Role of the Promyelocytic Leukemia Body in the Dynamic Interaction between the Androgen Receptor and Steroid Receptor Coactivator-1 in Living Cells

Omar J. Rivera, Chung S. Song, Victoria E. Centonze, James D. Lechleiter, Bandana Chatterjee and Arun K. Roy

Departments of Cellular & Structural Biology (O.J.R., V.E.C., J.D.L.) and Molecular Medicine (C.S.S., B.C., A.KR.), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207; and South Texas Veterans Health Care System (C.S.S., B.C.), San Antonio, Texas 78229

Address all correspondence and requests for reprints to: Arun K. Roy, Ph.D., Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 78245-3207. E-mail: roy{at}uthscsa.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2–1.0 µm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5{alpha}-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-{Delta}ligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t1/2) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANDROGEN RECEPTOR (AR) is a member of the nuclear receptor superfamily and mediates androgen signaling for development, growth, and function of the male reproductive system. Androgenic steroids also contribute to regulate a number of physiological processes associated with the male phenotype. Abnormal androgen action has been implicated in the proliferative pathogenesis of the prostate gland, a prevalent health problem of elderly men (1, 2, 3, 4). The unliganded AR is primarily localized in the cytoplasmic compartment of target cells and, upon androgen binding and concomitant conformational transition, it is translocated into the nucleus (5, 6, 7). After entering the nuclear compartment, the agonist-activated AR moves through different subnuclear sites and undergoes further processing through protein-protein interactions and possibly covalent modifications (6, 7, 8, 9, 10, 11). One of the critical steps within this multistep process of hormonal signaling is the recruitment of coactivator proteins such as the steroid receptor coactivator-1 (SRC-1) by the receptor (12). However, the subnuclear events leading to AR and SRC-1 interaction have not been clearly established, and it is uncertain whether the receptor interacts with the coactivator before or after the DNA binding.

We have used the spectral variants of the jellyfish fluorescent proteins as markers to follow the intracellular movements of AR and SRC-1 at different subnuclear locations by high-resolution confocal scanning microscopy in living cells. Fluorescence recovery after photobleaching (FRAP) analysis of the fluorescently labeled AR and SRC-1 at different subnuclear compartments has allowed us to evaluate the dynamics of the movement and interaction between these two proteins during androgen action (13, 14). Here we show that, in the absence of androgen signaling, the yellow fluorescent protein (YFP)-labeled SRC-1 is associated in approximately 40% of the transfected cells with a discrete subnuclear compartment variously known as the promyelocytic leukemia (PML) bodies, nuclear dots, and Kremer bodies. Structural organization of the PML body requires the PML gene product, a RING finger protein, and both the number and composition of this subnuclear compartment are cell cycle dependent. PML bodies gradually disassemble during progression of the cell cycle from the S to G2 phase (15, 16, 17, 18, 19, 20). The PML (promyelocytic leukemia) is a tumor suppressor protein that first received attention due to its role in the pathogenesis of acute promyelocytic leukemia. Many regulatory proteins are known to be associated with the PML body. These include p53, pRB, BRCA1, Daxx, cAMP response element binding protein (CBP), glucocorticoid interacting protein 1 (GRIP1), and Sp100 (21, 22). Thus, the PML body can potentially serve as a cross-road for regulatory factors involved in the transcriptional regulation of cell proliferation, cellular senescence, and apoptosis.

