• Genomics & Informatics
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• v.19 no.2
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• pp.17.1-17.13
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• 2021

Breast cancer is one of the leading causes of cancer in women all over the world and accounts for ~25% of newly observed cancers in women. Epigenetic modifications influence differential expression of genes through non-coding RNA and play a crucial role in cancer regulation. In the present study, epigenetic regulation of gene expression by in-silico analysis of histone modifications using chromatin immunoprecipitation sequencing (ChIP-Seq) has been carried out. Histone modification data of H3K4me3 from one normal-like and four breast cancer cell lines were used to predict miRNA expression at the promoter level. Predicted miRNA promoters (based on ChIP-Seq) were used as a probe to identify gene targets. Five triple-negative breast cancer (TNBC)-specific miRNAs (miR153-1, miR4767, miR4487, miR6720, and miR-LET7I) were identified and corresponding 13 gene targets were predicted. Eight miRNA promoter peaks were predicted to be differentially expressed in at least three breast cancer cell lines (miR4512, miR6791, miR330, miR3180-3, miR6080, miR5787, miR6733, and miR3613). A total of 44 gene targets were identified based on the 3'-untranslated regions of downregulated mRNA genes that contain putative binding targets to these eight miRNAs. These include 17 and 15 genes in luminal-A type and TNBC respectively, that have been reported to be associated with breast cancer regulation. Of the remaining 12 genes, seven (A4GALT, C2ORF74, HRCT1, ZC4H2, ZNF512, ZNF655, and ZNF608) show similar relative expression profiles in large patient samples and other breast cancer cell lines thereby giving insight into predicted role of H3K4me3 mediated gene regulation via the miRNA-mRNA axis.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.1
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• pp.37-44
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• 2017

Regulation of vascular smooth muscle cell (VSMC) phenotype plays an essential role in many cardiovascular diseases. In the present study, we provide evidence that $kr{\ddot{u}}ppel$-like factor 8 (KLF8) is essential for tumor necrosis factor ${\alpha}$ ($TNF{\alpha}$)-induced phenotypic conversion of VSMC obtained from thoracic aorta from 4-week-old SD rats. Stimulation of the contractile phenotype of VSMCs with $TNF{\alpha}$ significantly reduced the VSMC marker gene expression and KLF8. The gene expression of KLF8 was blocked by $TNF{\alpha}$ stimulation in an ERK-dependent manner. The promoter region of KLF8 contained putative Sp1, KLF4, and $NF{\kappa}B$ binding sites. Myocardin significantly enhanced the promoter activity of KLF4 and KLF8. The ectopic expression of KLF4 strongly enhanced the promoter activity of KLF8. Moreover, silencing of Akt1 significantly attenuated the promoter activity of KLF8; conversely, the overexpression of Akt1 significantly enhanced the promoter activity of KLF8. The promoter activity of SMA, $SM22{\alpha}$, and KLF8 was significantly elevated in the contractile phenotype of VSMCs. The ectopic expression of KLF8 markedly enhanced the expression of SMA and $SM22{\alpha}$ concomitant with morphological changes. The overexpression of KLF8 stimulated the promoter activity of SMA. Stimulation of VSMCs with $TNF{\alpha}$ enhanced the expression of KLF5, and the promoter activity of KLF5 was markedly suppressed by KLF8 ectopic expression. Finally, the overexpression of KLF5 suppressed the promoter activity of SMA and $SM22{\alpha}$, thereby reduced the contractility in response to the stimulation of angiotensin II. These results suggest that cross-regulation of KLF family of transcription factors plays an essential role in the VSMC phenotype.

• Journal of Life Science
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• v.33 no.1
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• pp.34-42
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• 2023

In a signal transduction network, from the perception of stress signals to stress-responsive gene ex- pression, binding of various transcription factors to cis-acting elements in stress-responsive promoters coordinate the adaptation of plants to abiotic stresses. Among the AP2/ERF transcription factor family genes, group VII ERF genes, such as RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/ HRE2, are known to be involved in the response to hypoxia stress in Arabidopsis. In this study, we dissected the HRE1 promoter to identify hypoxia-responsive region(s). The 1,000 bp upstream promoter region of HRE1 showed increased promoter activity in Arabidopsis protoplasts and transgenic plants under hypoxia conditions. Analysis of the promoter deletion series of HRE1, including 1,000 bp, 800 bp, 600 bp, 400 bp, 200 bp, 100 bp, and 50 bp upstream promoter regions, using firefly luciferase and GUS as reporter genes indicated that the -200 to -100 region of the HRE1 promoter is responsible for the transcriptional activation of HRE1 in response to hypoxia. In addition, we identified two putative hypoxia-responsive cis-acting elements, the ERF-binding site and DOF-binding site, in the -200 to -100 region of the HRE1 promoter, suggesting that the expression of HRE1 might be regulated via the ERF transcription factor(s) and/or DOF transcription factor(s). Collectively, our results suggest that HRE1 contains hypoxia-responsive cis-acting elements in the -200 to -100 region of its promoter.

