ISSN 0233-7657. Biopolymers and Cell. 2009. Vol. 25. N 3 ÃÅÍÎÌ²ÊÀ, ÒÐÀÍÑÊÐÈÏÒÎÌ²ÊÀ ² ÏÐÎÒÅÎÌ²ÊÀ Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells A. M. Slonchak, A. Chwieduk1, J. Rzeszowska-Wolny1, M. Yu. Obolenskaya Institute of Molecular Biology and Genetics NAS of Ukraine 150, Zabolotnogo str., Kyiv Ukraine, 03680 1 M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Gliwice Wybrzezhe Armiji Krajowej 15, 44-101, Gliwice, Poland firstname.lastname@example.org 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-B to the cognate site and ER in complex with unknown protein to the ARE site; the complex of ER with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ER with GSTP1 CRE site and indirect interaction of ER with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-B and ARE sites and the negative one via CRE site of the promoter. ER 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.
Introduction. Glutathione S-transferases comprise a participates in dinitrosyl-diglutathionyl-iron complex multigene superfamily of enzymes that catalyze the storage and metabolism . Aberrant expression of conjugation of electrophilic toxic compounds with GSTP1 is associated with carcinogenesis and developglutathione, playing a key role in phase II of ment of multidrug resistance (MDR).
CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS Despite the vast literature devoted to GSTP1 enzyme regions of the final constructs were confirmed by sequthe functional characteristics of responsive elements in encing in both directions in OIigo.pl DNA IBB PAN gene promoter and tissue-specific peculiarities of their Service (Poland).
regulation are poorly understood. Moreover the pre- Transient transfection assay. Me45 cells were vious investigations of molecular mechanisms involved grown in 24-well plates to 60 % confluence and transin the GSTP1 regulation were focused mainly on breast fected with 500 ng of pGSTP together with 25 ng of cancer, leukemia and prostate cancer cells. In present pRL-TK (plasmid with Renilla Luciferase and Thómiresearch we performed a functional analysis of GSTP1 dine kinase Promoter) plasmid («Promega», USA) per promoter in human melanoma cells Me45. We utilized well using LipofectamineTM LTX and PLUSTM reagents truncated promoter constructions to compare the («Invitrogen», USA). After 20 h the firefly and renilla functional role of different cis-acting promoter ele- luciferase activities were assessed using Dual Lucifements and identified transcription factors binding the rase® Reporter Assay System («Promega», USA).
responsive elements by competitive EMSA (electro- Electrophoretic mobility shift assay. Nuclear phoretic mobility shift assay) and supershift assay. extracts from Me45 cells were prepared by modified Matherials and methods. Cell culture. Human method of Dignam et al. . The following oligomelanona cell line Me45 was obtained from Polish Cell nucleotides and their complementary sequences were Bank and propagated in DMEM/F12 medium («Sig- used as probes in EMSA experiment: ARE (Antima», USA) supplemented with 588 µg/ml L-glutamine, oxidant Response Element of human GSTP1 promo0.16 % NaHCO3, 10 % heat inactivated fetal calf serum ter) 5'-CGCCGTGACTCAGCACTGGG-3', NF-B(«Gibco», USA) and 100 µg/ml gentamicine. Cells like (Nuclear Factor B-like site of human GSTP1 prowere grown at 37 °C in an atmosphere of 95 % air and moter) 5'-TCCGCGGGACCCTCCAGAAG-3', NF-B 5 % CO2. (Nuclear Factor B site of human GSTP1 promoter) Promoter deletion constructs. Fragments of GSTP1 5'-CTTAGGGAATTTCCCCCCGC-3', CRE (Cyclic gene promoter were prepared by PCR. The oligonuc- AMP Response Element site of human GSTP1 promoleotide 5'-ACTCACTGGTGGCGAAGACT-3' (positi- ter) 5'-GAGACTACGTCATAAAATAA-3', GATA on +15 to +35) was used as the downstream primer for (GATA-1 binding site of human GSTP1 promoter) all constructions. Each of the following oligonucleoti- 5'-GAGATCAATATCTAGAAATAA-3'. Probes des was used as upstream primers to amplify promoter (10 pmoles) were labeled with 20 pmoles [-32P]-ATP fragments: 5'-CATAAACACCA- ACCTCTTCCCC-3' 6000 Ci/mmole («Hartmann Analytic», Deutschland) (position –1379 to –1357) for pGSTP1415, 5'-ATAGC- by polynucleotide kinase («Roche», Switzerland).
CTAAGGCACAGCCAC-3' (position –1162 to –1142) Unincorporated nucleotides were removed by for pGSTP1197, 5'-TTTCCTTTCCTCTAAGCGGC-3' gel-filtration through Bio-gel® P-30 («Bio-Rad», (position –405 to –385) for pGSTP440, 5'-AGTCCGC- USA). Electrophoretic mobility shift assay was G GGACCCTCCAGA-3' (position –105 to –85) for performed using Electropforetic mobility shift assay kit pGSTP140 and 5'-AGAGCGGCCGGCGCCGTGAC- («Promega»). Consensus oligonucleotides for AP-3' (position –85 to –64) for pGSTP120. The amplified (Activator Protein 1), NF-B (Nuclear Factor B), products were subcloned into pCR®2.1-TOPO® vector CREB (Cyclic AMP Response Element Binding («Invitrogen», USA). The recombinant plasmids were protein), GATA, ER (Estrogen Receptor) and RAR sequenced and the orientation of inserts was deter- (Retinoic Acid Receptor), antibodies against human mined. Plasmids with directly oriented inserts were c-Jun, c-Fos, MafF/G/K, ER, Nrf3 (Nuclear erythroid submitted to digestion with KpnI and XhoI. Excised in- 2 p45 related factor 3), NF-B p50, NF-B p65 and serts were religated into pGL3-basic (plasmid with normal rabbit IgG were from «Santa Cruz BioGloTM Lu- ciferase 3 basic) plasmid («Promega», USA). technology» (USA).