In this article, we show that SRC-1 associated with PML bodies is recruited by the agonist-activated AR into another distinct subnuclear compartment characterized by its interlacing filamentous appearance, which has earlier been referred to as punctate foci/subnuclear speckles. Additionally, nuclear AR bound to a transcriptionally ineffective ligand (pure antagonist), fails to translocate SRC-1 from the PML bodies to nuclear speckles. Our results on the kinetics of movement determined by FRAP analysis suggested an active transport process of SRC-1 from its resting state in the PML body to the nuclear speckles by the activated AR. Additionally, our data for the first time reveal the surface features of the SRC-1-associated PML bodies within the living cell and shed new light on the mechanism of uptake of SRC-1 by these subnuclear structures. These results suggest that SRC-1-containing satellites that are associated with the PML bodies may serve as a transitory storage site for the coactivator.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Association of SRC-1 with the PML Bodies in the Absence of Androgen
Chimeric expression vectors containing YFP and cyan fluorescent protein (CFP) coding sequences ligated in frame at the 5'-end of the SRC-1 and AR cDNAs were used for cell transfection. Results presented in Fig. 1Go show that N-terminal conjugation of the fluorescent protein does not interfere with the coactivator function of SRC-1 on the CFP-AR-mediated transactivation of a promoter-reporter construct containing an androgen-responsive region of the probasin gene promoter ligated to the luciferase reporter gene. Under the particular transfection condition and at a CFP-AR to YFP-SRC1 ratio of 3:1, androgen (5{alpha}-dihydrotestosterone)-dependent transactivation function of AR was stimulated by YFP-SRC1 to a level that is approximately 3-fold higher than the vector control.



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Figure 1. Potentiation of AR Transactivation Function by the Chimeric YFP-SRC1

The first two bar graphs show transactivation activity of CFP-AR on the probasin-derived promoter-reporter construct in the absence (open bar) and presence (solid bar) of the androgen (DHT, 10-8 M). Transfection assay was performed in COS-1 cells. Unconjugated YFP expression vector was used as a negative control in place of YFP-SRC1. The next two bar graphs on the right show the same experiment except that the YFP plasmid was substituted with YFP-SRC1 expression plasmid. Averages of three experiments ± SD were used to construct the plot.

 
Live cell imaging of YFP-SRC1 shows that in an unsynchronized cell population, this coactivator is present within the nucleus as discrete subnuclear foci in about 40% of cells, whereas in the rest of the cells it is smoothly distributed (Fig. 2Go). Results that follow describe characterization of this focally distributed SRC-1 and its relevance to androgen action. The diameter of these discrete foci in 0.1-µm optical sections varied from 0.2–1.0 µm. The number of these SRC-1-containing foci differed from cell to cell with an approximate range of 30–80 foci per cell. That the focal pattern of distribution is not an artifact of overexpression or unique to COS-1 cells is indicated by a similar pattern of endogenous SRC-1 distribution in HEK293 cells immunostained with antibody against SRC-1 (Fig. 3Go). Both the number and size of these foci suggested a considerable similarity with the PML nuclear body.



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Figure 2. Pattern of Nuclear Distribution of YFP-SRC1 in Transfected COS-1 Cells

A, Percent of nuclei showing focal and homogenous distribution of the fluorescently labeled SRC-1. The graph represents the average of three independent transfection (n = 150, each) ± SD. B and C, Representative images of focal and smooth pattern of nuclear distribution of YFP-SRC1. Magnification bar, 2 µm.

 


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Figure 3. Focal Distribution of Endogenous SRC-1 in the Nucleus of a Single HEK293 Cell

The figure shows intranuclear distribution of endogenous SRC-1 in nontransfected HEK293 cells immunostained with antibody against SRC-1. Magnification bar, 3 µm.

 
PML bodies are thought to arise from a process of self-assembly of the PML protein after its conjugation with SUMO-1 (small ubiquitin-like modifier-1) (20, 23). One of the constitutive residents of the PML body is Sp100. It is a nuclear antigen initially identified as immunoreactive to the autoantibodies found in patients suffering from primary biliary cirrhosis (24). Immunolabeling of human kidney-derived HEK293 cells expressing either CFP-SRC1 or CFP-Sp100 with anti-PML antibodies showed colocalization of both SRC-1 and Sp100 with the PML foci (Fig. 4Go). Because ectopic expression of PML causes mitostasis and induces apoptosis, fluorescently labeled Sp100 instead of PML has been used to define PML bodies in living cells (25, 26). We have also used a chimeric expression vector containing the cyan fluorescent protein (CFP) cDNA fused in frame with the human Sp100 cDNA at its 5'-end to study the SRC-1-containing nuclear foci in living cells. Results presented in Fig. 5Go show that in living cells both of these chimeric proteins are associated with the same subnuclear foci.



