• Journal of Life Science
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• v.28 no.7
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• pp.765-771
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• 2018

Peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) is a key transcription factor that regulates adipogenesis, and epigenetic control of $PPAR{\gamma}$ is of great interest in obesity-inhibition research. Our previous study showed that CACUL1 (CDK2-associated cullin domain 1) acts as a corepressor that inhibits $PPAR{\gamma}$ transcriptional activity and adipocyte differentiation. Here, we investigated the roles of protein arginine methyltransferase 5 (PRMT5), a novel binding partner of CACUL1, in regulating $PPAR{\gamma}$. The interaction between PRMT5 and CACUL1 was shown by immunoprecipitation assay in vivo and GST pulldown assay in vitro. As shown by luciferase reporter assay, PRMT5 and CACUL1 cooperated to inhibit the transcriptional activity of $PPAR{\gamma}$. The suppressive role of PRMT5 in adipogenesis was examined by Oil Red O staining using 3T3-L1 cells, which stably overexpress or deplete PRMT5. Overexpression of PRMT5 suppresses $PPAR{\gamma}$-mediated adipogenesis, whereas PRMT5 knockdown increases lipid accumulation in 3T3-L1 cells. Consistently, PRMT5 attenuates the expression of Lpl and aP2, the target genes of $PPAR{\gamma}$, as demonstrated by RT-qPCR analysis. Overall, these results suggest that PRMT5 interacts with CACUL1 to impair the transcriptional activity of $PPAR{\gamma}$, leading to the inhibition of adipocyte differentiation. Therefore, the regulation of PRMT5 enzymatic activity may provide a clue to develop an anti-obesity drug.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10255-10262
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• 2015

High risk human papillomavirus (HR-HPV) E2 proteins play roles in transcriptional regulation and are commonly functionally disrupted when the HPV genome integrates into host chromosomes. Some 15-40% of cancer cases, however, contain an intact E2 gene or episomal HPV. In these cases, polymorphism of the E2 gene might be involved. This study aimed to determine polymorphisms of the E2 gene in episomal HPV16 detected in high grade squamous intraepithelial lesions and squamous cell carcinomas and altered functions compared to the E2 prototype. The E2 gene was amplified and sequenced. Two expression vectors containing E2 gene polymorphisms were constructed and transfected in SiHa and C33A cells, then E6 gene as well as Il-10 and TNF-${\alpha}$ expression was determined by quantitative RT-PCR. Expression vectors and reporter vectors containing the HPV16 long control region (LCR) were co-transfected and transcriptional activity was determined. The results showed that a total of 32 nucleotides and 23 amino acids were changed in all 20 cases of study, found in the transactivation (TA) domain, hinge (H) region and DNA binding (DB) domain with 14, 5 and 13 nucleotide positions. They mostly caused amino acid change. The expressing vectors containing different E2 gene polymorphisms showed E6 mRNA suppression, TNF-${\alpha}$ mRNA suppression and IL-10 induction but no statistically significant differences when compared to the E2 prototype. Moreover, promoter activity in HPV16 LCR was not affected by E2 protein with different gene polymorphisms, in contrast to nucleotide variations in LCR that showed an effect on transcription activity. These results demonstrated that E2 gene polymorphisms of episomal HPV16 did not affect transcriptional regulation and suggested that nucleotide variation as well as epigenetic modification of the LCR might play a role in inducing malignant transformation of cells containing episomal HPV16.

• BMB Reports
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• v.38 no.4
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• pp.500-506
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• 2005

HOX11 encodes a homeodomain-containing transcription factor which directs the development of the spleen during embryogenesis. While HOX11 expression is normally silenced through an unknown mechanism in all tissues by adulthood, the deregulation of HOX11 expression is associated with leukemia, such as T-cell acute lymphoblastic leukemia. The elucidation of regulatory elements contributing to the molecular mechanism underlying the regulation of HOX11 gene expression is of great importance. Previous reports of HOX11 regulatory elements mainly focused on the 5'-flanking region of HOX11 on the chromosome related to transcriptional control. To expand the search of putative cis-elements involved in HOX11 regulation at the post-transcriptional level, we analyzed HOX11 mRNA 3'-untranslated region (3'UTR) and found an AU-rich region. To characterize this AU-rich region, in vitro analysis of HOX11 mRNA 3'UTR was performed with human RNA-binding protein HuR, which interacts with AU-rich element (ARE) existing in the 3'UTR of many growth factors' and cytokines' mRNAs. Our results showed that the HOX11 mRNA 3'UTR can specifically bind with human HuR protein in vitro. This specific binding could be competed effectively by typical ARE containing RNA. After the deletion of the AU-rich region present in the HOX11 mRNA 3'UTR, the interaction of HOX11 mRNA 3'UTR with HuR protein was abolished. These findings suggest that HOX11 mRNA 3'UTR contains cis-acting element which shares similarity in the action pattern with RE-HuR interactions and may involve in the post-transcriptional regulation of the HOX11 gene.

