Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells
Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional...
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Інститут молекулярної біології і генетики НАН України
2009
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| Cite this: | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells / А.М. Slonchak, А. Cwieduk, J. Rzerzowska-Wolny, M.Yu. Obolenskaya // Біополімери і клітина. — 2009. — Т. 25, № 3. — С. 210–217. — Бібліогр.: 22 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859643292722921472 |
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| author | Slonchak, A.М. Cwieduk, A. Rzerzowska-Wolny, J. Obolenskaya, M.Yu. |
| author_facet | Slonchak, A.М. Cwieduk, A. Rzerzowska-Wolny, J. Obolenskaya, M.Yu. |
| citation_txt | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells / А.М. Slonchak, А. Cwieduk, J. Rzerzowska-Wolny, M.Yu. Obolenskaya // Біополімери і клітина. — 2009. — Т. 25, № 3. — С. 210–217. — Бібліогр.: 22 назв. — англ. |
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| description | Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational. We concentrated our attention on the transcriptional level of regulation. Methods. Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors, interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied. Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-kB to the cognate site and ERb in complex with unknown protein to the ARE site; the complex of ERb with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ERb with GSTP1 CRE site and indirect interaction of ERb with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-kB and ARE sites and the negative one via CRE site of the promoter. ERb is indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos with CRE site and via unknown protein with ARE site.
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ÃÅÍÎ̲ÊÀ, ÒÐÀÍÑÊÐÈÏÒÎ̲ÊÀ ² ÏÐÎÒÅÎ̲ÊÀ
Crosstalk between transcription factors in regulation of
the human glutathione S-transferase P1 gene expression
in Me45 melanoma cells
A. M. Slonchak, A. Cwieduk1, J. Rzerzowska-Wolny1, M. Yu. Obolenskaya
Institute of Molecular Biology and Genetics NAS of Ukraine
150, Zabolotnogo str., Kyiv Ukraine, 03680
1M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Gliwice
Wybrzezhe Armiji Krajowej 15, 44-101, Gliwice, Poland
elephass@gmail.com
Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant
expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The
GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and
post-translational. We concentrated our attention on the transcriptional level of regulation. Methods.
Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the
control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter
regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors,
interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied.
Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-kB to the cognate site
and ERb in complex with unknown protein to the ARE site; the complex of ERb with c-Fos negatively
regulates the gene expression via CRE site. The interaction of c-Fos/ERb with GSTP1 CRE site and indirect
interaction of ERb with GSTP1 ARE were identified. Conclusions. The positive regulation of the human
GSTP1 gene in Me45 melanoma cells is exerted via NF-kB and ARE sites and the negative one via CRE site
of the promoter. ERb is indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos
with CRE site and via unknown protein with ARE site.
Keywords: glutathione S-transferase, promoter, transcription factors, NF-kB, estrogen receptor,
melanoma, transcription regulation.
Introduction. Glutathione S-transferases comprise a
multigene superfamily of enzymes that catalyze the
conjugation of electrophilic toxic compounds with
glutathione, playing a key role in phase II of
detoxification [1]. The human GSTP1 isoform is a
major GST isoenzyme in most cell types, except hepa-
tocytes [2]. Besides its typical role in detoxification it
possesses other functions, including a ligandin
function [3], modulation of signaling pathways [4],
conjugation and transport of steroid hormones [5], and
participates in dinitrosyl-diglutathionyl-iron complex
storage and metabolism [6]. Aberrant expression of
GSTP1 is associated with carcinogenesis and develop-
ment of multidrug resistance (MDR).
The regulation of the GSTP1 gene expression is in
the focus of researchers and clinicians interests because
the stimulation of GSTP1 expression is expected to be
used as a preventive approach against cancer while its
down-regulation is in need to counteract the develop-
ment of MDR. The GSTP1 gene expression is regulated
at multiple levels including transcriptional, post-trans-
criptional and post-translational [7]. We concentrate
210
ISSN 0233-7657. Biopolymers and Cell. 2009. Vol. 25. N 3
Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2009
our attention on the transcriptional level of regulation.
