Dawn A. Walker

Rapid Periodic Binding and Displacement of the Glucocorticoid Receptor during Chromatin Remodeling

An ultrafast UV laser crosslinking assay has provided novel insights into the progression of the SWI/SNF-mediated chromatin-remodeling reaction and transcription factor binding in real time. We demonstrate site-specific crosslinking between the glucocorticoid receptor (GR), the hSWI/SNF chromatin-remodeling complex, and the mouse mammary tumor virus (MMTV) promoter assembled in an array of correctly positioned nucleosomes. GR first demonstrates rapid binding to the promoter and then is actively displaced from the template during the remodeling reaction. This displacement reaction requires the hSWI/SNF complex and ATP, is specific to the nucleoprotein template, and is accompanied by a core histone rearrangement. The hSWI/SNF complex associates with random positions on the chromatin template in the absence of GR but is recruited specifically to the B/C region when GR is included. These results indicate that enhancement of hSWI/SNF-mediated factor accessibility, a hallmark of chromatin remodeling, is in some cases transient, reversible, and periodic.

Dynamic behavior of transcription factors on a natural promoter in living cells

Through the use of photobleaching techniques, we examined the dynamic interaction of three members of the transcrsiption apparatus with a target promoter in living cells. The glucocorticoid receptor (GR) interacting protein 1 (GRIP‐1) exhibits a half maximal time for fluorescent recovery (τR) of 5 s, reflecting the same rapid exchange as observed for GR. In contrast, the large subunit (RPB1) of RNA polymerase II (pol II) required 13 min for complete fluorescence recovery, consistent with its function as a processive enzyme. We also observe a complex induction profile for the kinetics of GR‐stimulated transcription. Our results indicate that GR and GRIP‐1 as components of the activating complex are in a dynamic equilibrium with the promoter, and must return to the template many times during the course of transcriptional activation.

Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter

We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a ∼0.5-μm spot to form a fiber 1–10 μm long. Arrays later recondensed by 3–8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and α-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.

Ligand-Specific Dynamics of the Progesterone Receptor in Living Cells and during Chromatin Remodeling In Vitro

ABSTRACT Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

An estrogen receptor chimera senses ligands by nuclear translocation

We have developed a new mammalian cell-based assay to screen for ligands of the estrogen receptor. A fluorescently tagged chimera between the glucocorticoid and the estrogen receptors, unlike the constitutively nuclear estrogen receptor, is cytoplasmic in the absence of hormone and translocates to the nucleus in response to estradiol. The chimera maintains specificity for estrogen receptor alpha ligands and does not show cross-reactivity with other steroids, providing a clean system for drug discovery. Natural and synthetic estrogen receptor alpha agonists as well as phytoestrogens effectively translocate the receptor to the nucleus in a dose-dependent manner. Antagonists of the estrogen receptor can also transmit the structural signals that result in receptor nuclear translocation. The potency and efficacy of high-affinity ligands can be evaluated in our system by measuring the nuclear translocation of the fluorescently labeled receptor in response to increasing ligand concentrations. The chimera is transcriptionally competent on transient and replicating templates, and is inhibited by estrogen receptor antagonists. Interestingly, the nucleoplasmic mobility of the chimera, determined by FRAP analysis, is faster than that of the wild type estrogen receptor, and the chimera is resistant to ICI immobilization. The translocation properties of this chimera can be utilized in high content screens for novel estrogen receptor modulators.

Intranuclear trafficking and gene targeting by members of the steroid/nuclear receptor superfamily.

Upon binding to regulatory elements in mammalian chromosomes, steroid receptors induce specific transitions in the nucleoprotein structure of the template. These transitions reflect, in part, the reorganization of chromatin structure to permit interaction of secondary factors with target sequences in promoter regulatory regions. Steroid receptors represent a class of transcriptional activators that are able to interact with repressed nucleoprotein templates and recruit necessary activities for chromatin remodeling. The ligand-induced movement of nuclear receptors from inactive states, either in the cytoplasm or in the nucleus, to productive interactions with chromatin is complex and likely reflects the interaction with multiple protein complexes and subcellular structures. Regulation of gene expression by nuclear receptors is thus mediated through the subcellular distribution of inactive receptors, the redistribution of activated receptor complexes to appropriate nuclear domains, the reorganization of chromatin structures for interaction with soluble components of the nucleoplasm, and direct protein-protein contacts between receptors and the basal transcription apparatus.

A Novel In Situ Assay for the Identification and Characterization of Soluble Nuclear Mobility Factors

The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.