• The Korean Journal of Physiology and Pharmacology
• /
• 제24권3호
• /
• pp.203-212
• /
• 2020

Promyelocytic leukemia (PML) gene, through alternative splicing of its C-terminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.

• Molecules and Cells
• /
• 제38권11호
• /
• pp.959-965
• /
• 2015

Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

• 상하수도학회지
• /
• 제20권6호
• /
• pp.893-904
• /
• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.

• 한국식물생명공학회:학술대회논문집
• /
• 한국식물생명공학회 2004년도 생명공학 실용화를 위한 비젼
• /
• pp.144-144
• /
• 2004

• BMB Reports
• /
• 제43권1호
• /
• pp.17-22
• /
• 2010

In this study, a novel member of BTB-kelch proteins, named KBTBD7, was cloned from a human embryonic heart cDNA library. The cDNA of KBTBD7 is 3,008 bp long and encodes a protein product of 684 amino acids (77.2 kD). This protein is highly conserved in evolution across different species. Western blot analysis indicates that a 77 kD protein specific for KBTBD7 is wildly expressed in all embryonic tissues examined. In COS-7 cells, KBTBD7 proteins are localized to the cytoplasm. KBTBD7 is a transcription activator when fused to GAL4 DNA-binding domain. Deletion analysis indicates that the BTB domain and kelch repeat motif are main regions for transcriptional activation. Overexpression of KBTBD7 in MCF-7 cells activates the transcriptional activities of activator protein-1 (AP-1) and serum response element (SRE), which can be relieved by siRNA. These results suggest that KBTBD7 proteins may act as a new transcriptional activator in mitogen-activated protein kinase (MAPK) signaling.

• Biomolecules & Therapeutics
• /
• 제22권4호
• /
• pp.308-313
• /
• 2014

Activator protein-1 (AP-1) is an inducible transcription factor that contributes to the generation of chronic inflammation in response to oxidative and electrophilic stress. Previous studies have demonstrated that the PI3K/Akt1 pathway plays an important role in the transcriptional regulation of AP-1 expression. Although the histone post-translational modifications (PTMs) are assumed to affect the AP-1 transcriptional regulation by the PI3K/Akt pathway, the detailed mechanisms are completely unknown. In the present study, we show that heterochromatin 1 gamma ($HP1{\gamma}$) plays a negative role in TPA-induced c-Jun and c-Fos expression. We show that TPA-induced Akt1 directly phosphorylates $HP1{\gamma}$, abrogates its suppressive function and increases the interaction between histone H3 and 14-3-$3{\varepsilon}$. Collectively, these our data illustrate that the activation of PI3K/Akt pathway may play a permissive role in the recruitment of histone readers or other coactivators on the chromatin, thereby affecting the degree of AP-1 transcription.

• Molecules and Cells
• /
• 제40권8호
• /
• pp.533-541
• /
• 2017

Engineered DNA-binding domains provide a powerful technology for numerous biomedical studies due to their ability to recognize specific DNA sequences. Zinc fingers (ZF) are one of the most common DNA-binding domains and have been extensively studied for a variety of applications, such as gene regulation, genome engineering and diagnostics. Another novel DNA-binding domain known as a transcriptional activator-like effector (TALE) has been more recently discovered, which has a previously undescribed DNA-binding mode. Due to their modular architecture and flexibility, TALEs have been rapidly developed into artificial gene targeting reagents. Here, we describe the methods used to design these DNA-binding proteins and their key applications in biomedical research.

• BMB Reports
• /
• 제39권4호
• /
• pp.355-360
• /
• 2006

To gain a better understanding on the function of the potato Solanum tuberosum Multiprotein Bridging Factor 1 protein (StMBF1) its interaction with the TATA box binding protein (TBP) was demonstrated. In addition we reported that StMBF1 rescues the yeast mbf1 mutant phenotype, indicating its role as a plant co-activator. These data reinforce the hypothesis that MBF1 function is also conserved among non closely related plant species. In addition, measurement of StMBF1 protein level by Western blot using anti-StMBF1 antibodies indicated that the protein level increased upon $H_2O_2$ and heat shock treatments. However, the potato $\beta$-1,3-glucanase protein level was not changed under the same experimental conditions. These data indicate that StMBF1 participates in the cell stress response against oxidative stress allowing us to suggest that MBF1 genes from different plant groups may share similar functions.

• 미생물학회지
• /
• 제38권4호
• /
• pp.260-266
• /
• 2002

Ralstonia eutropha JMP134로부터 페놀대사의 조절에 관여하는 유전자부위를 cloning하여 염기서열을 파악하였으며 그 특성을 조사하였다. 염기서열 분석 결과 두 개의 open reading frame (ORF1 & ORF2)들을 발견하였다. ORF1은 페놀분해 구조유전자중의 마지막 인자인phlX의 stop codon으로부터 454 bp 아래에서 시작하여 총 501 개의 아미노산으로 구성되었으며 ORF2는 ORF1의 stop codon으로부터 1 bP 위쪽에서 4개의 염기쌍과 중첩된 상태에서 시작하여 총 232개의 아미노산으로 구성되어 있는 것으로 나타났다. 단백질 배열을 분석해본 결과 ORF1은 transcriptional activator로 작용하는 NtrC family에 속하는 것으로 확인되었으며, ORF2는 negative regulator로 알려진 GntR family에 속하는 것으로 나타났다. ORF1과 ORF2를 encoding하는 유전자를 각각 phlR2 와 phlA로 명명하였으며 이들의 가능한 조절기작을 고찰하였다.

• 한국미생물·생명공학회지
• /
• 제34권1호
• /
• pp.15-22
• /
• 2006

Ochrobactrum anthropi JW-2의 염색체 DNA로부터 paraquat 내성 유전자 pqrA를 포함하는 4,971 bp의 DNA 염기서열을 결정하였다. 염기서열 분석 결과 2개의 불완전한 ORF(orf1, orf5)와 4개의 완전한 ORF(orf2, pqrA, orf3, orf4)가 존재하는 것으로 나타났는데 orf1, pqrA, orf4, orf5는 direct strand에 orf2와 orf3은 reverse complementary strand 존재하였다. Orf1은 개시코돈이 결손된 불완전한 서열로서, response regulator receiver의 ATP binding region과 상동성을 나타내었다. Orf2는 tetR family에 속하는 transcription repressor와 높은 상동성을 나타내었고 H-T-H motif가 존재하는 것으로 나타났다. 따라서 orf2가 pqrA 유전자의 전사조절에 관여하는 repressor로 추정되어 pqrR2로 명명하였다. Orf3은 lysR type의 transcription activator와 높은 상동성을 나타내었고 N-terminal 부위에 H-T-H motif와 C-terminal 부위에 substrate binding domain이 존재하는 것으로 나타났다. 따라서 orf3은 pqrA의 전사조절에 관여하는 transcription activator로 추정되어 pqrR1로 명명하였다. Orf4는 amino acid ABC transporter의 periplasmic amino acid-binding protein과 상동성을 나타내었으며, orf5는 종결 코돈이 없는 불완전한 ORF로서 amino acid ABC transporter의 permease protein과 상동성을 나타내었다. 이와 같은 결과로 미루어 pqrA 유전자 주위에 존재하는 전사조절 유전자들이 paraquat 내성유전자인 pqrA의 발현조절을 통하여 paraquat에 대한 내성획득에 관여하는 것으로 판단되었다.