Despite the vast literature devoted to GSTP1 enzyme
the functional characteristics of responsive elements in
gene promoter and tissue-specific peculiarities of their
regulation are poorly understood. Moreover the pre-
vious investigations of molecular mechanisms involved
in the GSTP1 regulation were focused mainly on breast
cancer, leukemia and prostate cancer cells. In present
research we performed a functional analysis of GSTP1
promoter in human melanoma cells Me45. We utilized
truncated promoter constructions to compare the
functional role of different cis-acting promoter ele-
ments and identified transcription factors binding the
responsive elements by competitive EMSA (electro-
phoretic mobility shift assay) and supershift assay.
Matherials and methods. Cell culture. Human
melanona cell line Me45 was obtained from Polish Cell
Bank and propagated in DMEM/F12 medium («Sig-
ma», USA) supplemented with 588 mg/ml L-glutamine,
0.16 % NaHCO3, 10 % heat inactivated fetal calf serum
(«Gibco», USA) and 100 mg/ml gentamicine. Cells
were grown at 37 °C in an atmosphere of 95 % air and
5 % CO2.
Promoter deletion constructs. Fragments of GSTP1
gene promoter were prepared by PCR. The oligonuc-
leotide 5'-ACTCACTGGTGGCGAAGACT-3' (positi-
on +15 to +35) was used as the downstream primer for
all constructions. Each of the following oligonucleoti-
des was used as upstream primers to amplify promoter
fragments: 5'-CATAAACACCA- ACCTCTTCCCC-3'
(position –1379 to –1357) for pGSTP1415, 5'-ATAGC-
CTAAGGCACAGCCAC-3' (position –1162 to –1142)
for pGSTP1197, 5'-TTTCCTTTCCTCTAAGCGGC-3'
(position –405 to –385) for pGSTP440, 5'-AGTCCGC-
G GGACCCTCCAGA-3' (position –105 to –85) for
pGSTP140 and 5'-AGAGCGGCCGGCGCCGTGAC-
3' (position –85 to –64) for pGSTP120. The amplified
products were subcloned into pCR®2.1-TOPO® vector
(«Invitrogen», USA). The recombinant plasmids were
sequenced and the orientation of inserts was deter-
mined. Plasmids with directly oriented inserts were
submitted to digestion with KpnI and XhoI. Excised in-
serts were religated into pGL3-basic (plasmid with
GloTM Lu- ciferase 3 basic) plasmid («Promega», USA).
Resulted constructs were named pGSTPX, were X cor-
responds to the length of the inserted promoter frag-
ment and GSTP is the gene name. Sequences of relevant
regions of the final constructs were confirmed by sequ-
encing in both directions in OIigo.pl DNA IBB PAN
Service (Poland).
Transient transfection assay. Me45 cells were
grown in 24-well plates to 60 % confluence and trans-
fected with 500 ng of pGSTP together with 25 ng of
pRL-TK (plasmid with Renilla Luciferase and Thómi-
dine kinase Promoter) plasmid («Promega», USA) per
well using LipofectamineTM LTX and PLUSTM reagents
(«Invitrogen», USA). After 20 h the firefly and renilla
luciferase activities were assessed using Dual Lucife-
rase® Reporter Assay System («Promega», USA).
Electrophoretic mobility shift assay. Nuclear
extracts from Me45 cells were prepared by modified
method of Dignam et al. [8]. The following oligo-
nucleotides and their complementary sequences were
used as probes in EMSA experiment: ARE (Anti-
oxidant Response Element of human GSTP1 promo-
ter) 5'-CGCCGTGACTCAGCACTGGG-3', NF-kB-
like (Nuclear Factor kB-like site of human GSTP1 pro-
moter) 5'-TCCGCGGGACCCTCCAGAAG-3', NF-kB
(Nuclear Factor kB site of human GSTP1 promoter)
5'-CTTAGGGAATTTCCCCCCGC-3', CRE (Cyclic
AMP Response Element site of human GSTP1 promo-
ter) 5'-GAGACTACGTCATAAAATAA-3', GATA
(GATA-1 binding site of human GSTP1 promoter)
5'-GAGATCAATATCTAGAAATAA-3'. Probes
(10 pmoles) were labeled with 20 pmoles [g-32P]-ATP
6000 Ci/mmole («Hartmann Analytic», Deutschland)
by polynucleotide kinase («Roche», Switzerland).