Resulted constructs were named pGSTPX, were X cor- Results and discussion. Functional analysis of the responds to the length of the inserted promoter frag- GSTP1 promoter regions in Me45 cells. The structure ment and GSTP is the gene name. Sequences of relevant of GSTP1 promoter is summarized in fig. 1. To identify 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 the role of GSTP1 promoter regions in regulation of GATA-binding site, did not influence significantly the GSTP1 transcription in Me45 cells we utilized transient expression of the reporter gene. Deletion of the region transfection assay with reporter constructs containing from –1162 to –405, which contains CRE and ATAcomplete or truncated GSTP1 promoter fused to the AA-repeat, resulted in increase of f-luc expression firefly luciferase gene. For this purpose we designed approximately 1.8-fold in comparison with previous the reporter constructs each lacking the DNA fragment construct. Further deletion of the region from –405 to with one transcription factor binding site (fig. 2). The –105, containing NF-B site, reduced the reporter gene diagram in fig. 2 represents relative firefly luciferase expression 1.6-fold. Deletion of the region from –activities in lysates of Me45 cells transfected with re- to –85, known as an NF-B-like element, resulted in porter constructs. Each bar in the graph represents the 1.5-fold increase of f-luc expression.
average of 3 independent experiments with triplications Thereby, the results of the transient transfection in each. experiments suggest the presence of the negative Transfection of the largest vector (pGSTP1415) regulatory elements located in the regions from –containing the GSTP1 promoter fragment from –1379 to –405 and from –105 to –85. Also it provided the to +35 resulted in relatively high level of f-luc gene evidence for the presence of the strong positive expression in Me45 cells. Deletion of the GSTP1- regulatory element located from –405 to –105. The flanking region between –1379 and –1162, containing similar role of promoter sequences in the regulation of CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS the «negative» role of the NF- B-like element in GSTPtranscription 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 +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-, Nrf2 , ER  and RAR , depending on cell type. To identify the transcription factors acting on this site in Me45 cells we performed competitive Fig. 3. In vitro binding of Me45 nuclear proteins to GSTP1 promoter EMSA with consensus oligos for AP-1, Maf (the sites: A – electrophoretic mobility shift assay, demonstrating the DNA-binding component of Nrf2), ER and RAR ability of Me45 nuclear proteins to form complexes with ARE, NF- and supershift assay with antibodies for these transB, NF-B-like, CRE and GATA sites; B – results of competitive EMSA demonstrating, that protein binding to NF-B-like site is cription factors. Consistent with results shown in fig. 4, nonspecific; C – results of competitive EMSA demonstrating, that A, a 50- and 100-fold molar excess of unlabeled conprotein binding to GATA site is nonspecific; S – specific complex;
sensus oligonucleotides for AP-1, Maf, estrogen NS – nonspecific complex receptor beta (ER) and retinoic acid receptor (RAR) GSTP1 gene transcription was identified by Jhaveri and were not able to compete for the nuclear proteins Morrow  for breast cancer cells. binding to the ARE site. It means that AP-1, Maf, ER The study of ARE, NF-B-like, NF-B, CRE and and RAR do not interact with ARE site through their GATA binding sites interactions with nuclear proteins DNA-binding domains. To clarify these results the from Me45 cells. For identification of the transcription supershift experiment with polyclonal antibodies to factors interacting with the GSTP1 promoter the c-Jun (cross-reactive to JunB and JunD), c-Fos electrophoretic mobility shift assay (EMSA) was (cross-reactive to FosB, Fra1 and Fra2), MafF/G/K, ER applied. The ability of 20 bp promoter fragments, and Nrf3 (the placenta-specific homolog of Nrf2) was containing ARE, NF- B-like, NF-B, CRE and GATA performed. As indicated in fig. 4, A, neither sites to bind nuclear proteins from Me45 cells was transcription factors Jun, Fos nor Maf and Nrf3 prevent examined in this experiment. Fig. 3 shows that all the formation of specific complex of ARE site with a oligonucleotides form complexes with nuclear nuclear protein. Only ER antibody prevents the whole proteins. Specificity of the protein binding was as- complex formation resulting in appearance of a new sessed in a competition experiment, in which nuclear complex with higher electrophoretic mobility. This proteins were preincubated in 50- and 100-fold molar result clearly indicates that in Me45 nuclear extracts ER excess of unlabeled probe. In this experiment we binds to the GSTP1 ARE site through another yet determined, that ARE, NF-B and CRE sites unknown protein and DNA-binding domain of ER is specifically bind nuclear proteins while NF-B-like and not involved in these interactions.
GATA sites do not. One band observed in all The region of GSTP1 promoter from –405 to –elrctrophoregrams was non-specific because it was not contains NF-B site and positively regulates the eliminated in competitive experiments (fig. 3, A, B, C). reporter gene transcription in Me45 cells. This site Surprisingly, we did not find any proteins in- binds NF-B in K562 leukemia cells and mediates the teracting with NF-B-like element which was iden- gene induction by TNF . The results of the tified as a negative regulator of GSTP1 transcription in GSTP1 promoter NF-B site binding assay are the transient transfection experiment. We suppose that summarized in fig. 4, B. Two specific bands were 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 ER antibody;