Rapid glucocorticoid receptor exchange at a promoter is coupled to transcription and regulated by chaperones and proteasomes

Abstract

Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known system in which transcription factor cycling at a promoter is fast, occurring on a time scale of seconds. The mechanism and function of this rapid exchange are unknown. We provide evidence that proteasome activity is required for rapid GR exchange at a promoter. We also show that chaperones, specifically hsp90, stabilize the binding of GR to the promoter, complicating models in which the associated chaperone, p23, has been proposed to induce GR removal. Our results are the first to connect chaperone and proteasome functions in setting the residence time of a transcription factor at a target promoter. Moreover, our results reveal that longer GR residence times are consistently associated with greater transcriptional output, suggesting a new paradigm in which the rate of rapid exchange provides a means to tune transcriptional levels.

FIG. 1.

FRAPs at the MMTV array and elsewhere in the nucleus depend on bleach spot size and are well fit by an effective diffusion model. (a) 3617 cell after 30-min induction with 100 nM dexamethasone. The MMTV array is visible as a bright spot (circled) near one of the nucleoli. Scale bar, 10 μm. (b) For fast data collection during FRAP, images were collected only in the strip encompassed by the red rectangle in panel a. Selected time points (t) are shown. (c) Arrays were bleached with spot radii of either 0.9 or 1.8 μm. In all cases, the array was large enough to completely fill the bleach spot area. Larger bleach spots result in significantly slower recoveries. This indicates that diffusion contributes to the FRAP. For these and all succeeding FRAP curves, standard errors for all points were always <0.01 and therefore smaller than the dots used for plotting. (d) GFP-GR recovery elsewhere in the nucleus also shows dependence on bleach spot size, indicating a role for GFP-GR diffusion in nuclear mobility. (e) GFP-GR FRAP data both at the array and elsewhere in the nucleus were well fit by a single-parameter model for a diffusing molecule bleached by a circular spot with a recovery rate given by the fitting parameter, τ. Recoveries at the array are consistently slower than elsewhere in the nucleus. This difference is statistically significant, as indicated by a t test using the means for τ and their 95% confidence intervals. The computed P value (<0.0001) is highly significant. (f) Models for FRAPs that incorporate both diffusion and binding show that significant changes in binding parameters yield small changes in the FRAP curve. This is because diffusion remains unaffected after treatments that affect binding. Predicted curves are shown for a series of off rates.

FIG. 2.

GFP-GR exchange at the array is an energy-dependent process. A 30-min treatment with 10 mM sodium azide and deoxyglucose leads to a marked reduction in the exchange rate at the array (a) and elsewhere in the nucleus (b). Transferring the cells to a glucose-free medium (deoxyglucose only) for 60 to 90 min induces an ∼5% immobile fraction at the array (c), but the exchange at other sites in the nucleus does not show an immobile fraction (d). (e) Deoxyglucose washout eliminates the immobile fraction at the array.

FIG. 3.

Immunofluorescence detection of chaperone proteins at the MMTV array (circled). Antibodies against hsp90 (a to c), hsp70 (d to f), or p23 (g to i) stain the cytoplasm as expected, but within nuclei, they also consistently colocalize with GFP-GR at the MMTV array. Insets contain higher-magnification views of colocalization at the array. Scale bar, 10 μm.

FIG. 4.

Arrays (circled) disappear much more rapidly after geldanamycin treatment when corticosterone is the ligand than when dexamethasone is the ligand. In either case, geldanamycin induces arrays to disappear (red lines) faster than normal (black lines). An example of array disappearance for each case is shown in pseudocolor to accentuate the arrays. (a) When dexamethasone is the ligand, array disappearance is gradual and occurs over a 2-h time course. (b) When corticosterone is the ligand, the geldanamycin-induced disappearance of arrays is dramatically accelerated, occurring over a time course of 10 min. These results indicate that geldanamycin effects are exacerbated by corticosterone, presumably due to its more rapid exchange with GR. They also suggest that in the presence of geldanamycin, GR eventually becomes unliganded and incapable of binding to the MMTV sites. Scale bar, 5 μm.

FIG. 5.