Unincorporated nucleotides were removed by
gel-filtration through Bio-gel® P-30 («Bio-Rad»,
USA). Electrophoretic mobility shift assay was
performed using Electropforetic mobility shift assay kit
(«Promega»). Consensus oligonucleotides for AP-1
(Activator Protein 1), NF-kB (Nuclear Factor kB),
CREB (Cyclic AMP Response Element Binding
protein), GATA, ER (Estrogen Receptor) and RAR
(Retinoic Acid Receptor), antibodies against human
c-Jun, c-Fos, MafF/G/K, ERb , Nrf3 (Nuclear erythroid
2 p45 related factor 3), NF-kB p50, NF-kB p65 and
normal rabbit IgG were from «Santa Cruz Bio-
technology» (USA).
Results and discussion. Functional analysis of the
GSTP1 promoter regions in Me45 cells. The structure
211
CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS
of GSTP1 promoter is summarized in fig. 1. To identify
the role of GSTP1 promoter regions in regulation of
GSTP1 transcription in Me45 cells we utilized transient
transfection assay with reporter constructs containing
complete or truncated GSTP1 promoter fused to the
firefly luciferase gene. For this purpose we designed
the reporter constructs each lacking the DNA fragment
with one transcription factor binding site (fig. 2). The
diagram in fig. 2 represents relative firefly luciferase
activities in lysates of Me45 cells transfected with re-
porter constructs. Each bar in the graph represents the
average of 3 independent experiments with triplications
in each.
Transfection of the largest vector (pGSTP1415)
containing the GSTP1 promoter fragment from –1379
to +35 resulted in relatively high level of f-luc gene
expression in Me45 cells. Deletion of the GSTP1-
flanking region between –1379 and –1162, containing
GATA-binding site, did not influence significantly the
expression of the reporter gene. Deletion of the region
from –1162 to –405, which contains CRE and ATA-
AA-repeat, resulted in increase of f-luc expression
approximately 1.8-fold in comparison with previous
construct. Further deletion of the region from –405 to
–105, containing NF-kB site, reduced the reporter gene
expression 1.6-fold. Deletion of the region from –105
to –85, known as an NF-kB-like element, resulted in
1.5-fold increase of f-luc expression.
Thereby, the results of the transient transfection
experiments suggest the presence of the negative
regulatory elements located in the regions from –1162
to –405 and from –105 to –85. Also it provided the
evidence for the presence of the strong positive
regulatory element located from –405 to –105. The
212
SLONCHAK A. M. ET AL.
Fig. 1. Structure of the human GSTP1 gene 5'-regulatory region and potential transcription factors interacting with it [9–13]: «+» – positive
regulation; «–» – negative regulation; g – general transcription factors
Fig. 2. Schematic representation of the reporter constructs and their activities in transfected Me45 cells. Relative luciferase activity was
calculated as a ratio of firefly to renilla luciferase light emission. Cells cotransfected with pGL3-basic and pRL-TK vectors were as a
negative control
similar role of promoter sequences in the regulation of
GSTP1 gene transcription was identified by Jhaveri and
Morrow [14] for breast cancer cells.
The study of ARE, NF-kB-like, NF-kB, CRE and
GATA binding sites interactions with nuclear proteins
from Me45 cells. For identification of the transcription
factors interacting with the GSTP1 promoter the
electrophoretic mobility shift assay (EMSA) was
applied. The ability of 20 bp promoter fragments,
containing ARE, NF- kB-like, NF-kB, CRE and GATA
sites to bind nuclear proteins from Me45 cells was
examined in this experiment. Fig. 3 shows that all
oligonucleotides form complexes with nuclear
proteins. Specificity of the protein binding was as-
sessed in a competition experiment, in which nuclear
proteins were preincubated in 50- and 100-fold molar
excess of unlabeled probe. In this experiment we
determined, that ARE, NF-kB and CRE sites
specifically bind nuclear proteins while NF-kB-like and
GATA sites do not. One band observed in all
elrctrophoregrams was non-specific because it was not
eliminated in competitive experiments (fig. 3, A, B, C).
Surprisingly, we did not find any proteins in-
teracting with NF-kB-like element which was iden-
tified as a negative regulator of GSTP1 transcription in
the transient transfection experiment. We suppose that
the «negative» role of the NF- B-like element in GSTP1
transcription may be connected with the presence of
palindrome GGGACCCtc in the region that may hinder
an enchanceosome formation.
