Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATPdependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.The regulation of transcription by nuclear receptors is intimately associated with reorganization of the chromatin structures of target promoters. Receptors interact, either directly or indirectly, with a variety of chromatin-remodeling factors and recruit these activities for chromatin modification in the vicinity of the hormone response elements (HREs). There are two general classes of chromatin-remodeling factors: those that covalently modify components of the nucleoprotein structure (including histone acetylases and deacetylases, methylases, and kinases) (31, 45) and a second group comprised of ATP-dependent nucleosome-remodeling proteins, collectively referred to as the Swi/Snf family of factors (22,23,35,36,51,54).The current paradigm suggests that the purpose of these chromatin-modifying activities is to reorganize the nucleoprotein structures of regulated genes to provide greater access for both site-specific promoter binding proteins and general (or basal) transcription factors, both of which are excluded from their binding sites in nonremodeled chromatin. The nuclear receptors appear to be members of a restricted group of activators (pioneer proteins) with the ability to bind chromatin and initiate the remodeling process (18,26,27,44,48,49).A parallel theme of this model is the concept of a stable initiation complex. Steroid receptors, such as the glucocorticoid (GR), progesterone (PR), and estrogen receptors, require ligand to bind DNA and are believed to assist in the formation of large, multifactor complexes that reside on the template in the continued presence of hormone (55). By using a receptor tagged with a green...

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells

Nature [London] 324:375-377, 1986). Our present studies demonstrate that the mitogenic activity of the H-ras oncogene in H-ras p21-microinjected quiescent cells is markedly reduced under conditions in which PKC is downregulated by chronic phorbol ester treatment. The ability to reconstitute the mitogenic response upon microinjection of both H-ras p21 and PKC implies involvement of functional PKC in the mitogenic activity of the H-ras oncogene product.