• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.631-638
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• 2008

Tandem affinity purification (TAP) method combined with LC-MS/MS is the most accurate and reliable way to study the interaction of proteins or proteomics in a genome-wide scale. For the first time, we used a TAP-tag as a mutagenic tool to disrupt protein interactions at the specific site. Although lots of commonly used mutational tools exist to study functions of a gene, such as deletional mutations and site-directed mutagenesis, each method has its own demerit. To test the usefulness of a TAP-tag as a mutagenic tool, we applied a TAP-tag to RNA polymerase II, which is the key enzyme of gene expression and is controlled by hundreds of transcription factors even to transcribe a gene. Our experiment is based on the hypothesis that there will be interrupted interactions between Pol II and transcription factors owing to the TAP-tag attached at the C-terminus of each subunit of Pol II, and the abnormality caused by interrupted protein interactions can be observed by measuring a cell-cycle of each yeast strain. From ten different TAP-tagged strains, Rpb7- and Rpb12-TAP-tagged strains show severe defects in growth rate and morphology. Without a heterodimer of Rpb4/Rpb7, only the ten subunits Pol II can conduct transcription normally, and there is no previously known function of Rpb7. The observed defect of the Rpb7-TAP-tagged strain shows that Rpb7 forms a complex with other proteins or compounds and the interruption of the interaction can interfere with the normal cell cycle and morphology of the cell and nucleus. This is a novel attempt to use a TAP-tag as a proteomic tool to study protein interactions.

• Molecules and Cells
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• 제45권9호
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• pp.660-672
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• 2022

The target of rapamycin complex (TORC) plays a key role in plant cell growth and survival by regulating the gene expression and metabolism according to environmental information. TORC activates transcription, mRNA translation, and anabolic processes under favorable conditions, thereby promoting plant growth and development. Tomato fruit ripening is a complex developmental process promoted by ethylene and specific transcription factors. TORC is known to modulate leaf senescence in tomato. In this study, we investigated the function of TORC in tomato fruit ripening using virus-induced gene silencing (VIGS) of the TORC genes, TOR, lethal with SEC13 protein 8 (LST8), and regulatory-associated protein of TOR (RAPTOR). Quantitative reverse transcription-polymerase chain reaction showed that the expression levels of tomato TORC genes were the highest in the orange stage during fruit development in Micro-Tom tomato. VIGS of these TORC genes using stage 2 tomato accelerated fruit ripening with premature orange/red coloring and decreased fruit growth, when control tobacco rattle virus 2 (TRV2)-myc fruits reached the mature green stage. TORC-deficient fruits showed early accumulation of carotenoid lycopene and reduced cellulose deposition in pericarp cell walls. The early ripening fruits had higher levels of transcripts related to fruit ripening transcription factors, ethylene biosynthesis, carotenoid synthesis, and cell wall modification. Finally, the early ripening phenotype in Micro-Tom tomato was reproduced in the commercial cultivar Moneymaker tomato by VIGS of the TORC genes. Collectively, these results demonstrate that TORC plays an important role in tomato fruit ripening by modulating the transcription of various ripening-related genes.

• Nutrition Research and Practice
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• 제1권1호
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• pp.19-28
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• 2007

To identify regulatory molecules which play key roles in the development of obesity, we investigated the transcriptional profiles in 3T3-L1 cells at early stage of differentiation and analyzed the promoter sequences of differentially regulated genes. One hundred and sixty-one (161) genes were found to have significant changes in expression at the 2nd day following treatment with differentiation cocktail. Among them, 86 transcripts were up-regulated and 75 transcripts were down-regulated. The 161 transcripts were classified into 10 categories according to their functional roles; cytoskeleton, cell adhesion, immune, defense response, metabolism, protein modification, protein metabolism, regulation of transcription, signal transduction and transporter. To identify transcription factors likely involved in regulating these differentially expressed genes, we analyzed the promoter sequences of up- or - down regulated genes for the presence of transcription factor binding sites (TFBSs). Based on coincidence of regulatory sites, we have identified candidate transcription factors (TFs), which include those previously known to be involved in adipogenesis (CREB, OCT-1 and c-Myc). Among them, c-Myc was also identified by our microarray data. Our approach to take advantage of the resource of the human genome sequences and the results from our microarray experiments should be validated by further studies of promoter occupancy and TF perturbation.