Effects of hsp90 inhibition. FRAPs at MMTV arrays are faster after treatment with 2.5 μg of geldanamycin/ml (a) or 5 μg of radicicol/ml (c) than in control cells, but the speed-up is detectable immediately in corticosterone (e) while it appears only after 30 to 60 min in dexamethasone (a). (b) These changes are not caused by a generic effect on protein mobility, as cells transfected with GFP-HP1α show no effect on FRAPs after geldanamycin treatment. (Note that the GFP-HP1α recoveries are not fit by effective diffusion [data not shown], so a t test to compute a P value cannot be performed.) (d) Hormone withdrawal experiments demonstrate that, compared to dexamethasone, corticosterone exchanges much more rapidly with GR. Shown are the average transcriptional levels measured from 35 cells by RNA FISH. Cells were induced by 100 nM corticosterone (CORT) or dexamethasone (DEX) for 15 min and then washed three times over a 5-min period in hormone-free medium and left in that medium for 45 min. With corticosterone as a ligand, transcription is abolished after a 5-min wash, indicating complete exchange of the ligand with GR during the wash time. In the same wash period, a significant amount of dexamethasone remains bound, since transcription drops by only 50%. (f) Treatment with geldanamycin induces a progressive loss of GFP-GR from the array and a decrease in size, and this is accompanied by a loss of chaperones. Shown is the loss of p23 from the array following geldanamycin (GELD) treatment with dexamethasone as a ligand. Scale bar, 5 μm.

FIG. 6.

Nuclear GFP-GR aggregates. (a to c) Geldanamycin treatment combined with stress (heat, cold, or prolonged imaging) causes the disappearance of arrays and the formation of GFP-GR spots, which colocalize with a proteasome antibody. (d) In these spots, a fraction of GFP-GR is immobilized compared to other regions of the nucleus. During imaging, these aggregates appear more rapidly with corticosterone than with dexamethasone. Scale bar, 5 μm.

FIG. 7.

Immunofluorescence detection of the proteasome at the MMTV array (circled) and FRAPs after perturbation of proteasome function. Considerable proteasome staining was found in the cytoplasm. (a to c) Within the nucleus, colocalization with the MMTV array was consistently observed. The inset contains a higher-magnification view of colocalization at the array. (d and e) Cells induced with dexamethasone or corticosterone and exposed to the proteasome inhibitor MG-132 exhibited slower FRAP at MMTV arrays. (f) This difference was not caused by a generic retardation of protein mobilities in the nucleus, as MG-132 treatment did not alter the recovery of GFP-HP1α. (Again, these GFP-HP1α recoveries are not fit by effective diffusion, so neither a recovery rate, τ, nor a P value for the comparison can be calculated.) Scale bar, 10 μm.

FIG. 8.

Effects of either geldanamycin treatment on proteasomes or MG-132 treatment on chaperones. (a to f) Progressive loss of the 19S proteasome is seen at the MMTV array (circled) with longer geldanamycin (GELD) treatment. Clear proteasomal staining is seen after 15 min of geldanamycin treatment (b), whereas much fainter staining at levels close to nuclear background is detected after 1 h of geldanamycin treatment (e). (g to l) MG-132 does not affect the levels of p23 with either dexamethasone (DEX) (g to i) or corticosterone (CORT) (j to l). Only one time point is shown for each ligand, since unlike geldanamycin treatment, there is no loss of GFP-GR from the array after proteasome inhibition. Scale bar, 5 μm.

FIG. 9.

Accelerated GFP-GR exchange is associated with less transcription. (a) Examples of a condensed array and a decondensed array are shown with the corresponding RNA FISH signals. (b) FRAPs of condensed arrays were consistently faster. (c and d) RU486 (100 nM) and corticosterone (100 nM) also yielded faster FRAPs than dexamethasone. Scale bar, 1 μm.

FIG. 10.

Model for proteasome-chaperone interaction at the MMTV template and association with transcription. Liganded GR binding to MMTV ultimately leads to initiation complex formation. Formation of the complex may be enhanced by longer GR residence at the MMTV template. The duration of GR occupancy at the template is determined in part by competition between proteasome and chaperone functions. Proteasome inhibition favors GR occupancy, leading to a slower FRAP with an immobile fraction. Chaperone inhibition by geldanamycin favors GR loss, leading to a faster FRAP. The equilibrium between these two components helps to set the transcriptional level and may be mediated in part by one or more factors that are known to couple chaperone and proteasome activities (5, 21).