Analysis of the mechanism of steroid hormone receptor-dependent gene activation in cell-free systems

Steroid hormones regulate the expression of specific gene networks and thereby exert a wide variety of effects on growth, development, and differentiation in higher eukaryotes (for reviews see Refs. 1–6). The hormonal signals are transduced to the target genes through specific, intracellular receptors. The ligand-free receptor is functionally inactive. The hormone response is triggered by the binding of a steroidal ligand to its cognate receptor, followed by the interaction of the hormonereceptor complex with specific enhancer sequences referred to as steroid response elements (SREs) at the target gene. The receptor regulates gene transcription presumably by interacting with the transcription machinery at the target promoter. During the last several years, good progress has been made toward unravelling the molecular mechanism of signal transduction by steroid hormones. The molecular cloning of virtually all known steroid receptor genes has been accomplished, and precise definition of various SREs has been achieved through gene transfection experiments. However, the mechanisms underlying key molecular events in the steroid-induced gene activation pathway, such as ligand-induced activation of receptors and transactivation of target promoters by the activated receptor, have remained unresolved. One approach to analyzing these mechanisms is to reconstitute hormone and receptor-dependent gene activation in vitro. In a cell-free system one can precisely define the minimal combination and appropriate concentration of components necessary to achieve hormone and tissue-specific gene induction by careful manipulation of well-defined DNA templates, purified receptor and other transcription factors, and hormonal ligand. For many years the establishment of a steroid receptor-regulated gene expression system eluded the researchers in this area. Recently a number of such receptor- and ligand-regulated in vitro gene expression systems have been devised in our laboratory as well as in others (7–11). In this review we will describe how these systems have been exploited to gain useful insights into the mechanism of gene activation by steroid receptors.