• Molecules and Cells
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• v.19 no.2
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• pp.239-245
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• 2005

Factor for inversion stimulation (FIS), the Escherichia coli protein, is a positive regulator of the transcription of genes that encode stable RNA species, such as rRNA and tRNA. Transcription of the rnpB gene encoding M1 RNA, the catalytic subunit of E. coli RNase P, rapidly declines under stringent conditions, as does that of other stable RNAs. There are multiple putative FIS binding sites upstream of the rnpB promoter. We tested whether FIS binds to these sites, and if so, how it affects rnpB transcription. In vitro binding assays revealed specific binding of FIS to multiple sites in the rnpB promoter region. Interestingly, FIS bound not only to the upstream region of the promoter, but also to the region from +4 to +18. FIS activated rnpB transcription in vitro, but the level of activation was much lower than that of the rrnB promoter for rRNA. We also examined the effects of FIS on rnpB transcription in vivo using isogenic $fis^+$ and $fis^-$ strains. rnpB transcription was higher in the $fis^-$ than the $fis^+$ cells during the transitions from lag to exponential phase, and from exponential to stationary phase.

• Molecules and Cells
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• v.23 no.1
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• pp.23-29
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• 2007

Cyclic AMP-responsive element binding protein (CREB) is known to be associated with angiogenesis. In the present study we investigated the possible role of CREB in the expression of vascular endothelial growth factor (VEGF) by mouse macrophages. Over-expression of CREB increased VEGF secretion by cells of the RAW264.7 mouse macrophage cell line. It also increased the promoter activity of a mouse reporter driven by the VEGF promoter, while a dominant negative CREB (DN-CREB) abrogated the activity, suggesting that CREB mediates VEGF transcription. Forskolin, an adenylyl cyclase activator, stimulated VEGF transcription, and the PKA inhibitor H89 abolished this effect. IFN-${\gamma}$, a potent cytokine, stimulated VEGF expression only in part through the PKA-CREB pathway. These results indicate that PKA phosphorylates CREB and so induces VEGF gene expression. An analysis of mutant promoters revealed that one of the putative CREB responsive elements (CREs), at -399 ~ -388 in the promoter, is critical for CREB-mediated VEGF promoter activity, and the significance of this CRE was confirmed by chromatin immunoprecipitation assays.

• International Journal of Oral Biology
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• v.40 no.1
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• pp.19-25
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• 2015

Osteocytes may function as mechanotransducers by regulating local osteoclastogenesis. Reduced availability of oxygen, i.e. hypoxia, could occur during disuse, bone development, and fracture. Receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) is an osteoblast/stromal cell derived essential factor for osteoclastogenesis. The hypoxia induced osteoclastogenesis via increased RANKL expression in osteoblasts was demonstrated. Hypoxic regulation of gene expression generally involves activation of the hypoxia-inducible factor (HIF) transcription pathway. In the present study, we investigated whether hypoxia regulates RANKL expression in murine osteocytes and HIF-$1{\alpha}$ mediates hypoxia-induced RANKL expression by transactivating RANKL promoter, to elucidate the role of osteocyte in osteoclastogenesis in the context of hypoxic condition. The expression levels of RANKL mRNA and protein, as well as hypoxia inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) protein, were significantly increased in hypoxic condition in MLO-Y4s. Constitutively active HIF-$1{\alpha}$ alone significantly increased the levels of RANKL expression in MLO-Y4s under normoxic conditions, whereas dominant negative HIF-$1{\alpha}$ blocked hypoxia-induced RANKL expression. To further explore to find if HIF-$1{\alpha}$ directly regulates RANKL transcription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity, whereas mutations of putative HIF-$1{\alpha}$ binding elements in RANKL promoter prevented this hypoxia-induced RANKL promoter activity in MLO-Y4s. These results suggest that HIF-$1{\alpha}$ mediates hypoxia-induced up-regulation of RANKL expression, and that in osteocytes of mechanically unloaded bone, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in osteocytes.

