In vitro regulation of reporter gene transcription by the androgen receptor AF1 domain

2004 ◽  
Vol 32 (6) ◽  
pp. 1103-1106 ◽  
Author(s):  
M.A. Choudhry ◽  
I.J. McEwan
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Reporter Gene ◽  
Protein Interactions ◽  
Transcription Initiation ◽  
Muscular Atrophy ◽  
Activation Function ◽  
Regulation Of Transcription ◽  
Gene Assay

The androgen receptor (AR) is a ligand-activated transcription factor that regulates gene expression in response to the steroids testosterone and dihydrotestosterone. AR-dependent gene expression is likely to play an important role in a number of receptor-associated disorders, such as prostate cancer, spinal bulbar muscular atrophy, male type baldness and hirsutism. The AR contains two transactivation domains, termed AF1 (activation function 1) located in the N-terminus and AF2 (activation function 2) in the C-terminal ligand-binding domain. AF2 exhibits weak transcriptional activity, whereas AF1 is a strong regulator of transcription. Transcriptional regulation by AF1 is thought to be modulated by a number of proteins that interact with this region, and by post-translational modifications. Our focus is on the N-terminal-interacting proteins and their regulation of transcription via interaction with the receptor. To better understand the mechanism of AR-AF1 action, we have reconstituted AR activity in HeLa nuclear extracts using a unique dual reporter gene assay. Multiple LexA-binding sites in the promoter allow transcription to be driven by a recombinant AR-AF1–Lex fusion protein. The findings from initial experiments suggest an increase in transcription initiation and elongation rates by AR-AF1–Lex. The role of protein–protein interactions involving co-activators and basal transcription factors and AR-AF1 activity are discussed.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

scholarly journals In vitro bioanalytical assessment of toxicity of wetland samples from Spanish Mediterranean coastline

2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Alberto Celma ◽  
Geeta Mandava ◽  
Agneta Oskarsson ◽  
Juan Vicente Sancho ◽  
Lubertus Bijlsma ◽  
...  
Keyword(s):  
Water Quality ◽  
Reporter Gene ◽  
Biological Activities ◽  
Reporter Gene Assay ◽  
Water Bodies ◽  
Good Tool ◽  
Adverse Health Effects ◽  
Aryl Hydrocarbon ◽  
Gene Assay

Abstract Background Fresh water bodies represent less than 1% of overall amount of water on earth and ensuring their quality and sustainability is pivotal. Although several campaigns have been performed to monitor the occurrence of micropollutants by means of chemical analysis, this might not cover the whole set of chemicals present in the sample nor the potential toxic effects of mixtures of natural and anthropogenic chemicals. In this sense, by selecting relevant toxicity endpoints when performing in vitro bioanalysis, effect-based methodologies can be of help to perform a comprehensive assessment of water quality and reveal biological activities relevant to adverse health effects. However, no prior bioanalytical study was performed in wetland water samples from the Spanish Mediterranean coastline. Methods Eleven samples from relevant water bodies from the Spanish Mediterranean coastline were collected to monitor water quality on 8 toxicity endpoints. Aryl hydrocarbon receptor (AhR), androgenicity (AR+ and AR−), estrogenicity (ER+ and ER−), oxidative stress response (Nrf2) and vitamin D receptor (VDR+ and VDR−) reporter gene assays were evaluated. Results AhR was the reporter gene assay showing a more frequent response over the set of samples (activated by 9 out of 11 samples), with TCDD-eq in the range 7.7–22.2 pM. For AR, ER and VDR assays sporadic activations were observed. Moreover, no activity was observed on the Nrf2 reporter gene assay. Wastewater and street runaway streams from Valencia could be responsible for enhanced activities in one of the water inputs in the Natural Park ‘L’Albufera’. Conclusions Water quality of relevant wetlands from the Spanish Mediterranean coastline has been evaluated. The utilization of a panel of 5 different bioassays to cover for different toxicity endpoints has demonstrated to be a good tool to assess water quality.

Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay

2005 ◽  
Vol 15 (4) ◽  
pp. 271-280 ◽  
Author(s):  
Mihoko Kojima ◽  
Kenji Fukunaga * ◽  
Mari Sasaki ◽  
Masafumi Nakamura ◽  
Motohiro Tsuji ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Gene Assay

Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements

1993 ◽  
Vol 13 (4) ◽  
pp. 2104-2112
Author(s):  
A S Alberts ◽  
T Deng ◽  
A Lin ◽  
J L Meinkoth ◽  
A Schönthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Protein Phosphatase ◽  
Signal Transduction Pathways ◽  
Marker Genes ◽  
Direct Mechanism ◽  
Phosphatase 2A ◽  
Beta Galactosidase ◽  
Regulatory Sites

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.

scholarly journals Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

1995 ◽  
Vol 15 (6) ◽  
pp. 3442-3449 ◽  
Author(s):  
M S Donoviel ◽  
N Kacherovsky ◽  
E T Young
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Reporter Gene ◽  
Copy Number ◽  
Glucose Repression ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Upstream Regulatory Region ◽  
Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis

2015 ◽  
Vol 49 (10) ◽  
pp. 6319-6326 ◽  
Author(s):  
Masaru Ihara ◽  
Tomokazu Kitamura ◽  
Vimal Kumar ◽  
Chang-Beom Park ◽  
Mariko O. Ihara ◽  
...  
Keyword(s):  
Chemical Analysis ◽  
Reporter Gene ◽  
Estrogenic Activity ◽  
Reporter Gene Assay ◽  
Gene Assay

scholarly journals Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
Gwang Sik Kim ◽  
Young Chul Lee
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Reporter Gene Expression ◽  
Temperature Sensitive ◽  
Internal Deletion ◽  
Cell Extracts ◽  
Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

scholarly journals Integrative In Silico and In Vitro Transcriptomics Analysis Revealed Gene Expression Changes and Oncogenic Features of Normal Cholangiocytes after Chronic Alcohol Exposure

2019 ◽  
Vol 20 (23) ◽  
pp. 5987
Author(s):  
Suthipong Chujan ◽  
Tawit Suriyo ◽  
Jutamaad Satayavivad
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Function Analysis ◽  
Alcohol Exposure ◽  
Gene Expression Omnibus ◽  
Regulation Of Transcription ◽  
Chronic Alcohol ◽  
Chronic Alcohol Exposure

Cholangiocarcinoma (CCA) is a malignant tumor originating from cholangiocyte. Prolonged alcohol consumption has been suggested as a possible risk factor for CCA, but there is no information about alcohol’s mechanisms in cholangiocyte. This study was designed to investigate global transcriptional alterations through RNA-sequencing by using chronic alcohol exposure (20 mM for 2 months) in normal human cholangiocyte MMNK-1 cells. To observe the association of alcohol induced CCA pathogenesis, we combined differentially expressed genes (DEGs) with computational bioinformatics of CCA by using publicly gene expression omnibus (GEO) datasets. For biological function analysis, Gene ontology (GO) analysis showed biological process and molecular function related to regulation of transcription from RNA polymerase II promoter, while cellular component linked to the nucleoplasm. KEGG pathway presented pathways in cancer that were significantly enriched. From KEGG result, we further examined the oncogenic features resulting in chronic alcohol exposure, enhanced proliferation, and migration through CCND-1 and MMP-2 up-regulation, respectively. Finally, combined DEGs were validated in clinical data including TCGA and immunohistochemistry from HPA database, demonstrating that FOS up-regulation was related to CCA pathogenesis. This study is the first providing more information and molecular mechanisms about global transcriptome alterations and oncogenic enhancement of chronic alcohol exposure in normal cholangiocytes.

scholarly journals σIfromBacillus subtilis: Impact on Gene Expression and Characterization of σI-Dependent Transcription That Requires New Types of Promoters with Extended −35 and −10 Elements

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Olga Ramaniuk ◽  
Martin Převorovský ◽  
Jiří Pospíšil ◽  
Dragana Vítovská ◽  
Olga Kofroňová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Iron Metabolism ◽  
Transcription Initiation ◽  
Iron Homeostasis ◽  
Model Organism ◽  
Content Type ◽  
Σ Factor

ABSTRACTThe σIsigma factor fromBacillus subtilisis a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI. Further analysis revealed that the majority of these genes were affected indirectly by σI. The σIregulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (thedhbandykuoperons) are involved in iron metabolism. The involvement of σIin iron metabolism was confirmed phenotypically. Next, we set up anin vitrotranscription system and defined and experimentally validated the promoter sequence logo that, in addition to −35 and −10 regions, also contains extended −35 and −10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organismB. subtilis.IMPORTANCEIn bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σIregulon from the industrially important model Gram-positive bacteriumBacillus subtilis. We reveal that σIaffects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of theB. subtilistranscription machinery.

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