The region spanning nucleotides from –85 to +35
which is shown to be able to support the transcription of
the reporter gene in Me45 cell at the level even higher
than the full-length promoter is known to be a minimal
promoter essential for the GSTP1 gene expression. This
minimal promoter region contains ARE site which
interacts with different transcription factors – AP-1
[15], Nrf2 [16], ERb [17] and RARa [18], depending
on cell type. To identify the transcription factors acting
on this site in Me45 cells we performed competitive
EMSA with consensus oligos for AP-1, Maf (the
DNA-binding component of Nrf2), ERb and RARa
and supershift assay with antibodies for these trans-
cription factors. Consistent with results shown in fig. 4,
A, a 50- and 100-fold molar excess of unlabeled con-
sensus oligonucleotides for AP-1, Maf, estrogen
receptor beta (ERb) and retinoic acid receptor (RAR)
were not able to compete for the nuclear proteins
binding to the ARE site. It means that AP-1, Maf, ERb
and RARa do not interact with ARE site through their
DNA-binding domains. To clarify these results the
supershift experiment with polyclonal antibodies to
c-Jun (cross-reactive to JunB and JunD), c-Fos
(cross-reactive to FosB, Fra1 and Fra2), MafF/G/K, ER
b and Nrf3 (the placenta-specific homolog of Nrf2) was
performed. As indicated in fig. 4, A, neither
transcription factors Jun, Fos nor Maf and Nrf3 prevent
the formation of specific complex of ARE site with a
nuclear protein. Only ERb antibody prevents the whole
complex formation resulting in appearance of a new
complex with higher electrophoretic mobility. This
result clearly indicates that in Me45 nuclear extracts ER
b binds to the GSTP1 ARE site through another yet
unknown protein and DNA-binding domain of ERb is
not involved in these interactions.
The region of GSTP1 promoter from –405 to –105
contains NF-kB site and positively regulates the
reporter gene transcription in Me45 cells. This site
binds NF-kB in K562 leukemia cells and mediates the
gene induction by TNFa [11]. The results of the
GSTP1 promoter NF-kB site binding assay are
213
CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS
Fig. 3. In vitro binding of Me45 nuclear proteins to GSTP1 promoter
sites: A – electrophoretic mobility shift assay, demonstrating the
ability of Me45 nuclear proteins to form complexes with ARE, NF-
kB, NF-kB-like, CRE and GATA sites; B – results of competitive
EMSA demonstrating, that protein binding to NF-kB-like site is
nonspecific; C – results of competitive EMSA demonstrating, that
protein binding to GATA site is nonspecific; S – specific complex;
NS – nonspecific complex
summarized in fig. 4, B. Two specific bands were
observed in the reaction of genuine NF-kB site
containing oligo with nuclear extract. The unlabeled
NF-kB consensus was able to efficiently compete for
the nuclear proteins from both specific complexes
leading to suggestion that NF- kB binds to GSTP1
NF-kB site in this cell line. To clarify the matter,
nuclear extract was incubated with polyclonal
antibodies to p50 and p65 subunits of NF- kB before the
probe was added to the EMSA reaction. In the
supershift assay of NF-kB site two new bands were
observed after the incubation with p50 antibody – one
originated from the lower and one from the upper
complex, providing the evidence that both complexes
contain p50. The upper complex of nuclear proteins and
NF-kB site contains the p50/p65 heterodimer, while the
lower complex observed in binding reaction is the
p50/p50. These data together with the results of
transient transfection assay strongly suggest that NF-
kB interacts with the human GSTP1 NF-kB site and
up-regulates gene transcription in Me45 cells.
The negative regulatory element –1162 … –404
contains a CRE site and ATAAA-repeated sequence. It
was previously reported that CRE site of GSTP1
mediates gene response to cAMP by interacting with
CREB in Calu-6 lung cancer cells [19]. Competitive
EMSA was also conducted to determine which protein
is a part of the DNA-protein complex formed by CRE
214
SLONCHAK A. M. ET AL.
