• BMB Reports
• /
• v.42 no.3
• /
• pp.154-159
• /
• 2009

JARID1B (jumonji AT rich interactive domain 1B) is a large nuclear protein that is highly expressed in breast cancers and is proposed to function as a repressor of gene expression. In this paper, a phage display screen using the N-terminus of JARID1B as bait identified one of the JARID1B interacting proteins, namely PcG protein (Polycomb group) hPc2. We demonstrated that the C-terminal region, including the COOH box, was required for the interaction with the N-terminus of JARID1B. In a reporter assay system, co-expression of JARID1B with hPc2 significantly enhanced the transcriptional repression. These results support a role for hPc2 acting as a transcriptional co-repressor.

• BMB Reports
• /
• v.44 no.3
• /
• pp.199-204
• /
• 2011

Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insight into the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.

• Biomolecules & Therapeutics
• /
• v.17 no.1
• /
• pp.12-16
• /
• 2009

One of the WHSC1/MMSET/NSD2 variant RE-IIBP is a histone H3-K27 methyltransferase with transcriptional repression activity. Overexpression of RE-IIBP in various types of leukemia suggests it's role in leukemogenesis. Here we identify two proteins, KRAB zinc finger protein ZNF 350 and enolase-1 as RE-IIBP interacting proteins by yeast two-hybrid screening and confirmed direct interaction in vivo and in vitro. Both proteins have been known for their role in transcriptional repression. Reporter assays using transient transfection demonstrated that both ZNF 350 and enolase-1 proteins synergistically repressed transcription with RE-IIBP, respectively. These results indicate both proteins have roles in RE-IIBP mediated transcriptional repression by involving co-repressor complex.

• BMB Reports
• /
• v.51 no.2
• /
• pp.92-97
• /
• 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.

• Molecules and Cells
• /
• v.20 no.2
• /
• pp.241-246
• /
• 2005

PDK-1 activates PI3-kinase/Akt signaling and regulates fundamental cellular functions, such as growth and survival. NF-${\kappa}B$ is involved in the induction of a variety of cellular genes affecting immunity, inflammation and the resistance to apoptosis induced by some anti-cancer drugs. Even though the crucial involvement of the PI3-kinase/Akt pathway in the anti-apoptotic activation of NF-${\kappa}B$ is well known, the exact role of PDK-1 as well as PI3-kinase/Akt in NF-vactivation is not understood. Here we demonstrate that PDK-1 plays a pivotal role in transcriptional activation of NF-${\kappa}B$ by dissociating the transcriptional co-repressor HDAC1 from the p65 subunit of NF-${\kappa}B$. The association of CBP with p65 was not directly modulated by PDK-1 or by PI3-kinase. Etoposide activated NF-${\kappa}B$ through PI3-kinase/Akt, and the transcription activation domain (TAD) of p65 was further activated by wild-type PDK-1. Overexpression of a dominant negative PDK-1 mutant decreased etoposide-induced NF-${\kappa}B$ transcription and further down-regulated the ectopic HDAC1-mediated decrease in NF-${\kappa}B$ transcriptional activity. Thus activation of PDK-1 relieves the HDAC1-mediated repression of NF-${\kappa}B$ that may be related to basal as well as activated transcription by NF-${\kappa}B$. This effect may also explain the role of the PI3-kinase/PDK-1 pathway in the anti-apoptotic function of NF-${\kappa}B$ associated with the chemoresistance of cancer cells.

• BMB Reports
• /
• v.52 no.6
• /
• pp.403-408
• /
• 2019

Dorsoventral patterning of body axis in vertebrate embryo is tightly controlled by a complex regulatory network of transcription factors. Ventx1.1 is known as a transcriptional repressor to inhibit dorsal mesoderm formation and neural differentiation in Xenopus. In an attempt to identify, using chromatin immunoprecipitation (ChIP)-Seq, genome-wide binding pattern of Ventx1.1 in Xenopus gastrulae, we observed that Ventx1.1 associates with its own 5'-flanking sequence. In this study, we present evidence that Ventx1.1 binds a cis-acting Ventx1.1 response element (VRE) in its own promoter, leading to repression of its own transcription. Site-directed mutagenesis of the VRE in the Ventx1.1 promoter significantly abrogated this inhibitory autoregulation of Ventx1.1 transcription. Notably, Ventx1.1 and Xcad2, an activator of Ventx1.1 transcription, competitively co-occupied the VRE in the Ventx1.1 promoter. In support of this, mutation of the VRE down-regulated basal and Xcad2-induced levels of Ventx1.1 promoter activity. In addition, overexpression of Ventx1.1 prevented Xcad2 from binding to the Ventx1.1 promoter, and vice versa. Taken together, these results suggest that Ventx1.1 negatively regulates its own transcription in competition with Xcad2, thereby fine-tuning its own expression levels during dorsoventral patterning of Xenopus early embryo.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.6
• /
• pp.1249-1255
• /
• 2004

This study has developed Corynebacterium ammoniagenes (c. ammoniagenes) purine gene transcriptional reporters (purF-lacZ and purE-lacZ) that function in Escherichia coli (E. coli) DH5a. After transformation of a C. ammoniagenes gDNA library into E. coli cells harboring either purF-lacZ or purE-lacZ, C. ammoniagenes clones were obtained that repress purF-lacZ and purE-lacZ gene expression. The potential purE and purF regulatory genes are homologous to the genes encoding transcription regulators, the regulatory subunit of RNA polymerase, and genes for purine nucleotide biosynthesis of various bacteria. The C. ammoniagenes purE-lacZ and purF-lacZ reporters were repressed by adenine and guanine within E. coli, indicating similarity in the regulatory mechanism of purine biosynthesis in C. ammoniagenes and E. coli. Gene regulation of pur-lacZ by adenine and guanine was partly abolished in cells expressing potential purine regulatory genes, indicating functionality of the purine gene regulators in repression of purE-lacZ and purF-lacZ. The purE-lacZ and purF-lacZ reporters can be used for the screening of genes involved in the regulation of the de novo synthesis of the purine nucleotides.

• International Journal of Oral Biology
• /
• v.32 no.4
• /
• pp.119-125
• /
• 2007

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3(wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation.

• Molecules and Cells
• /
• v.38 no.3
• /
• pp.259-266
• /
• 2015

The regulation of flowering time has crucial implications for plant fitness. MicroRNA156 (miR156) represses the floral transition in Arabidopsis thaliana, but the mechanisms regulating its transcription remain unclear. Here, we show that two AGAMOUS-like proteins, AGL15 and AGL18, act as positive regulators of the expression of MIR156. Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression. Histochemical analysis further indicated that the double mutants showed a reduction in MIR156 promoter strength. The double mutants also showed reduced abundance of pri-miR156a and pri-miR156c, two of the primary transcripts from MIR156 genes. Electrophoretic mobility shift assays demonstrated that AGL15 directly associated with the CArG motifs in the MIR156a/c promoters. AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15. GFP reporter assays and bimolecular fluorescence complementation (BiFC) showed that AGL15 and AGL18 co-localize in the nucleus and confirmed their in vivo interaction. Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts. Taking these data together, we present a model for the transcriptional regulation of MIR156. In this model, AGL15 and AGL18 may form a complex along with other proteins, and bind to the CArG motifs of the promoters of MIR156 to activate the MIR156 expression.