• BMB Reports
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• 제51권2호
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• pp.92-97
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• 2018

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the $NF-{\kappa}B$ family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity.

• BMB Reports
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• 제44권4호
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• pp.267-272
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• 2011

ZAS3 is a large zinc finger transcription repressor that binds the ${\kappa}B$-motif via two signature domains of ZASN and ZASC. A loss-of-function study showed that lack of ZAS3 protein induced accelerated cell proliferation and tumorigenesis. Conversely, gain-of-function studies showed that ZAS3 repressed $NF{\kappa}B$-activated transcription by competing with $NF{\kappa}B$ for the ${\kappa}B$-motif. Based on these observations, we hypothesize that ZAS3 promotes apoptosis by interrupting anti-apoptotic activity of $NF{\kappa}B$. Here, we present evidence that upon $TNF{\alpha}$ stimulation, ZAS3 inhibits $NF{\kappa}B$-mediated cell survival and promotes caspase-mediated apoptosis. The inhibitory effect of ZAS3 on $NF{\kappa}B$ activity is mediated by neither direct association with $NF{\kappa}B$ nor disrupting nuclear localization of $NF{\kappa}B$. Instead, ZAS3 repressed the expression of two key anti-apoptotic genes of $NF{\kappa}B$, TRAF1 and TRAF2, thereby sensitizing cells to $TNF{\alpha}$-induced cell death. Taken together, our data suggest that ZAS3 is a tumor suppressor gene and therefore serves as a novel therapeutic target for developing anti-cancer drugs.

• 한국미생물·생명공학회지
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• 제23권3호
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• pp.288-296
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• 1995

To investigate high-level expression of Serratia marcescens metalloprotease (SMP) in Escherichia coli and S. marcescens, we constructed various recombinant plasmids: pSP2, containing SMP gene and lac promoter; pKSP2, containing SMP gene and tac promoter; pTSP2, containing SMP gene, trc99a promoter, and lacI$^{q}$. The recombinant E. coli (pKSP2) strain expressed SMP to a high-level, about 36% of total cellular proteins but accumulated inactive SMP precursors intracellularly, which indicated that E. coli does not have activation and secretion system for SMP. To overproduce active SMP, we transformed S. marcescens with the recombinant plasmids by a modified CaCl$_{2}$ method. The recombinant S. marcescens ATCC27117 (pSP2) containing lac promoter for SMP transcription produced 530 U/ml of active SMP on LB broth, which is about 5.1 times of the SMP yield, 105 U/ml of a control strain, S. marcescens ATCC27117 (pUC19). However, S. marcescens ATCC27117 (pKSP2) containing tac promoter for SMP transcription did not grow healthy and hardly produced SMP. To overcome a harmful effect of the strong tac promoter, we constructed a regulatory plasmid pTSP2 containing a strong trc99a promoter and its repressor gene lacI$^{q}$. When S. marcescens ATCC27117 (pTSP2) was induced with 1.0 mM IPTG after 9 hr cultivation, 2,200 U/ml of SMP was obtained in LB broth, which is about 21 times of that of a control strain.

• Molecules and Cells
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• 제21권2호
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• pp.261-268
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• 2006

The Drosophila methuselah (mth) mutant has an approximately 35 percent increase in average lifespan, and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat. To examine the transcriptional regulation of mth, we used luciferase assays employing Drosophila S2 cells. Two positive control elements were found at -542 ~ -272 (PE1) and +28 ~ +217 (PE2), where putative binding sites for transcription factors including Dorsal (Dl) were identified. Cotransfection of a Dl expression plasmid with a mth-luciferase reporter plasmid resulted in decreased reporter activity. PE1 and PE2, the minimal elements for strong promoter activity, were required for maximal repression by Dl protein. The N-terminal Rel homology domain (RHD) of Dl was not sufficient for repression of mth. We demonstrated by chromatin affinity precipitation (ChAP) assays in S2 cells that Dl bound to the putative PE1 binding site. Unexpectedly, semi-quantitative RT-PCR analysis revealed that the level of mth transcripts was reduced in dl flies. However, the in vivo result support the view that mth expression is regulated by dl, since it is well known that Dl functions as both a transcriptional activator and repressor depending on what other transcription factors are present. These findings suggest that both innate immunity and resistance to stress are controlled by Dl protein.

• Molecules and Cells
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• 제23권3호
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• pp.316-322
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• 2007

Zinc finger transcription factor genes are a significant fraction of the genes in the vertebrate genome. Here we report the isolation and characterization of a human zinc finger-containing gene, ZNF435, from a fetal brain cDNA library. ZNF435 cDNA is 1290 base pairs in length and contains an open reading frame encoding 349 amino acids with four C2H2-type zinc fingers at its carboxyl terminus and a SCAN motif at its amino terminus. RT-PCR results showed that ZNF435 was expressed in all tested tissues. A ZNF435-GFP fusion protein was located in the nucleus and the four zinc fingers acted as nuclear localization signals (NLSs). ZNF435 was found to be capable of homo-association, and this effect was independent of its zinc fingers. Furthermore, ZNF435 proved to be a transcription repressor as its overexpression in AD293 cells inhibited the transcriptional activities of AP-1.

