• BMB Reports
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• 제32권6호
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• pp.594-598
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• 1999

The Jun and Fos families of eukaryotic transcription factors form heterodimers capable of binding to their cognate DNA enhancer elements. We are interested in searching for inhibitors or antagonists of the binding of the Jun-Fos heterodimer to the activator protein-1 (AP-1) site. The basic-region leucine zipper (bZIP) domain of c-Fos was expressed as a fusion protein with glutathione S-transferase, and allowed to form a heterodimer with the bZIP domain of c-Jun. The heterodimer was bound to glutathione-agarose, to which were added radiolabeled AP-1 nucleotides. After thorough washing, the gel-bound radioactivity was counted. The assay is faster than the coventional electrophoretic mobility shift assay because the gel electrophoresis step and the autoradiography step are eliminated. Moreover, the assay is very sensitive, allowing the detection of picomolar quantities of nucleotides, and is not affected by up to 50% dimethylsulfoxide, a solvent for hydrophobic inhibitors. Curcumin and dihydroguaiaretic acid, recently known inhibitors of Jun-Fos-DNA complex formation, were applied to this Jun-GST-fused Fos system and revealed to decrease the dimer-DNA binding.

• 생명과학회지
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• 제15권1호
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• pp.55-60
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• 2005

생쥐의 도파민 수용체 조절인자 (DRRF)유전자는 몇몇 Spl 결합부위를 가지고 TATA가 없는 프로모터로부터 전사된다. 본 연구에서는 이 유전자의 발현을 조절하는 기능성 조절인자들을 밝힌다. $D_2$ 도파민 수용체를 발현하는 NB41A3 세포에서 Spl은 pCAT-DRRF-l153/+17에 포함된 DRRF 프로모터부터 의 전사를 촉진시키지만 DRRF는 전사를 억제시켰다. -1153과 -1122 사이의 31 bp 단편의 결손에 의해 전사활성은 약 $60\%$ 정도 감소하였다. 이 단편은 기능성 AP1 결합부위를 포함하고 있다 게다가, -901과 -772사이의 129 bp영역의 결손에 의해 전사활성이 더욱 더 감소하였다. 이 영역은 기능성 AP2 결합부위를 가진다. DRRF_AP1 (bases -1153 to -1121) 탐침을 이용한 gel shift실험에서 특정 벤드가 관찰되었고, 이 벤드는 API 상보성 경쟁자에 의해 효과적으로 사라졌다. 더욱이, DRRF_AP2 (bases -873 to -846) 탐침을 이용한 gel shift실험에서도 특정 벤드가 관찰되었고, 이 벤드도 AP2상보성 경쟁자에 의해 효과적으로 사라졌다. 본 연구결과로, Spl과 DRRF가 DRRF 프로모터를 효과적으로 조절한다는 사실과, AP1과 AP2 역시 이 유전자를 조절한다는 사실을 알 수 있었다.

• Molecules and Cells
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• 제28권2호
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• pp.87-92
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• 2009

Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study were: 1) to verify if GATA and AP-1 sites could bind GATA2 and c-Jun/c-Fos factors, respectively; 2) to investigate whether such sites modulate the enhancer activity of the SIRT3-VNTR alleles. DAPA assay proved that GATA2 and c-Jun/c-Fos factors are able to bind the corresponding sites. Moreover, co-transfection experiments showed that the over-expression of GATA2 and c-Jun/c-Fos factors boosts the VNTR enhancer activity in an allelic-specific way. Furthermore, we established that GATA2 and c-Jun/c-Fos act additively in modulating the SIRT3-VNTR enhancer function. Therefore, GATA2 and AP-1 are functional sites and the T > C transition of the second VNTR repeat affects their activity.

• Bulletin of the Korean Chemical Society
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• 제20권8호
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• pp.925-928
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• 1999

The process of transcription is the major point at which gene expression is regulated. The jun and fos families of eukaryotic transcription factor heterodimerize to form complexes capable of binding 5'-TGAGTCA-3'DNA elements (AP-1 binding site). To search for the inhibitors of the jun-fos-DNA complex formation, several natural products extracts were screened and methanol extract of tanshen (the dried roots of Salvia miltiorrhiza Bunge) showed remarkable inhibitory activity. The active compounds of the extracts were purified using re-peated column chromatography and recrystallization. Their structures were identified as tanshinone I and tanshinone IIA. Through the electrophoresis mobility shift assay and cell cytotoxicity test, tanshinone I and tanshinone IIA were identified as inhibitors that suppress not only AP-1 function but also the cell proliferation. Tanshinone I also suppressed the jun-fos-DNA complex formation in TPA-induced NIH 3T3 cells.

• Bulletin of the Korean Chemical Society
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• 제22권10호
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• pp.1131-1135
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• 2001

• BMB Reports
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• 제34권1호
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• pp.28-32
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• 2001

The Fos-Jun heterodimers are part of the regulatory network of gene expression and nuclear proteins encoded by proto-oncogenes. The activation of Fos-Jun is important in the transmission of the tumor-promoting signal from the extracellular environment to the nuclear transcription mechanism. To search for the inhibitors of the Fos-Jun DNA complex formation, several natural products were screened and water-soluble paeoniflorin reduced the binding activity of the Fos-Jun heterodimer. This active compound was purified by silica gel column chromatography and HPLC. The electrophoresis mobility shift assay and reverse-phase HPLC test showed that paeoniflorin reduced the AP-l function. The cytotoxic effect of paeoniflorin was observed in HL-60. These results indicate that paeoniflorin blocks the Fos-Jun heterodimer-binding site of the AP-l DNA and it also has cytotoxic effects on human leukemia cell lines.