Fig. 4. Analysis of the complexes formed by ARE, NF- B and CRE sites
from the human GSTP1 promoter: A – ARE-protein complex formation
was inhibited by unlabeled ARE site (cold probe) and by ERb antibody;
B – NF-kB site forms two complexes with the nuclear proteins from Me45
cells; both complexes were disrupted by the cold probe and NF-kB
consensus and supershifted by p50 antibody; p65 antibody disrupted only
the upper complex; C – CRE site from the human GSTP1 promoter
interacts with Me45 nuclear proteins and the complex formation can be
inhibited by the cold probe; AP-1, but not CRE consensus compete with
the CRE for the nuclear proteins and antibodies to Fos and ERb supershift
the complexes
site in Me45. Regarding the ability of CRE sites in
different genes to interact with CREB [19] and
AP-1[20] proteins, consensus oligonucleotides for both
transcription factors were utilized in the competitive
EMSA. A representative autoradiograph in fig. 4, C,
shows, that CREB consensus oligonucleotide could not
compete with GSTP1 promoter CRE for protein
binding, however genuine oligonucleotide CRE and
AP-1 consensus competed successfully. This suggests
that CRE site forms the complex with AP-1 in Me45
cells. The supershift experiment with antibodies against
the transcription factors known to interact directly or
indirectly with CREs of other genes was performed to
verify the results. Antibodies to c-Jun (cross-reactive to
JunB and JunD), c-Fos (cross-reactive to FosB, Fra1
and Fra2), MafF/G/K, ERb and Nrf3 were utilized in
this assay. The supershifted bands were observed after
the incubation of nuclear extracts with Fos and ERb
antibodies. The supershift analysis indicates that ERb
together with Fos protein interacts with the human
GSTP1 CRE in Me45 cells and this interaction has a
negative regulatory effect.
The phenomenon that protein binding sites can be
shared between different transcription factors is called
a transcription factor crosstalk [20]. It can be realized
by interaction of a «noncanonical» transcription factor
directly with a DNA sequence which has a partial
homology to the binding sites of this and another
transcription factors [20] or by protein-protein inter-
actions of «noncanonical» transcription factor with a
«genuine» protein bound to its recognition site. In case
of the human GSTP1 promoter both types of crosstalk
are present – noncanonical c-Fos together with ERb
crosstalks with CREB at CRE site and ERb together
with an unknown protein crosstalks with AP-1 at ARE
site. In both cases CREB and Fos/Jun has an opposite
effect on gene transcription. In case of the GSTP1
promoter this negative effect is seems to be potentiated
by ERb binding which is known to repress Fos-driven
transcription [21]. In the present finding we identified
ERb indirectly interacting with two promoter ele-
ments – CRE and ARE sites. It evidences for the
importance of this protein for the formation of the
enchanceosome on GSTP1 promoter.
The ER signaling mechanisms discussed until now
provide an explanation for the regulation of genes
lacking estrogen response element and requiring a
second DNA-binding transcription factor to mediate
ER association with the DNA. ERa and ERb have been
shown to act in opposite ways at Fos/Jun-binding sites.
In the presence of E2 ERa activates transcription via its
AF-1 and AF-2 transactivating domains while ERb-E2
which lacks a functional AF-1inhibits the Fos/Jun-
dependent transcription [22]. We suggest that ERb
exerts the similar inhibitory effect at CRE site of
GSTP1 promoter. The role of ERb associated with an
unknown protein at ARE site is different and may
activate transcription. The dual function of ERb in
regulation of different promoter elements may be
considered in context of enchanceosome formation.
Conclusions. In the present research the
transcriptional mechanisms controlling the basal level
of GSTP1 expression in Me45 cells have been analyzed
for the first time. The obtained data indicate that the
GSTP1 transcription in this cell type is positively
regulated by binding of NF-kB to –323 site and ERb in
complex with unknown protein binding to the ARE
site; the complex of ERb with c-Fos at CRE site nega-
tively regulates the gene expression. The interaction of
c-Fos/ERb with GSTP1 CRE site and indirect inter-
action of ER with GSTP1 ARE site have been
discovered.
The regulation of GSTP1 transcription in Me45
melanoma cells has been examined in details also for
the first time. Several transcription factors – NF-kB in
p50/p50 homodimer and p50/p65 heterodimer, ERb
and c-Fos regulate GSTP1 transcription in these cells.
Positive regulation is exerted via NF-kB and ARE sites
and negative via CRE site. ERb is indirectly involved
in regulation of GSTP1 transcription. It is bound via
c-Fos with CRE site and via unknown protein with
ARE site.