• Animal cells and systems
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• v.4 no.2
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• pp.181-186
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• 2000

The expression of $p21^{WAF1/ClP1/SDl1}$, one of the cyclin-dependent kinase inhibitors, is regulated by a variety of transcription factors including p53 and STAT. Heat shock induces the expression of p21 in a temperature- and time-dependent manner. Although the p21 induction by heat shock has been reported to be controlled by p53, a p53-independent mechanism Is also involved. To understand the p53-independent regulation of heat shock-induced p21 expression, we searched the promoter region of p21 gene and found one or two heat shock element (HSE)-like sequences in human, rat, and mouse. Electromobility shift assay (EMSA) showed that heat shock factor (HSF) could bind to these HSE-like sequences In response to heat shock, even though to a lesser extent than to HSE. In addition, p21 promoter deletion analysis revealed that heat shock activated a p21 deletion promoter construct containing the HSE-like sequences but lacking p53-binding sites, but not a promoter construct containing neither HSE-like sequences nor the p53-responsive element. Furthermore, the p21 induction by heat shook was significantly inhibited in confluent cells in which heat shock-induced HSF activation was reduced. These results suggest that the transcriptional regulation of p21 by heat shock may be mediated through activation and binding to HSE-like sequences of HSF.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.120-120
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• 1995

The total size of the pF AR1, a genomic clone of Frankia FaCI, was estimated to be about 44Kb by summation of the individual fragment length generated by single or double restriction enzymes. Southern hybridization analyses with Azotobacter vinelandii nif-genes as probes and partial sequencing analyses of the subclones revealed that organization of the nif-gene in the FaCI strain was nifV, H, D, K, E, N, X, W, B. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes but the positioning of the nifV-like gene relative to the nifHDK cluster differs. A consensus nif-promoter-like sequence, found at 5' of nifH, was not detected upstream of the niJV-like gene. nifV-like gene contained a ORF of 1206 NT encoding 401 amino acids. The nucleotide sequence and deduced amino acid sequence of the gene exhibit homology value of 65% and 41% with that from A vinelandii, respectively. The putative Shine-Dargamo sequences were present preceding nitK, nifH, D, K, and nifE, and in nitK gene putative start codon GTG was detected instead of A TG. The nucleotide and amino acid sequence of niIK of FaCI showed 82% and 76% homolgy with those of Frankia HFPCc 13, respectively. Amino acid sequence of niIK showed 69% and 61% homology with those of A vinelandii, Klebsiella pnewnoniae, respectively, while that of nifE 73% and 71%, respecti vely.i vely.

• BMB Reports
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• v.37 no.2
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• pp.177-184
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• 2004

The Crustacean hyperglycemic hormone (CHH) has been shown to exist as multiple molecular forms in several crustacean species. In Penaeus monodon, a gene encoding CHH (so-called Pem-CHH1) was recently described. In this study, the molecular structures of two other CHH genes (Pem-CHH2 and Pem-CHH3) are reported. Both the Pem-CHH2 and Pem-CHH3 genes contain three exons that are separated by two introns that are similar to the structure of other genes in the same family. An analysis of the upstream nucleotide sequences of each Pem-CHH gene has identified the putative promoter element (TATA box) and putative binding sites for several transcription factors. The binding sites for CREB, Pit-1, and AP-1 were found upstream of all three Pem-CHH genes. A Southern blot analysis showed that at least one copy of each Pem-CHH gene was located within the same 10 kb genomic DNA fragment. These results suggest that the CHH genes are arranged in a cluster in the genome of P. monodon, and that their expression may be modulated by similar mechanisms.

• Journal of Life Science
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• v.27 no.8
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• pp.857-863
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• 2017

The FBJ murine osteosarcoma viral oncogene homolog B (FosB) gene is located at chromosome 19, and encodes 43 Kda protein. Functionally, the FosB gene is important for differentiation, development, and pathogenesis. Furthermore, the FosB gene is suggested as possible biomarker for tracing disease prognosis. In this study, we constructed plasmid containing a FosB promoter region and evaluate its promoter activity. We analyzed the putative promoter region in FosB genomic DNA using bioinformatics program, and we found important regulatory elements in 1 Kb upstream from transcription start site (TSS). Therefore, we performed polymerase chain reaction (PCR) amplification on region from-1,555 upstream to +73 of the FosB genomic DNA, and PCR product was inserted into TA vector to create the $TA-1^{st}FosBp$ plasmid. We then prepared the primer sets, which contain a restriction enzyme site for Kpn1 and Nhe1, in order to reinsert into the TA vector to prepare $TA-2^{nd}FosBp$ plasmid. It was finally subcloned into pGL3-luc vector after enzyme cutting. To evaluate whether the cloned plasmid is useful in cell based experiment, we performed luciferase assay with pGL3-FosBp-luctransfection. FosB promoter activity was increased compared to empty vector, and this activity was significantly increased by treatment of doxorubicin and taxol. We obtained consistent data on regulation of FosB gene expression after anticancer drug treatment using Western blot analysis. The results suggest that promoter cloning of the human FosB gene is very useful for studying gene expression and analyzing biomarkers.