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B - NF-B site forms two complexes with the nuclear proteins from Mecells; both complexes were disrupted by the cold probe and NF-B 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 ER supershift the complexes observed in the reaction of genuine NF-B site NF-B site contains the p50/p65 heterodimer, while the containing oligo with nuclear extract. The unlabeled lower complex observed in binding reaction is the NF-B consensus was able to efficiently compete for p50/p50. These data together with the results of the nuclear proteins from both specific complexes transient transfection assay strongly suggest that NFleading to suggestion that NF- B binds to GSTP1 B interacts with the human GSTP1 NF-B site and NF-B site in this cell line. To clarify the matter, up-regulates gene transcription in Me45 cells.

nuclear extract was incubated with polyclonal The negative regulatory element Ц1162 Е Цantibodies to p50 and p65 subunits of NF- B before the contains a CRE site and ATAAA-repeated sequence. It probe was added to the EMSA reaction. In the was previously reported that CRE site of GSTPsupershift assay of NF-B site two new bands were mediates gene response to cAMP by interacting with observed after the incubation with p50 antibody - one CREB in Calu-6 lung cancer cells [19]. Competitive originated from the lower and one from the upper EMSA was also conducted to determine which protein complex, providing the evidence that both complexes is a part of the DNA-protein complex formed by CRE contain p50. The upper complex of nuclear proteins and site in Me45. Regarding the ability of CRE sites in CROSSTALK BETWEEN TRANSCRIPTION FACTORS IN REGULATION OF THE HUMAN GSTP1 GENE EXPRESSION IN Me45 CELLS different genes to interact with CREB [19] and second DNA-binding transcription factor to mediate AP-1[20] proteins, consensus oligonucleotides for both ER association with the DNA. ER and ER have been transcription factors were utilized in the competitive shown to act in opposite ways at Fos/Jun-binding sites.

EMSA. A representative autoradiograph in fig. 4, C, In the presence of E2 ER activates transcription via its shows, that CREB consensus oligonucleotide could not AF-1 and AF-2 transactivating domains while ER-Ecompete with GSTP1 promoter CRE for protein which lacks a functional AF-1inhibits the Fos/Junbinding, however genuine oligonucleotide CRE and dependent transcription [22]. We suggest that ER AP-1 consensus competed successfully. This suggests exerts the similar inhibitory effect at CRE site of that CRE site forms the complex with AP-1 in Me45 GSTP1 promoter. The role of ER associated with an cells. The supershift experiment with antibodies against unknown protein at ARE site is different and may the transcription factors known to interact directly or activate transcription. The dual function of ER in indirectly with CREs of other genes was performed to regulation of different promoter elements may be verify the results. Antibodies to c-Jun (cross-reactive to considered in context of enchanceosome formation.

JunB and JunD), c-Fos (cross-reactive to FosB, Fra1 Conclusions. In the present research the and Fra2), MafF/G/K, ER and Nrf3 were utilized in transcriptional mechanisms controlling the basal level this assay. The supershifted bands were observed after of GSTP1 expression in Me45 cells have been analyzed the incubation of nuclear extracts with Fos and ER for the first time. The obtained data indicate that the antibodies. The supershift analysis indicates that ER GSTP1 transcription in this cell type is positively together with Fos protein interacts with the human regulated by binding of NF-B to Ц323 site and ER in GSTP1 CRE in Me45 cells and this interaction has a complex with unknown protein binding to the ARE negative regulatory effect. site; the complex of ER with c-Fos at CRE site negaThe phenomenon that protein binding sites can be tively regulates the gene expression. The interaction of shared between different transcription factors is called c-Fos/ER with GSTP1 CRE site and indirect intera transcription factor crosstalk [20]. It can be realized action of ER with GSTP1 ARE site have been by interaction of a noncanonical transcription factor discovered.

directly with a DNA sequence which has a partial The regulation of GSTP1 transcription in Mehomology to the binding sites of this and another melanoma cells has been examined in details also for transcription factors [20] or by protein-protein inter- the first time. Several transcription factors - NF-B in actions of noncanonical transcription factor with a p50/p50 homodimer and p50/p65 heterodimer, ER genuine protein bound to its recognition site. In case and c-Fos regulate GSTP1 transcription in these cells.

of the human GSTP1 promoter both types of crosstalk Positive regulation is exerted via NF-B and ARE sites are present - noncanonical c-Fos together with ER and negative via CRE site. ER is indirectly involved crosstalks with CREB at CRE site and ER together in regulation of GSTP1 transcription. It is bound via with an unknown protein crosstalks with AP-1 at ARE c-Fos with CRE site and via unknown protein with site. In both cases CREB and Fos/Jun has an opposite ARE site.

effect on gene transcription. In case of the GSTP1 Acknowledgement. The experimental part of the promoter this negative effect is seems to be potentiated work was carried out in the Institute of Oncology in by ER binding which is known to repress Fos-driven Gliwice and was financed by the Polish Ministry of transcription [21]. In the present finding we identified Education and Science, grant number N 401 ER indirectly interacting with two promoter ele- 32/3043.

ments - CRE and ARE sites. It evidences for the A. M. Слончак, A. Кведук, Й. Жешовска-Вольни, importance of this protein for the formation of the М. Ю. Оболенська enchanceosome on GSTP1 promoter.

Переговори мж транскрипцйними факторами у регуляц The ER signaling mechanisms discussed until now експрес гена Р1 глутатон-S-трансферази людини у клтинах provide an explanation for the regulation of genes меланоми Меlacking estrogen response element and requiring a SLONCHAK A. M. ET AL.

с сайтом ARE. Выводы. Позитивная регуляция гена GTазыРРeзюмe человека в клетках меланомы Me45 осуществляется через NFМета.Глутатон-S-трансфераза (GTаза) людини головним B и ARE-сайты, а негативная - через CRE-сайт промотора.

ферментом II фази детоксикац у бльшост типв клтин.

ER опосредованно участвует в регуляции транскрипции GTаЗмна рвня експрес гена повТязана з канцерогенезом форзыР1: через c-Fos он связывается с CRE-сайтом и через неизмуванням численно карсько стйкост. Експреся GTазиРвестный белок - с ARE-сайтом.

регулються на транскрипцйному, поcттранскрипцйному та Ключевые слова: глутатион-S-трансфераза, промотор, посттрансляцйному рвнях. У данй робот ми зосередилися транскрипционные факто ры, NF-B, эстрагеновый рецептор, на транскрипцйнй регуляц гена. Методи. Трансфекцю меланома, регуляция транскрипции.

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