• Journal of Life Science
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• v.25 no.2
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• pp.136-150
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• 2015

Pseudomonas syringae pathovar tabaci is a plant pathogenic bacterium that causes wildfire disease in tobacco plants. In P. syringae pv. tabaci, PsyI, a LuxI-type protein, acts as an AHL synthase, while primary and secondary sequence analysis of PsyR has revealed that it is a homolog of the LuxR-type transcriptional regulator that responds to AHL molecules. In this study, using phenotypic and genetic analyses in P. syringae pv. tabaci, we show the effect of PsyR protein as a quorum-sensing (QS) transcriptional regulator. Regulatory effects of PsyR on swarming motility and production of siderophores, tabtoxin, and N-acyl homoserine lactones were examined via phenotypic assays, and confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Further qRT-PCR showed that PsyR regulates expression of these virulence genes in response to environmental signals. However, an upstream region of the gene was not bound with purified MBP-PsyR protein; rather, PsyR was only able to shift the upstream region of psyI. These results suggested that PsyR may be indirectly controlled via intermediate-regulatory systems and that auto-regulation by PsyR does not occur.

• Journal of the korean academy of Pediatric Dentistry
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• v.29 no.3
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• pp.337-344
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• 2002

Runx2, a Runt-related osteoblast-specific transcription factor, is essential for osteoblast differentiation and function. Runx2 was identified as a key regulator of osteoblast-specific gene expression through its binding to the OSE2 element present in these genes. However, little is known about the signaling mechanism regulating Runx2 activity. This study examines the role of protein kinase C (PKC) pathway and mitogen-activated protein kinase (MAPK) pathway in regulating Runx2 and bone marker genes (osteopontin; OP, osteocalcin; OC). Luciferase assay and Northern blot analysis suggested that the stimulation of PKC by PMA increased transcription activity of Runx2 and bone marker genes (OP and OC) and also increased expression of Runx2. The stimulation of MAPK by okadaic acid increased transcription activity of Runx2 and bone marker genes (OP and OC). Pretreatment with PD98059 (Erk pathway inhibitor) and SB203580 (P38 pathway inhibitor) prior to PMA treatment decreased PMA stimulated Runx2 activity. Together these results indicate that both PKC and MAPKs are involved in the regulation of Runx2 activity and also the stimulation of Runx2 transcriptional activity by the PKC pathway is through activation of MAPK pathway.

• Animal cells and systems
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• v.6 no.3
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• pp.271-275
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• 2002

Rph1 and Gisl are damage-responsive repressors involved in PHR1 expression. They have two $C_{2}$H/ sub 2/ zinc finger motifs as putative DNA binding domains and N-terminal conserved domain with unknown function. They are also found in the human retinoblastoma binding protein 2 and the mouse jumonji- encoded protein. The repressors are able to bind to A $G_{4}$ sequence within a 39-bp sequence called upstream repressing sequence of PHR1 promoter (UR $S_{PHR1}$) responsible for the damage-response of PHR1. We report here that Rph1 is predominantly localized in the nucleus as examined by fluorescence microscopic analysis with GFP-Rph1 fusion protein. On the basis of the fact that the A $G_{4}$ sequence that is recognized by Rph1 and Gisl is also recognized by Msn2 and Msn4 in a process of stress response, we a1so tried to examine the in vivo function of A $G_{4}$ and the role of Msn2 and Msn4 in PHR1 expression. Our results demonstrate that Msn2 and Msn4 are actually required for the basal transcription of PHR1 expression but not for its damage induction. When A $G_{4}$ sequence was inserted into the minimal promoter of the cyc1-LacZ reporter, the increased LacZ expression was observed indicating its involvement in transcriptional activation. The data suggest that the A $G_{4}$ is primarily required for basal transcriptional activation of PHR1 or CYC1 promoter through the possible involvement of Msn2 and Msn4. However, since the A $G_{4}$ is also involved in the repression of PHR1 via Rphl and Gisl, it is proposed that A $G_{4}$ functions as either URS or upstream activating sequence (UAS) depending on the promoter context.t.

