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Transactivation Activity (transactivation + activity)
Selected AbstractsModulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domainsFEBS JOURNAL, Issue 10 2006Pei-Yao Liu Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions. [source] Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2003Kwok Keung Chan Abstract SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder. © 2003 Wiley-Liss, Inc. [source] SEI family of nuclear factors regulates p53-dependent transcriptional activationGENES TO CELLS, Issue 8 2005Rie Watanabe-Fukunaga SEI family proteins, p34SEI-1 and SEI-2(TRIP-Br2), are nuclear factors that are implicated in cell cycle regulation through interaction with CDK4/CyclinD and E2F-1/DP-1 complexes. Here we report that the SEI family proteins regulate transcriptional activity of p53 tumor suppressor protein. Expression of SEI-1, SEI-2 or SEI-3 strongly stimulates p53-dependent gene activation in HeLa and U2OS cells but not in p53-deficient Saos2 or p53-knockdown HeLa cells. SEI proteins possess an intrinsic transactivation activity, interact with the coactivator CREB-binding protein, and cooperate synergistically with the ING family of chromatin-associated proteins to stimulate the transactivation function of p53. Doxycycline-induced expression of SEI proteins results in activation of the p21 gene and inhibition of cell growth, but the growth arrest was not suppressed by the siRNA-mediated knockdown of the endogenous p53 protein. These results indicate that the SEI family of nuclear proteins regulates p53 transcriptional activity and a p53-independent signaling pathway leading to growth inhibition. [source] Analysis of a non-functional HNF-1, (TCF1) mutation in Japanese subjects with familial type 1 diabetesHUMAN MUTATION, Issue 4 2001Issei Yoshiuchi Abstract Mutations in the transcription factor hepatocyte nuclear factor-1, (HNF-1,; gene symbol TCF1) cause maturity-onset diabetes of the young type 3 (MODY3), a form of diabetes mellitus characterized by autosomal dominant inheritance, early onset, and pancreatic ,-cell dysfunction. Recent genetic studies, however, also found mutations in patients diagnosed with idiopathic (non-autoimmune based) type 1 diabetes. We identified a novel frameshift mutation (142delG) in the TCF1 gene in a family with a strong family history of type 1 diabetes and examined the functional properties of the mutant HNF 1,. The expression of the mutant protein was not detected in COS-7 cells by Western blot analysis after transfection of the mutant cDNA. This is the first case of an unstable mutant HNF-1, protein. Reporter gene analysis indicated that the mutant HNF-1, had no transactivation activity in HeLa and MIN6 cells. Haploinsufficiency for HNF-1, may lead to severe forms of diabetes like type 1 diabetes. Hum Mutat 18:345,351, 2001. © 2001 Wiley-Liss, Inc. [source] Expression of estrogen receptor alpha increases leptin-induced STAT3 activity in breast cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2010Nadine A. Binai Abstract Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ER, (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ER,. Downregulation of ER, using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ER, ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ER, ligands. We also detected ER, binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ER,-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ER,-dependent development of malignant diseases. [source] MEK/ERK Signaling Controls Osmoregulation of Nucleus Pulposus Cells of the Intervertebral Disc by Transactivation of TonEBP/OREBP,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Tsung-Ting Tsai Abstract Earlier studies have shown that intervertebral disc cells express TonEBP, a transcription factor that permits adaptation to osmotic stress and regulates aggrecan gene expression. However, the mechanism of hyperosmotic activation of TonEBP in disc cells is not known. Results of this study show that hypertonic activation of ERK signaling regulates transactivation activity of TonEBP, modulating its function. Introduction: In an earlier report, we showed that tonicity enhancer binding protein (TonEBP) positively regulates aggrecan gene expression in disc cells, thereby autoregulating its osmotic environment. Although these studies indicated that the cells of the nucleus pulposus were optimally adapted to a hyperosmotic state, the mechanism by which the cells transduce the osmotic stress was not delineated. The primary goal of this study was to test the hypothesis that, in a hyperosmotic medium, the extracellular signal-regulated kinase (ERK) signaling pathway regulated TonEBP activity. Materials and Methods: Nucleus pulposus cells were maintained in isotonic or hypertonic media, and MAPK activation and TonEBP expression were analyzed. To study the role of MAPK in regulation of TonEBP function, gel shift and luciferase reporter assays were performed. ERK expression in cells was modulated by using expression plasmids or siRNA, and transactivation domain (TAD)-TonEBP activity was studied. Results: We found that hypertonicity resulted in phosphorylation and activation of ERK1/2 proteins and concomitant activation of C terminus TAD activity of ELK-1, a downstream transcription factor. In hypertonic media, treatment with ERK and p38 inhibitors resulted in downregulation of TonE promoter activity