• Journal of Nutrition and Health
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• 제51권1호
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• pp.23-30
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• 2018

Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn-($1{\mu}M$ Zn) or Zn+($15{\mu}M$ Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.53-54
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• 2001

Expression of HAND genes in sympathetic adrenal lineage suggests that HAND genes may regulate Mash-I independent neuronal genes. HAND genes are also expressed in other cell types, e.g. Cardiac cells, trophoblasts, and decidua, suggesting that HAND genes are not cell fate determination factors. It is unclear how HAND genes function specifically in different types of cells. Combinational actions of HANDs with other cell-lineage specific transcription factor may determine each cell fate and differentiation processes. Identifying the transcription target genes of HANDs and Mash-I will be important to elucidate the function of these bHLH factors in SNS factors in SNS development. (omitted)

• 자연과학논문집
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• 제14권1호
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• pp.51-61
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• 2004

박테리오 파아지 T7 RNA 중합효소는 어떤 전사인자도 관여하지 않는 간단한 시스템으로 파아지 RNA 중합효소와 전사촉진제만의 단백질-DNA 상호작용에 의해 전사가 진행된다. T7 파아지의 숙주 세포의 파괴에 관여하는 T7 파아지 lysozyme은 전사를 억제하고, 전사종결에 영향을 미친다. 따라서 T7 파아지 lysozyme 유전자를 대장균 발현 벡터에 삽입하여 pT7lys를 얻었고, 발현시켜 Ni-NTA column chromatography로 순수 분리하였다. T7 파아지 lysozyme은 SDS-gel에서 단일 밴드로 확인하였으며, amidase 활성 역시 확인하였다. 염기서열 특이적 전사 종결에 미치는 T7 파아지 lysozyme의 역할을 알아보기 위하여, rrnB T1 전사종결 인자 부근에서의 전사연장 복합체 제조에 미치는 T7 파아지 lysozyme의 영향력을 조사하였다. 이 결과 T7 파아지 lysozyme 존재 하에 형성되는 전사연장 복합체는 불안정함을 알 수 있었다.

• BMB Reports
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• 제55권11호
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• pp.535-540
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• 2022

Ribosomes, acting as the cellular factories for protein production, are essential for all living organisms. Ribosomes are composed of both proteins and RNAs and are established through the coordination of several steps, including transcription, maturation of ribosomal RNA (rRNA), and assembly of ribosomal proteins. In particular, diverse factors required for ribosome biogenesis, such as transcription factors, small nucleolar RNA (snoRNA)-associated proteins, and assembly factors, are tightly regulated by various post-translational modifications. Among these modifications, small ubiquitin-related modifier (SUMO) targets lots of proteins required for gene expression of ribosomal proteins, rRNA, and snoRNAs, rRNA processing, and ribosome assembly. The tight control of SUMOylation affects functions and locations of substrates. This review summarizes current studies and recent progress of SUMOylation-mediated regulation of ribosome biogenesis.

• BMB Reports
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• 제46권12호
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• pp.567-574
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• 2013

Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting post-translational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed.

• 한국초지조사료학회지
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• 제43권1호
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• pp.42-49
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• 2023

The plant-specific NAC transcription factors control various biological processes, including plant development and stress responses. We have isolated an ANAC032 gene, one of the NAC transcription factor family, which was highly activated by multi-abiotic stresses, including high salt and drought in Arabidopsis. Here, we generated transgenic plants constitutively expressing ANAC032 and its knockout to identify the functional roles of ANAC032 in Arabidopsis under abiotic stress responses. The ANAC032-overexpressing plants showed enhanced tolerance to salinity and drought stresses. The anac032 knockout mutants were observed no significant changes under the high salt and drought conditions. We also monitored the expression of high salt and drought stress-responsive genes in the ANAC032 transgenic plants and anac032 mutant. The ANAC032 overexpression upregulated the expression of stress-responsive genes, RD29A and ERD10, under the stresses. Thus, our data identify that transcription factor ANAC032 plays as an enhancer for salinity and drought tolerance through the upregulation of stress-responsive genes and provides useful genetic traits for generating multi-abiotic stress-tolerant forage crops.

• IMMUNE NETWORK
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• 제16권3호
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• pp.176-182
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• 2016

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.