• 생명과학회지
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• 제15권6호
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• pp.928-934
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• 2005

ATF2는 c-Fos와 c-Jun과 같은 전사인자이며 이들은 루신지퍼 단백질이다. 루신지퍼 단백질은 동형이합체 혹은 이형이합체를 형성하며 promoter 영역에 결합하여 전사조절에 중요한 역할을 한다. 세포의 전사인자 ATF2는 ATF/CRE site에 결합하며 특히 선택적인 interfamily 이형이량체를 형성함으로써 전사 조절에 있어서 다양한 메카니즘을 제공할 수 있다. 본 연구에서는 ATF2 cDNA를 6xHis를 가진 expression vector에 subcloning하여 E.cnli BL2l에서 발현시켰다. 6xHis tag은 nickel-chelating chromatography를 가능하게 하였다 발현된 ATF2는 In vitro binding pull-down assay에서 동형이합체를 이를 뿐만 아니라 AP-1 그룹의 인자들과 선택적인 이형이합체를 형성함을 보여 주었다. BATF와는 강하게 결합하였으며 c-Jun과도 안정된 이합체를 형성하였다. 그러나 c-Fos와는 이합체를 형성하지 않으므로서 AP-1그룹 내에서도 이합체 형성이 선택적으로 이루어짐을 알 수 있다.

• 대한한의학회지
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• 제23권1호
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• pp.50-60
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• 2002

Objectives: Sunghyangjunggisan (SHJS) is a commonly prescribed drug with a wide neuropharmacological spectrum. The water extracts of SHJS were found to be protective against neurotoxicity elicited by deprivation of serum and glucose. Methods: The morphological examination and Hoechst staining of nucleus also clearly showed that the extracts attenuated the cell shrinkage, membrane blebbing, representing typical neuronal apoptotic phenomena and nucleosome-sized fragmentation under the microscope in PC 12 rat pheochromocytoma cells. Results: Activation of protein kinase A (PKA) with dibutyl-cAMP and forskolin also protected during glucose deprivation, although it was not additive with the effect provided by phorbol ester. Interestingly, treatment with the protein kinase A inhibitor, KT5720, was not neuroprotective in the presence of SHJS. Electrophoretic mobility shift assays were used to characterize the neuroprotective binding of nuclear proteins to consensus sequences for AP-l, nuclear factor kappa B ($NF-{\kappa}B$) after glucose deprivation. When PC 12 cells are induced to undergo apoptosis by serum deprivation, AP-l and $NF-{\kappa}B$ DNA binding activity transiently increases to a slight degree. This stimulation is blocked by the water extracts of SHJS. The site of action of the drugs appeared to involve specific inhibition of AP-1 and nuclear factor kB binding activity. Conclusions: Taken together, these results suggested the possibility that the extracts of SHJS might provide a neurotrophic-like activity in PC 12 cells.

• 대한한방내과학회지
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• 제20권1호
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• pp.99-110
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• 1999

Effects of Chiyangtang(CYT) on H. pylori-induced increase of interleukin 8 and interleukin 1 gene expression was studied in Kato Ⅲ cell line, a human stomach epithelial cell line. Treatment of H. pylori to the cell culture signifant!y increased IL-8 and IL-1 mRNA synthesis. When CYT was added along with H. pylori, the increase of IL-8 and IL-1 mRNA synthesis was blocked. Activation of transcription factor $NF-{\kappa}B$ and AP-1 which were known to important in IL-8 and IL-1 gene expression was also studied using chloramphenicol acetyltransferase(CAT) assay. Treatment of H. pylori increased activation of $NF-{\kappa}B$ and AP-l and CYT effectively protected the activation. Electrophoretic mobility shift assay suggested that CYT effectively inhibited DNA binding of $NF-{\kappa}B$ and AP-l to their cognate site. These results suggested that CYT could prevent stomach diseases through the down regulation of IL -8 and IL-l gene expression which might be mediated by the inhibition of $NF-{\kappa}B$ and AP-1 activities and their binding to DNA.

• 생명과학회지
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• 제33권1호
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• pp.34-42
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• 2023

환경 스트레스 신호 인지부터 스트레스 반응 유전자의 발현에 이르는 신호전달 네트워크에 있어서, 스트레스 반응 프로모터의 cis-조절 요소와 거기에 결합하는 다양한 전사인자는 환경 스트레스에 대한 식물의 적응을 조절한다. 애기장대 AP2/ERF 전사인자 패밀리 중 그룹 VII ERF는 RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, AtERF71/HRE2 유전자를 포함하며, 저산소 스트레스 반응에서 중요한 역할을 하는 것으로 알려져 있다. 본 연구에서는 HRE1 프로모터의 저산소 반응 부위를 동정하였다. 이를 위해 1,000 bp, 800 bp, 600 bp, 400 bp, 200 bp, 100 bp, 그리고 50 bp HRE1 프로모터 부위를 포함하는 벡터를 제작하여 프로모 터 활성을 분석한 결과, -200에서 -100 프로모터 부위가 HRE1의 저산소 반응에서 중요함을 확인하였다. HRE1의 -200에서 -100 프로모터 부위에는 저산소 반응 cis-조절 요소로 알려진 ERF-결합 부위와 DOF-결합 부위가 존재하는데, 이는 HRE1의 발현이 ERF 전사인자와 DOF 전사인자에 의해 조절될 수 있음을 시사한다. 전체적으로, 본 연구를 통해 HRE1 의 저산소 스트레스 반응에는 -200에서 -100 프로모터 부위에 존재하는 cis-조절 요소가 중요한 역할을 한다는 것을 확인하였다.