• BMB Reports
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• v.42 no.9
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• pp.593-598
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• 2009

Cell cycle progression is regulated by both transcriptional and post-transcriptional mechanisms. MicroRNAs (miRNAs) emerge as a new class of small non-coding RNA regulators of cell cycle as recent evidence suggests. It is hypothesized that expression of specific miRNAs oscillates orderly along with cell cycle progression. However, the oscillated expression patterns of many candidate miRNAs have yet to be determined. Here, we describe miRNA expression profiling in double-thymidine synchronized HeLa cells as cell cycle progresses. Twenty-five differentially expressed miRNAs were classified into five groups based on their cell cycle-dependent expression patterns. The cyclic expression of six miRNAs (miR-221, let-7a, miR-21, miR-34a, miR-24, miR-376b) was validated by real-time quantitative RT-PCR (qRT-PCR). These results suggest that specific miRNAs, along with other key factors are required for maintaining and regulating proper cell cycle progression. The study deepens our understanding on cell cycle regulation.

• Molecules and Cells
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• v.27 no.1
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• pp.15-19
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• 2009

Notch signaling is controlled at multiple levels. In particular, stabilized Notch receptor activation directly affects the transcriptional activations of Notch target genes. Although some progress has been made in terms of defining the regulatory mechanism that alters Notch stability, it has not been determined whether Notch1/NICD stability is regulated by $GSK-3{\alpha}$. Here, we show that Notch1/NICD levels are significantly regulated by $GSK-3{\beta}$ and by $GSK-3{\alpha}$. Treatment with LiCl (a specific GSK-3 inhibitor) or the overexpression of the kinase-inactive forms of $GSK-3{\alpha}/{\beta}$ significantly increased Notch1/NICD levels. Endogenous NICD levels were also increased by either $GSK-3{\alpha}/{\beta}$- or $GSK-3{\alpha}$-specific siRNA. Furthermore, it was found that $GSK-3{\alpha}$ binds to Notch1. Deletion analysis showed that at least three Thr residues in Notch1 (Thr-1851, 2123, and 2125) are critical for its response to LiCl, which increased not only the transcriptional activity of endogenous NICD but also Hes1 mRNA levels. Taken together, our results indicate that $GSK-3{\alpha}$ is a negative regulator of Notch1/NICD.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.164-169
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• 2008

Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, 24 and 48h in human neuroblastoma SH-SY5Y cell line. Some of the identified genes by MeHg treatment were significant at early time points (3h), while that of others was at late time points (48h). The early response genes that may represent those involved directly in the MeHg response included pantothenate kinase 3, a kinase (PRKA) anchor protein (yotiao) 9, neurotrophic tyrosine kinase, receptor, type 2 gene, associated with NMDA receptor activity regulation or perturbations of central nervous system homeostasis. Also, when SH-SY5Y cells were subjected to a longer exposure (48h), a relative increase was noted in a gene, glutamine-fructose-6-phosphate transaminase 1, reported that overexpression of this gene may lead to the increased resistance to MeHg. To confirm the alteration of these genes in cultured neurons, we then applied real time-RT PCR with SYBR green. Thus, this result suggests that a neurotoxic effect of the MeHg might be ascribed that MeHg alters neuronal receptor regulation or homeostasis of neuronal cells in the early phase. However, in the late phase, it protects cells from neurotoxic effects of MeHg.

• Journal of Microbiology
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• v.40 no.4
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• pp.305-312
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• 2002

The effects of two natural polyamines (putrescine and spermidine) on the synthesis of ArcA, a response regulator of the Arc two-component signal transduction system, were studied using an E. coli mutant deficient in polyamine biosynthesis. Endogenous polyamine deficiency of the mutant resulted in marked reduction in the ArcA level determined by Western blot analysis. Putrescine supplement to the growth medium effectively increased the ArcA level of the mutant in a concentration-dependent manner. Spermidine also stimulated the ArcA level in the mutant to a greater degree than putrescine. Expression of arcA'::lacZ operon fusion in the mutant was stimulated 6-fold and 10-fold by putrescine and spermidine at a 1mM concentration, respectively, indicating that the stimulatory effect of the polyamines on ArcA synthesis is due to transcriptional induction, and that spermidine is a more potent arcA inducer than putrescine. The polyamine-dependent arcA'::lacZ induction was growth-phase-dependent and independent of either arcA or fnr which are two regulators involved in anaerobic stimulation of the Arch level. These results suggested that putrescine and spermidine polyamines may be potential intracellular signal molecules in the control of arcA expression, and thereby may play an important role in cellular metabolism.