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Figure 4. Immunolabeling of CFP-SRC1 and CFP-Sp100-Expressing Cells with anti-PML Antibody

Human kidney-derived cells (HEK293) were transfected with either CFP-SRC1 or CFP-Sp100 expression vectors, and fixed cells were immunostained with antibody to human PML protein. A, Colocalization of SRC-1 (cyan); with PML protein (red). B, Colocalization of Sp100 (cyan) with the PML protein (red). Magnification bar, 3 µm.

 


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Figure 5. Association of SRC-1 with Sp100-Containing PML Bodies

Photographs show a representative z-section from a cell that was cotransfected with CFP-Sp100 and YFP-SRC1 expression vectors. Left frame shows the Sp100 fluorescence (cyan), the middle shows SRC-1 (yellow), and the right shows the merged image. Magnification bar, 3 µm.

 
Transport of SRC-1 from the PML Bodies to Subnuclear Speckles by the Agonist-Activated AR
We have previously reported that ligands capable of promoting the transactivation function of AR can not only translocate the receptor into the nucleus but also transport the receptor into a subnuclear compartment with a distinct punctate appearance. However, a pure antagonist such as casodex can translocate the receptor into the nucleus but fails to induce transactivation of the target genes and also fails to transfer the receptor to these subnuclear speckles (6). This observation has suggested distinct functional roles of these subnuclear compartments directing various steps that ultimately lead to hormonal signaling for gene expression. Because SRC-1 plays an important role in the mediation of androgen action, we have used the PML body associated SRC-1 to trace its intranuclear movement after ligand-receptor interaction.

Figure 6AGo presents an image of a COS-1 cell cotransfected with YFP-SRC1 and CFP-AR after casodex (antagonist)-mediated translocation of the CFP-AR into the nuclear compartment. Under this condition, despite the availability of the AR in its vicinity, SRC-1 is still associated with the PML bodies. However, when the physiological androgen agonist, 5{alpha}-dihydrotestosterone (DHT) was used to transport the receptor into the nucleus, YFP-SRC1 is removed from the PML bodies and is transferred to the AR-containing subnuclear speckles (Fig. 6BGo). When these cells coexpressing both CFP-AR and YFP-SRC1 were not treated with DHT, the CFP-AR remained in the cytoplasm and YFP-SRC1 maintained its focal pattern of distribution (Fig. 6CGo).



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Figure 6. Differential Effects of AR Agonist and Antagonist on the Translocation of SRC-1 from the PML Body

A, Images of a cell nucleus treated with casodex (10-6 M), a pure androgen antagonist. B, Images of a cell nucleus treated with the physiological agonist DHT (10-8 M). Magnification bar, 3 µm. C, Images of a cell transfected with CFP-AR but not treated with any ligand. Under this condition CFP-AR resides predominantly in the cytoplasm, whereas the focal distribution of YFP-SRC1 remains unaltered.

 
The progressive movement of SRC-1 from the PML bodies to the subnuclear speckles was authenticated further through imaging of a single cell at various time points after addition of DHT into the culture medium (Fig. 7Go, A and B). Agonist-mediated translocation of CFP-AR from the cytoplasmic compartment was followed by removal of the YFP-SRC1 from the PML bodies to another nuclear compartment (nuclear speckles). Cotransfection of YFP-SRC1-expressing cells with an unlabeled constitutively active mutant of AR [AR-{Delta}ligand-binding domain (LBD)] caused removal of SRC-1 from the PML bodies to the nuclear speckles in all cells (representative pattern presented in Fig. 7CGo). Thus, the absence of any PML body-associated SRC-1 in cells expressing the constitutively active mutant of the AR confirms our conclusion that this coactivator is actively removed from the PML body during androgen action. The mutant AR without its ligand-binding domain that we have used for this experiment caused about the same degree of transcriptional activation of the probasin-luciferase promoter-reporter construct as the wild-type AR in the presence of 10-8 M DHT (Rivera, D., and A. K. Roy, unpublished).



