Steroid-regulated promoters

Scientific aspects

Among the chemically-inducible gene expression systems for plants and animals are the ones based on steroid hormone receptors. The mammalian glucocorticoid receptor (GR) is a member of the family of animal steroid hormone receptors. GR also acts as a transcription factor by activating gene transcription from promoters containing glucocorticoid response elements (GRE). In the cellular environment, GR exists in a  complex in the cytoplasm with the 90-kilodaltons (kDa) heat shock protein (HSP90), which dissociates once GR binds to its ligand (hormone).

A system comprised of the GR and GREs resulted in transient expression in tobacco cells in the presence of dexamethasone, a strong synthetic glucocorticoid. In stably transformed Arabidopsis plants, however, the system did not induce expression.

The hormone binding domain (HBD) of GR and other steroid receptors can also be used to regulate heterologous proteins in cis, that is, operatively linked to protein-encoding sequences upon which it acts. Thus, the HBD of GR, estrogen receptor (ER) and an insect ecdysone receptor have shown relatively tight control and high inducibility.

The HBD of the rat GR has been linked to the DNA-binding domain of the yeast GAL4 transcription factor and to the acidic transactivating domain of the herpes viral protein VP16. This chimeric transcriptional factor, named GVG, has driven the expression of a number of genes in several plants in the presence of the ligand dexamethasone. In some cases however, it appears to be toxic, which is caused by a high concentration of and extended exposure to dexamethasone and induction of untargeted genes (e.g. defense-related genes).

An ER-based inducible system for use in transgenic plants includes a chimeric transactivator called XVE, assembled by fusion of the regulatory region of the human ER, the DNA-binding domain of the bacterial repressor LexA and the acidic transactivating domain of VP16. In transgenic tobacco and ArabidopsisXVE has induced the expression of genes in the presence of estradiol to levels 8-fold higher than observed when expression is driven by a constitutive promoter, such as 35S CaMV. The XVE system does not exhibit the toxic effects found with the GVG system, but in some legumes (e.g., soybean) it appears to be deregulated, presumably due to the presence of phyto-estrogens.

Since none of the systems described are induced by compounds suitable for agricultural use, a transcription system inducible by non-steroidal agrochemicals was developed based on the insect ecdysone receptorEcdysones are insect steroidal hormones that trigger the expression of critical genes during larval development and have been proposed as safer alternatives to pesticides. An advantage of the ecdysone receptor is that can also bind non-steroidal ecdysone agonists (agonists are molecules that improve the activity of a different molecule). One hybrid transactivator system contains the DNA-binding domain and the receptor activation of GR and the hormone-regulatory domain of the Heliothis virescensER. In stable transgenic tobacco plants, the system induced the expression of a reporter gene over 400-fold greater than the activity of the 35S CaMV promoter. The system is highly responsive to RH5992 (tebufenozide), a non-steroidal ecdysone agonist that lacks phytotoxicity and is used as a lepidopteran control agent on vine and horticulture plants. A drawback of the system is a relatively high background expression.