Transcriptional activation assay

Metcalfe C.D., Metcalfe T.L., Kiparissis Y., et al., 2001. Environ. Toxicol. Chem., 20, 297. Murk A.J., Legler J., Van Lipzig M.M.H., et al., 2002. Environ. Toxicol. Chem., 21, 16. NICEATM, 2002. Background Review Document Current Status of Test Methods for Detecting Endocrine Disruptors In vitro Estrogen Receptor Transcriptional Activation Assays. 

A RED approach was used to screen a library of natural products against PPARy, a target involved in many diseases, including type II diabetes mellitus. HTD was performed with GLIDE [61] onto two PPARy crystal structures (cf Figure 9.3), and it revealed 29 potential agonists [62]. Six flavonoids were identified in a PPARy transcriptional activity assay and one of these six displayed low micromolar activity,... 

PIAS Proteins. Figure 1 Reporter gene assay with an interferon-y-driven artificial promoter demonstrating the inhibitory transcriptional activity of increasing amounts of PIAS on STAT1 signal transduction. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

To study the transcriptional activity of pel and pern genes during pathogenesis, a culture of L37 cells harboring one of the pelA , pelB , pelC , pelD and pelE uidA fusions was inoculated into the leaf parenchyma of African violet pottexi plants. GUS activity, produced by the bacteria present in extractable fluids, was assayed at 3 hour-intervals over a 72 h period after inoculation. 

As expected, in vitro transcription assays involving PARP-1, NAD, and PARC illustrate these predicted outcomes (Kim et al, 2004). Even when driven by a transcriptional activator, such as estradiol-bound estrogen receptor, transcription is repressed when PARP-1 is added to chromatin templates. The repression is reversed by NAD+, and the NAD+-dependent effects are reversed by PARC (Kim et al, 2004). This system for transcriptional control shifts new importance onto the enzymes responsible for synthesis of NAD+ in the nucleus, such as nicotinamide mononucleotide adenylyltransferase-1 (Magni et al, 2004). Because NAD+ facilitates the decompaction of chromatin and the derepression of transcription, nuclear NAD+ biosynthetic enzymes may play critical roles as cofactors. 

Chromatin fractionation approaches including ChIP assays have provided evidence for and against uH2A and uH2B being associated with transcriptionally active chromatin [270,281-285]. Most evidence supports uH2A being associated... 

OECD. 2006e. Stably transfected transcriptional activation TA) assay for detecting estrogenic activity of chemicals - The human estrogen receptor alpha mediated reporter gene assay using HeLa-hER-9903 cell line. Version October 2006. Paris OECD, http //www.oecd.org/dataoecd/49/55/37531918.pdf... 

The potential impact of the chromatin structure on ERa- and ER/1-mediated transcriptional activities was investigated using an in vitro chromatin assembly assay. These experiments have shown that the AF-1 domain of ERa, but not of ER/1, contains a transferable activation domain, which permits the ERa to efficiently activate transcription on chromatin templates [58]. Furthermore, the co-activators CBP/p300 and SRC have to be recruited to the ERa in order to maximally enhance transcription on ERa-susceptible chromatin templates. The p300/CBP-SRC complex, when interacting with the AF-1 of the ERa, is primarily involved in the stable formation of the preinitiation complex of transcription [59]. 

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