Dynamics of gene targeting and chromatin remodelling by nuclear receptors

Activation of the murine-mammary-tumour virus (MMTV) promoter by the glucoc orticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). We hav e reconstituted this nucleoprotein transition with chromatin assembled on M MTV LTR DNA with Drosophila embryo extracts, purified GR, and HeLa nuclear extract. Chromatin remodelling in vitro is ATP-dependent and maps to a regi on identical with that found in vivo. We demonstrate specific, glucocortico id response element dependent, binding of purified GR to a large, multi-nuc leosome MMTV chromatin array and show that GR-dependent chromatin remodelli ng is a multistep process. In the absence of ATP, GR binds to multiple site s on the chromatin array and inhibits nuclease access to GR recognition sit es. On the addition of ATP, GR induces remodelling resulting in a large inc rease in access of enzymes to their sites within the transition region. The se findings are complemented by studies in living cells; using a tandem arr ay of MMTV-Ras reporter elements and a form of GR labelled with the green f luorescent protein, we have observed direct targeting of the receptor to re sponse elements in live mouse cells. Whereas the ligand-activated receptor is associated with the MMTV promoter for observable periods, photobleaching experiments provide direct evidence that the hormone-occupied receptor und ergoes rapid exchange between chromatin and the nucleoplasmic compartment. The results both in vitro and in vivo are consistent with a dynamic model ( 'hit and run') in which GR first binds to chromatin after ligand activation , recruits a remodelling activity and is then lost from the template.