A. M. Ñëîí ÷àê, A. Êâå äóê, É. Æå øîâ ñêà-Âîëüíè,
Ì. Þ. Îáî ëå íñüêà
Ïå ðå ãî âî ðè ì³æ òðàíñ êðèïö³éíè ìè ôàê òî ðà ìè ó ðå ãó ëÿö³¿
åêñïðåñ³¿ ãåíà Ð1 ãëó òàò³îí-S-òðàíñ ôå ðà çè ëþ äè íè ó êë³òè íàõ
ìå ëà íî ìè Ìå45
Ðeçþìe
Ìåòà.Ãëó òàò³îí-S-òðàíñ ôå ðà çà (GTà çà) ëþ äè íè º ãî ëîâ íèì
ôåð ìåí òîì II ôàçè äå òîê ñè êàö³¿ ó á³ëüøîñò³ òèï³â êë³òèí.
Çì³íà ð³âíÿ åêñïðåñ³¿ ¿¿ ãåíà ïî â’ÿ çàíà ç êàí öå ðî ãå íå çîì ³ ôîð -
ìó âàí íÿì ÷èñ ëåí íî¿ ë³êà ðñüêî¿ ñò³éêîñò³. Åêñïðåñ³ÿ GTà çèÐ1
215
CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS
ðå ãó ëþºòüñÿ íà òðàíñ êðèïö³éíî ìó, ïî còòðà íñêðèïö³éíî ìó òà
ïî ñòòðàí ñëÿö³éíî ìó ð³âíÿõ. Ó äàí³é ðî áîò³ ìè çî ñå ðå äè ëè ñÿ
íà òðàíñ êðèïö³éí³é ðå ãó ëÿö³¿ ãåíà. Ìå òî äè. Òðàí ñôåêö³þ
êë³òèí ìå ëà íî ìè Ìå45 êî íñòðóêö³ÿìè, ÿê³ ì³ñòÿòü ãåí ëþ öè -
ôå ðà çè ï³ä êîí òðî ëåì ïî âíî ãî àáî âêî ðî ÷å íî ãî ïðî ìî òî ðà
GTà çèÐ1, âè êî ðèñ òà íî äëÿ âñòà íîâ ëåí íÿ ðîë³ ð³çíèõ ä³ëÿ íîê
ïðî ìî òî ðà â ðå ãó ëÿö³¿ òðàíñ êðèïö³¿ ãåíà GTà çè Ð1 ó êë³òè íàõ
Ìå45. Ùîá âèç íà ÷è òè òðàíñ êðèïö³éí³ ôàê òî ðè, ÿê³ âçàº-
ìîä³þòü ç ïðî ìî òî ðîì ãåíà GTà çèÐ1, âè ÿâ ëÿ ëè çì³íè åëåê òðî -
ôî ðå òè÷ íî¿ ðóõ ëè âîñò³ ÄÍÊ-á³ëêî âèõ êîì ïëåêñ³â çà ïðè ñóò -
íîñò³ àí òèò³ë ³ êîí êó ðåí òíèõ îë³ãî íóê ëå î òèä³â. Ðå çóëü òà òè.
Òðà íñêðèïö³ÿ ãåíà GTà çèÐ1 ó êëè òè íàõ Me45 ïî çè òèâ íî ðå ãó -
ëþºòüñÿ ÷å ðåç çâ’ÿ çó âàí íÿ NF-kB ³ç ñàé òîì –323 òà çà ðà õó íîê
çâ’ÿ çó âàí íÿ ERb y êîì ïëåêñ³ ç íåâ³äî ìèì á³ëêîì – ç ARE-ñàé -
òîì; êîì ïëåêñ ERb ç c-Fos íå ãà òèâ íî ðå ãó ëþº åêñïðåñ³þ ãåíà.
Âñòà íîâ ëå íî òà êîæ ïðÿ ìó âçàºìîä³þ c-Fos/ERb ³ç ñàé òîì
CRE ãåíà GTà çèÐ1 ³ íå ïðÿìy – ERb ³ç ñàé òîì ARE. Âèñ íîâ êè.
Ïî çè òèâ íà ðå ãó ëÿö³ÿ ãåíà GTà çèÐ1 ëþ äè íè â êë³òè íàõ ìå ëà íî -
ìè Me45 çä³éñíþºòüñÿ ÷å ðåç NF-kB ³ ARE-ñàé òè, à íå ãà òèâ íà –
÷å ðåç CRE-ñàéò ïðî ìî òî ðà. ERb îïî ñå ðåä êî âà íî áåðå ó÷àñòü ó
ðå ãó ëÿö³¿ òðàíñ êðèïö³¿ GTà çèÐ1: ÷å ðåç c-Fos â³í çâ’ÿ çóºòüñÿ ç
CRE-ñàé òîì ³ ÷å ðåç íåâ³äî ìèé á³ëîê – ç ARE-ñàé òîì.