of TauT and HSP-70 and decreased binding of TonEBP to TonE motif. Similarly, forced expression of DN-ERK and DN-p38 in nucleus pulposus cells suppressed TauT and HSP-70 reporter gene activity. Finally, we noted that ERK was needed for transactivation of TonEBP. Expression of DN-ERK significantly suppressed, whereas, WT-ERK and CA-MEK1 enhanced, TAD activity of TonEBP. Experiments performed with HeLa cells indicated that the ERK signaling pathway also served a major role in regulating the osmotic response in nondiscal cells. Conclusions: Together, these studies showed that adaptation of the nucleus pulposus cells to their hyperosmotic milieu is dependent on activation of the ERK and p38- MAPK pathways acting through TonEBP and its target genes. [source] Autocrine TGF, signaling mediates vitamin D3 analog-induced growth inhibition in breast cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2001Limin Yang In this study, we address whether TGF, signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGF, was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGF,. MCF7L TGF,RII-transfected cells, which have autocrine TGF, activity, were more sensitive to EB1089 than MCF7L cells. TGF, neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGF, signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGF, isoforms and/or TGF, receptors was induced by the analogs in the vitamin D3 and TGF, sensitive cells. Vitamin D3 analogs did not induce TGF, or TGF, receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGF, activity through increasing expression of TGF, isoforms and/or TGF, receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGF, signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGF, signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGF,/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGF, and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGF, and vitamin D signaling is also PI 3-kinase pathway dependent. © 2001 Wiley-Liss, Inc. [source] Immobilized HIV-1 Tat protein promotes gene transfer via a transactivation-independent mechanism which requires binding of Tat to viral particlesTHE JOURNAL OF GENE MEDICINE, Issue 11 2009Filomena Nappi Abstract Background Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism. Methods Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay. Results Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env,CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants. Conclusions Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy. Copyright © 2009 John Wiley & Sons, Ltd. [source] The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteinsTHE PLANT JOURNAL, Issue 4 2004Ken-ichiro Taoka Summary In higher plants, although several genes involved in shoot apical meristem (SAM) formation and organ separation have been isolated, the molecular mechanisms by which they function are largely unknown. CUP-SHAPED COTYLEDON (CUC) 1 and CUC2 are examples of two such genes that encode the NAC domain proteins. This study investigated the molecular basis for their activities. Nuclear localization assays indicated that green fluorescent protein (GFP)-CUC proteins accumulate in the nucleus. Yeast one-hybrid and transient expression assays demonstrated that the C-terminal domain (CTD) of the CUC has transactivation activity. Domain-swapping experiments revealed that the functional specificity of the CUC for promoting adventitious shoot formation resides in the highly conserved NAC domain, not in the CTD in which motifs specific to the CUC subfamily are located. Taken together, these observations suggest that CUC proteins transactivate the target genes involved in SAM formation and organ separation through a specific interaction between the NAC domain and the promoter region of the target genes. [source] The hepatitis E virus ORF3 protein stabilizes HIF-1, and enhances HIF-1-mediated transcriptional activity through p300/CBPCELLULAR MICROBIOLOGY, Issue 9 2009Syed M. Moin Summary The hepatitis E virus (HEV) causes hepatitis E and is an important human pathogen. We have previously shown that the HEV open reading frame 3 (ORF3) protein promotes survival of the host cell. Here we report finding increased expression of glycolytic pathway enzymes in ORF3-expressing cells. Promoter analysis of these genes revealed the ubiquitous presence of hypoxia inducible factor (HIF) responsive element (HRE). Dominant-negative and siRNA studies showed increased expression of glycolytic pathway genes by the ORF3 to be mediated by the HIF-1 transcription factor. Our results showed that HIF-1,, a highly unstable subunit of the HIF-1, was stabilized in ORF3-expressing cells. This was through phosphatidylinositol-3-kinase (PI3K) mediated activation of Akt/protein kinase B. Enhanced binding to the consensus HRE and increased transactivation activity of HIF-1 were also observed in ORF3-expressing cells. The HIF complex recruits the transcriptional adapter/histone acetyltransferase protein p300/CBP to target gene promoters and p300/CBP phosphorylation is required for this interaction. We show that ORF3-mediated extracellularly regulated kinase (Erk) activation was responsible for the observed increase in phosphorylation and transactivation activity of p300/CBP. Our results reveal a two-pronged strategy through which the ORF3 protein might modulate the energy homeostasis in HEV infected cells and thus contribute to pathogenesis. [source] | |||||||||||||