• Mycobiology
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• 제51권5호
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• pp.372-378
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• 2023

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.

• Journal of Microbiology and Biotechnology
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• 제18권10호
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• pp.1672-1677
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• 2008

IscR (iron-sulfur cluster regulator) has been reported to be a repressor of the iscRSUA operon, and in vitro transcription reactions have revealed that IscR has a repressive effect on the iscR promoter in the case of [$Fe_{2}S_{2}$] cluster loading. In the present study, the iscR gene from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, and then purified by one-step affinity chromatography to homogeneity. The molecular mass of the IscR was 18 kDa by SDS-PAGE. The optical and EPR spectra results for the recombinant IscR confirmed that an iron-sulfur cluster was correctly inserted into the active site of the protein. However, no [$Fe_{2}S_{2}$] cluster was assembled in apoIscR with ferrous iron and sulfide in vitro. Therefore, the [$Fe_{2}S_{2}$] cluster assembly in IscR in vivo would appear to require scaffold proteins and follow the Isc "AUS" pathway.

• BMB Reports
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• 제49권11호
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• pp.587-589
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• 2016

The circadian clock system enables organisms to anticipate the rhythmic environmental changes and to manifest behavior and physiology at advantageous times of the day. Transcriptional/translational feedback loop (TTFL) is the basic feature of the eukaryotic circadian clock and is based on the rhythmic association of circadian transcriptional activator and repressor. In Drosophila, repression of dCLOCK/CYCLE (dCLK/CYC) mediated transcription by PERIOD (PER) is critical for inducing circadian rhythms of gene expression. Pacemaker neurons in the brain control specific circadian behaviors upon environmental timing cues such as light and temperature cycle. We show that amino acids 657-707 of dCLK are important for the transcriptional activation and the association with PER both in vitro and in vivo. Flies expressing dCLK lacking AA657-707 in $Clk^{out}$ genetic background, homologous to the mouse Clock allele where exon 19 region is deleted, display pacemaker-neuron-dependent perturbation of the molecular clockwork. The molecular rhythms in light-cycle-sensitive pacemaker neurons such as ventral lateral neurons ($LN_vs$) were significantly disrupted, but those in temperature-cycle-sensitive pacemaker neurons such as dorsal neurons (DNs) were robust. Our results suggest that the dCLK-controlled TTFL diversify in a pacemaker-neuron-dependent manner which may contribute to specific functions such as different sensitivities to entraining cues.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.81-81
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• 1997

We previously demonstrated an enhancer-like positive regulatory element within a 259-bp sequence (-2352 to-2094 bp) of the human CYP1A2 gene in HepG2 cells. Three protein binding sites were identified by DNase I footprint analyses within the 259-bp sequence: protected region A PRA ( -2283 to-2243 bp), PRB (-2218 to-2187 bp), and PRC (-2124 to-2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995). In the present study, the functional significance of those protected regions was examined. Transfection experiments with deletion and substitution mutants defined the PRB and PRC as containing positive and negative regulatory elements, respectively. Human breast carcinoma MCF-7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter-or thymidine kinase promoter-luciferase remoter gene constructs. HNF-1, which contributes to the liver specificity of genes, enhanced reporter gene activity in a PRC sequence-dependent manner. These results suggested that PRC could exist bound to a repressor which was displaceable by other transcription factors such as HNF-1. Results obtained by transfection of HepG2 hepatoma cells with various PRB substitution mutant-luciferase gene fusion constructs indicated that the entire sequence of PRB was necessary for promoter activity. Consequently, the regulation of CYP1A3 expression is very complex, requiring a number of both positive and negative regulatory factors.

• Animal cells and systems
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• 제13권2호
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• pp.119-125
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• 2009

Mammalian hibernation is a type of natural adaptation that allows organisms to avoid harsh environment and to increase the possibility of survival. To investigate the molecular link between circadian and hibernating rhythms in the greater tube-nosed bats, Murina leucogaster, we set out to identify circadian genes that are expressed in bats, with specific focus on the PAS domain by using PCR-based screens. We could isolate a eDNA clone, designated as LPAS1, that encodes a protein of 521 amino acid residues. LPAS1 is closely related with CLOCK family with the highest homology to human CLOCK. Based on RT-PCR analyses, LPAS1 transcripts are ubiquitously present in tissues from both summer active and winter dormant periods. Given that LPAS1 is a member of the bHLH-PAS protein superfamily but lacks polyglutamine transactivation domains, it is likely to function as a repressor for endogenous CLOCK to hinder its roles in promoting transcription. Our result will open a new avenue to further examine the functional interconnection between the circadian clock and the circannual clock such as mammalian hibernation.