Êëþ ÷îâ³ ñëî âà: ãëó òàò³îí-S-òðàíñ ôå ðà çà, ïðî ìî òîð,
òðàíñ êðèïö³éí³ ôàê òî ðè, NF-kB, åñ òðà ãå íî âèé ðå öåï òîð, ìå -
ëà íî ìà, ðå ãó ëÿö³ÿ òðàíñ êðèïö³¿.
A. M. Ñëîí ÷àê, A. Êâå äóê, É. Æå øîâ ñêà-Âîëü íû,
Ì. Þ. Îáî ëåí ñêàÿ
Ïå ðå ãî âî ðû ìåæ äó òðàíñ êðèï öè îí íû ìè ôàê òî ðà ìè â
ðå ãó ëÿ öèè ýêñ ïðåñ ñèè ãåíà Ð1 ãëó òà òè îí-S-òðàíñ ôå ðà çû
÷å ëî âå êà â êëåò êàõ ìå ëà íî ìû Ìå45
Ðeçþìe
Öåëü. Ãëó òà òè îí-S-òðàíñ ôå ðà çà (GTà çà) ÷å ëî âå êà ÿâ ëÿ åò ñÿ
ãëàâ íûì ôåð ìåí òîì II ôàçû äå òîê ñè êà öèè â áîëü øè íñòâå òè -
ïîâ êëå òîê. Èçìå íå íèå óðîâ íÿ ýêñ ïðåñ ñèè åå ãåíà ñâÿ çà íî ñ êàí -
öå ðî ãå íå çîì è ôîð ìè ðî âà íè åì ìíî æåñ òâåí íîé ëå êà ðñòâåí-
íîé óñòîé ÷è âîñ òè. Ýêñïðåñ ñèÿ GTà çûÐ1 ðå ãó ëè ðó åò ñÿ íà
òðàíñ êðèï öè îí íîì, ïî còòðà íñêðèï öè îí íîì è ïî ñòòðàí ñëÿ -
öè îí íîì óðîâ íÿõ.  äàí íîé ðà áî òå ìû ñî ñðå äî òî ÷è ëèñü íà
òðàíñ êðèï öè îí íîé ðå ãó ëÿ öèè ãåíà. Ìå òî äû. Òðàí ñôåê öèÿ êëå -
òîê ìå ëà íî ìû Ìå45 êî íñòðóê öè ÿ ìè, ñî äåð æà ùè ìè ãåí ëþ öè -
ôå ðà çû ïîä êîí òðî ëåì ïî ëíî ãî èëè óêî ðî ÷åí íî ãî ïðî ìî òî ðà
GTà çûÐ1, èñ ïîëü çî âà íà äëÿ âû ÿñ íå íèÿ ðîëè ðàç íûõ ó÷àñ òêîâ
ïðî ìî òî ðà â ðå ãó ëÿ öèè òðàíñ êðèï öèè ãåíà GTà çû Ð1 â êëåò êàõ
Ìå45. ×òî áû âû ÿ âèòü òðàíñ êðèï öè îí íûå ôàê òî ðû, âçà è ìî -
äå éñòâó þ ùèå ñ ïðî ìî òî ðîì ãåíà GTà çûÐ1, îïðå äå ëÿ ëè èç ìå -
íå íèÿ ýëåê òðî ôî ðå òè ÷åñ êîé ïîä âèæ íîñ òè ÄÍÊ-áåë êî âûõ
êîì ïëåê ñîâ â ïðè ñó òñòâèè àí òè òåë è êîí êó ðåí òíûõ îëè ãî íóê -
ëå î òè äîâ. Ðå çóëü òà òû. Òðà íñêðèï öèÿ ãåíà GTà çûÐ1 â êëåò êàõ
Me45 ïî çè òèâ íî ðå ãó ëè ðó åò ñÿ ÷å ðåç ñâÿ çû âà íèå NF-kB ñ ñàé -
òîì –323 è ÷å ðåç ñâÿ çû âà íèå ERb â êîì ïëåê ñå ñ íå èç âåñ íûì áåë -
êîì – ñ ARE-ñàé òîì; êîì ïëåêñ ERb ñ c-Fos íå ãà òèâ íî ðå ãó ëè-
ðóåò ýêñ ïðåñ ñèþ ãåíà. Óñòà íîâ ëå íî òàê æå ïðÿ ìîå âçà è ìî äåé-
ñòâèå c-Fos/ERb ñ ñàé òîì CRE ãåíà GTà çûÐ1 è íå ïðÿ ìîå – ERb
ñ ñàé òîì ARE. Âû âî äû. Ïî çè òèâ íàÿ ðå ãó ëÿ öèÿ ãåíà GTà çûÐ1
÷å ëî âå êà â êëåò êàõ ìå ëà íî ìû Me45 îñó ùå ñòâëÿ åò ñÿ ÷å ðåç NF-
kB è ARE-ñàé òû, à íå ãà òèâ íàÿ – ÷å ðåç CRE-ñàéò ïðî ìî òî ðà.
