Novel gene switches for targeted and timed expression of proteins of interest.

This article reports on the construction and analysis in vitro and in vivo of novel gene switches that can be used to achieve spatial as well as temporal control over the expression of a transgene of interest. The switches are expected to be functional in virtually any tissue and cell type. They consist of (a) a foreign or modified transactivator expressed under the dual control of a promoter or promoter cassette that is responsive to heat and the transactivator and (b) a promoter responsive to the transactivator for controlling the transgene of interest. A preferred gene switch of this type incorporated a mifepristone-dependent transactivator. This gene switch could be activated by a transient heat treatment in the presence of mifepristone. Activity increased with the intensity of the activating heat treatment and was found to persist for more than 6 days. The gene switch was essentially inactive prior to an activating heat treatment, in the absence or presence of mifepristone. Activated gene switch could be silenced by removal/withdrawal of mifepristone.

DAXX interacts with heat shock factor 1 during stress activation and enhances its transcriptional activity.

DAXX, a modulator of apoptosis and a repressor of basal transcription, was identified in a two-hybrid screen as a protein capable of interacting with a trimeric form of human heat shock factor 1 (HSF1). In human cells, DAXX interacted with HSF1 essentially only during stress, i.e., when factor trimerization occurred. Several lines of experimentation suggested that DAXX is an important mediator of HSF1 activation: (i) overexpression of DAXX enhanced basal transactivation competence of HSF1 in the absence of a stress; (ii) a DAXX fragment exerted dominant-negative effects on HSF1 activation by different types of stress; (iii) induction of heat shock or stress protein (HSP)70 by heat stress was defective in a cell line lacking functional DAXX; and (iv) RNA interference depletion of DAXX also substantially reduced heat induction of HSF1 activity and HSP70 expression. HSF1 transactivation competence is repressed by an HSP90-containing multichaperone complex that interacts with trimeric factor. Overexpressed HSF1, known to be largely trimeric, only marginally increased HSF1 activity on its own but potentiated the activating effect of DAXX overexpression. Expression of a nonnative protein capable of competing for multichaperone complex also synergistically enhanced activation of HSF1 by DAXX. These observations suggest a model in which DAXX released from its nuclear stores during stress opposes repression of HSF1 transactivation competence by multichaperone complex through its interaction with trimerized HSF1. Our identification of DAXX as a mediator of HSF1 activation raises the question whether DAXX produces some of its pleiotropic effects through modulation of HSP levels.