Magnitude Of Transcriptional Activation Research Articles

Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity.

Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification of their ligands. In this chapter we describe in detail the assays performed to investigate LXR activity in a macrophage-like cell line (RAW 267.4). These can be easily adapted to other nuclear receptors and transcription factors.

Estrogen receptor beta acts as a dominant regulator of estrogen signaling.

The physiological effects of estrogens are mediated by two intracellular transcription factors, the estrogen receptors (ERs), that regulate transcription of target genes through binding to specific DNA target sequences. Here we describe alterations in cellular responses to different ER agonists and to the anti-estrogenic compound tamoxifen resulting from co-expression of the two ERs in transient co-transfection experiments. Our results demonstrate that ERbeta can act as a negative or positive dominant regulator of ER activity. This is manifested through reduced transcriptional activity at low concentrations of estradiol (E2); increased antagonistic effects of tamoxifen on E2 stimulated activity; and enhanced agonistic action of the phytoestrogenic compound genistein. Furthermore, using chimeric proteins lacking the N-terminal activation function 1 (AF-1), we show that the differential responses of ERalpha and ERbeta to different agonists and antagonists are primarily dictated by inherent differences in the C-terminal ligand-binding domains of the receptors, whereas the magnitude of transcriptional activity is influenced by ERalpha AF-1, but not ERbeta AF-1. The ERalpha AF-1 activity appears to be modulated upon co-expression of both ERs. The alterations in transcriptional activity resulting from co-expression of ERalpha and ERbeta are probably due to the formation of alpha/beta heterodimeric complexes. This study demonstrates that co-localization and subsequent heterodimerization of ERalpha and ERbeta may result in receptor activity distinct from that of ER homodimers.