ERb îïîñ ðå äî âàí íî ó÷àñ òâó åò â ðå ãó ëÿ öèè òðàíñ êðèï öèè GTà -
çûÐ1: ÷å ðåç c-Fos îí ñâÿ çû âà åò ñÿ ñ CRE-ñàé òîì è ÷å ðåç íå èç -
âåñ òíûé áå ëîê – ñ ARE-ñàé òîì.
Êëþ ÷å âûå ñëî âà: ãëó òà òè îí-S-òðàíñ ôå ðà çà, ïðî ìî òîð,
òðàíñ êðèï öè îí íûå ôàê òî ðû, NF-kB, ýñ òðà ãå íî âûé ðå öåï òîð,
ìå ëà íî ìà, ðå ãó ëÿ öèÿ òðàíñ êðèï öèè.
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ÓÄÊ 577.152.28
Íàä³éøëà äî ðå äàêö³¿ 23.03.09
217
CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS
|
| id | nasplib_isofts_kiev_ua-123456789-5659 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T13:24:17Z |
| publishDate | 2009 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Slonchak, A.М. Cwieduk, A. Rzerzowska-Wolny, J. Obolenskaya, M.Yu. 2010-02-01T16:22:53Z 2010-02-01T16:22:53Z 2009 Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells / А.М. Slonchak, А. Cwieduk, J. Rzerzowska-Wolny, M.Yu. Obolenskaya // Біополімери і клітина. — 2009. — Т. 25, № 3. — С. 210–217. — Бібліогр.: 22 назв. — англ. 0233-7657 https://nasplib.isofts.kiev.ua/handle/123456789/5659 577.152.28 Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational. We concentrated our attention on the transcriptional level of regulation. Methods. Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors, interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied. Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-kB to the cognate site and ERb in complex with unknown protein to the ARE site; the complex of ERb with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ERb with GSTP1 CRE site and indirect interaction of ERb with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-kB and ARE sites and the negative one via CRE site of the promoter. ERb is indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos with CRE site and via unknown protein with ARE site. en Інститут молекулярної біології і генетики НАН України Геноміка, транскриптоміка і протеоміка Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells Переговори між транскрипційними факторами у регуляції експресії гена Р1 глутатіон-S-трансферази людини у клітинах меланоми Ме45 Переговоры между транскрипционными факторами в регуляции экспрессии гена Р1 глутатион-S-трансферазы человека в клетках меланомы Ме45 Article published earlier |
| spellingShingle | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells Slonchak, A.М. Cwieduk, A. Rzerzowska-Wolny, J. Obolenskaya, M.Yu. Геноміка, транскриптоміка і протеоміка |
| title | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells |
| title_alt | Переговори між транскрипційними факторами у регуляції експресії гена Р1 глутатіон-S-трансферази людини у клітинах меланоми Ме45 Переговоры между транскрипционными факторами в регуляции экспрессии гена Р1 глутатион-S-трансферазы человека в клетках меланомы Ме45 |
| title_full | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells |
| title_fullStr | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells |
| title_full_unstemmed | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells |
| title_short | Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells |
| title_sort | crosstalk between transcription factors in regulation of the human glutathione s-transferase p1 gene expression in me45 melanoma cells |
| topic | Геноміка, транскриптоміка і протеоміка |
| topic_facet | Геноміка, транскриптоміка і протеоміка |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/5659 |
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