

cells and Na-medium vs. Li-medium samples) in case

of both vectors. Incubation of cells transfected with

pFOS-TurboGFP in the presence of ouabain and in

the Li-medium increased the content of endogenous

FOS mRNA 17 and 10 times, respectively (Fig.  4).

It is important to note that introduction of plas-

mid DNA into the cells was accompanied by the in-

crease in the content of Na

ions, but had no effect

on the amount of endogenous FOS mRNA (data not

shown). This indicates that the elevated expression

of TurboGFP-dest1 under the FOS promoter was not

related to the experimental conditions, but rather re-

sulted from the dysregulation of the FOS promoter

activity and/or the presence of the TurboGFP-dest1

coding sequence in the used constructs. The levels of

TurboGFP-dest1 mRNA were 1000-3000 times higher

than the levels of endogenous FOS mRNA in all ex-

perimental and control samples. We believe that these

results indicate overactivation of the FOS promoter

in the pFOS-TurboGFP vector, so that its activity was

comparable to that of promoters of the housekeep-

ing genes (e.g., RPLP0). This might be the reason why

the observed effect of the experimental treatments

on the activity of the FOS promoter in the content

of the reporter vector was insignificant.

Taking into account the above data, we can con-

clude that the plasmid carrying the coding sequence

for TurboGFP-dest1 under control of the FOS promoter

is not suitable for studying the role of G-quadruplexes

GORBUNOV et al.586

BIOCHEMISTRY (Moscow) Vol. 90 No. 5 2025

Fig.  2. Effect of different experimental treatments on the intracellular content of monovalent metal cations in

HEK293T cells. a)Transfection of cells with the pFOS-TurboGFP and pRPLP0-TurboGFP vectors increased the content

of Na

by 60 and 40%, respectively, but had no significant effect on the content of K

. Transfection with Lipofect-

amine 2000 alone did not affect the Na

and K

content. b)Incubation of cells in the presence of 1μM ouabain for

3 h resulted in ~12-fold increase in the Na

content, but significantly decreased the content of K

. c) Incubated of

cells in the Li-medium (135 mM Li

, 5.5 mM K

) for 5 h led to a considerable intracellular accumulation of Li

(up

to 740 nmol/mg protein) and reduction in the Na

and K

content compared to cells incubated in the Na-medium

(135 mM Na

and 5.5 mM K

). d) Phase-contrast images of cells incubated in the presence of 1 μM ouabain for 3 h,

in Na-medium for 5 h, or in Li-medium for 5 h. The data are presented as individual experimental values (n =6-8)

and box plots with whiskers equal to 1.5 interquartile range. Statistically significant differences were identified

using the t-test or ANOVA followed by the Tukey’s test (*  p < 0.05).

FOS PROMOTER IS HYPERACTIVATED OUTSIDE OF GENOME CONTENT 587

BIOCHEMISTRY (Moscow) Vol. 90 No. 5 2025

Fig. 3. Expression of the TurboGFP-dest1 reporter protein under control of the FOS or RPLP0 promoters. a)Confocal

images of cells transfected with pFOS-TurboGFP and pRPLP0-TurboGFP. Two days after transfection, the cells were

incubated in the presence of 1 μM ouabain for 3 h, in Li-medium for 5 h, or in Na-medium for 5 h. Scale bars:

100 µm. b) Distribution of TurboGFP-dest1 mean fluorescence intensity in individual cells (n = 500-1000). Statisti-

cal analysis revealed a small but significant increase in the reporter protein fluorescence in response to ouabain

compared to the control (DMEM). A similar effect was observed in the cells incubated in the Li-medium compared

to the Na-medium. Statistically significant differences were detected using the Mann–Whitney U-test (* p < 0.05).

in the regulation of FOS expression by monovalent

cations. Apparently, it is necessary to create a genetic

construct that, in addition to the FOS promoter and

5′-UTR, would include the FOS-coding sequence and

the 3′-UTR rather than the reporter gene.

The effect of Na

ratio on the FOS promot-

er activity has been studied before. Using a vector

containing the firefly luciferase gene under control

of the FOS 5′-UTR (−1264 to +103 bp), Haloui et al.

found that ouabain did not affect the expression of

luciferase [4], which is consistent with our results. In

another study, ouabain caused a significant increase

in the FOS transcription level (in this case, the full-

length human FOS gene was used) [9]. Based on the

results of these studies and our findings, we hypoth-

esize that regulatory elements in the FOS coding se-

quence may deactivate transcription during the cell

resting state. The first intron of the FOS gene con-

tains a strong transcription elongation block (+363 to

+387 bp) [19-22], which suppressed the basal expres-

sion of not only the FOS promoter, but also of the

“strong” metallothionein-2 gene promoter placed in

the vector together with the FOS coding sequence [21].

It is likely that the high activity of the FOS promot-

er in plasmid DNA can be explained by the absence

of regulatory elements normally located at a distance

from the FOS gene in the genome or by the differenc-

es in the modification of plasmid and genomic DNAs.

GORBUNOV et al.588

BIOCHEMISTRY (Moscow) Vol. 90 No. 5 2025

Fig.  4. Expression of endogenous FOS mRNA and

TurboGFP-dest1 mRNA under control of the FOS and

RPLP0 promoters in cells transfected with the pFOS-

TurboGFP or pRPLP0-TurboGFP vectors. Two days after

transfection, the cells were incubated in the presence

of 1 μM ouabain for 3 h, in Li-medium for 5 h, or in

Na-medium for 5 h. The levels of endogenous FOS and

TurboGFP-dest1 mRNAs were measured in cell lysates by

qPCR. The data are presented as geometric mean (GM)

with whiskers from GM×SD to GM/SD and individual ex-

perimental values (overlapping; n = 4). Statistical analy-

sis revealed a significant increase in the levels of endog-

enous FOS mRNA in response to ouabain and Li-medium

compared to the control and Na-medium, respective-

ly. Statistically significant differences were detected

using the t-test (* p < 0.05).

It is also important to take into account possible

differences in the post-transcriptional regulation of

the TurboGFP-dest1 and FOS expression, for exam-

ple, different stability of the reporter protein mRNA.

However, these assumptions need further investigation.

CONCLUSIONS

Using a genetic construct encoding TurboGFP-dest1

under control of the FOS promoter, we demonstrated

that ouabain increased the activity of the FOS promot-

er in HEK293T cells and caused intracellular accumu-

lation of Li

ions. The levels of the TurboGFP-dest1

gene expression in the cells transfected with the ob-

tained construct were extremely high and insensitive

to changes in the intracellular ion composition. It is

likely that repression of the FOS expression during

the cell resting state requires regulatory elements

located outside of the FOS promoter region. We also

found that lipofection of HEK293T cells with the ob-

tained genetic constructs resulted in the Na

accumu-

lation, which may be due to the activation of cellular

response to foreign DNA or a nonspecific effect of

TurboGFP-dest1 on the ion transport.

Supplementary information. The online version

contains supplementary material available at https://

doi.org/10.1134/S0006297925600371.

Contributions. E.A.K. developed the concept, su-

pervised the study, and edited the manuscript; A.M.G.,

D.A.F., and O.E.K. planned and performed experiments

and prepared the manuscript; O.D.L. developed the

study conception and edited the manuscript.

Funding. The study was conducted under the

State Assignment to the Lomonosov Moscow State Uni-

versity.

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest.

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