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Figure 7. Redistribution of SRC-1 Foci from PML Bodies to Subnuclear Speckles by Activated AR and a Constitutively Active AR Mutant (LBD Deleted)

Cells were cotransfected with YFP-SRC1 and CFP-AR and were imaged using YFP and CFP filters. A, Images showing the time-dependent movement of the SRC-1 from the PML bodies to subnuclear speckles after androgen treatment. The cell was imaged at 0, 30, 60, and 90 min after addition of DHT (10-8 M) into the culture medium. B, The same cell (shown in A) displaying movement of CFP-AR from the cytoplasm to the nucleus after androgen treatment. C, A typical cell cotransfected with YFP-SRC1 and AR-{Delta}LBD in the absence of hormone treatment (left panel). The right panel shows maintenance of the focal pattern of SRC-1 in a cell nucleus when cotransfected with YFP-SRC1 and wild-type AR, also in the absence of hormone treatment. Magnification bar, 2 µm.

 
The distinctive morphological characteristics of the SRC-1-containing PML bodies and the subnuclear speckles containing both SRC-1 and the agonist-activated AR were defined by reconstruction of the three-dimensional (3-D) images through stacking of 0.2-µm z-sections and processing them through a Carl Zeiss surface-rendering algorithm. Reconstructed 3-D images presented in Fig. 8AGo show that most of the SRC-1-associated PML bodies in living cells are round to elliptical in shape and, in some cases, more than one of these nuclear bodies are fused with each other. A number of attached microsatellites are also seen on the PML body surface. Many of these microsatellites contain YFP-SRC1, and some of them seem to be in the process of fusing with the Sp100-containing PML bodies. After agonist treatment both AR and SRC-1 move to the same subnuclear structure (speckles). The surface feature of the subnuclear speckles, where both SRC-1 and AR migrate after agonist activation, appears as interconnected filamentous structures that do not show any definite geometric shape (Fig. 8Go, B and C).



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Figure 8. 3-D Images of CFP-Sp100/YFP-SRC1 and CFP-AR/YFP-SRC1 in Living Cells with and without Androgen Treatment

A, Cell nucleus cotransfected with CFP-Sp100, YFP-SRC1, and wild-type AR showing the association of SRC-1 (yellow) with Sp100 (cyan) containing PML bodies in the absence of any hormonal stimulation. B and C, Images of a cell cotransfected with CFP-AR and YFP-SRC1 60 min after treatment with DHT (10-8 M). Both SRC-1 and AR appear to colocalize at the subnuclear speckles after agonist activation.

 
Relative Dynamics of SRC-1 and AR in Different Subnuclear Locations
The equilibrium kinetics and mobility of SRC-1 and AR in the living cell were probed through FRAP analysis. This approach is based on the kinetics of replacement of proteins containing the photo-destructed fluorophore with fluorescent proteins from the surrounding intranuclear space. Proteins that are not anchored to any nuclear structure are highly mobile, as indicated by the extremely rapid recovery of the free YFP in cells transfected with an YFP expression vector (pCMV-YFP) (Fig. 9AGo). After photobleaching with a laser light (514-nm line) of a small area (4.5 x 0.8 µm, indicated by the rectangular box on an optical section), replacement of the bleached molecules occurs even during the period of photobleaching with a concomitant decline of the total nuclear fluorescence. Thus, the dynamics of movement were so rapid that we failed to observe any bleached zone, and the partial recovery of the total nuclear fluorescence observed at 90 sec after photobleaching may be due to the movement of unbleached cytoplasmic YFP into the nucleus. The CFP-AR, when translocated into the nuclear compartment due to its interaction with a pure antagonist, i.e. casodex, recovers 50% of the fluorescence intensity within about 7 sec after photobleaching an area of the same dimension (Fig. 9Go, B and D). This difference in the recovery time between free YFP and CFP-AR suggests that, even in the absence of its transactivating capability, the antagonist-bound nuclear AR may be associated with certain nuclear structures, which retards its intranuclear mobility. However, when CFP-AR is activated by an agonist, i.e. DHT, and is associated with the subnuclear speckles, its mobility is further restrained with a half-time (t1/2) of about 12 sec (Fig. 9Go, C and D). We have also performed a comparative analysis of the rate of replacement of YFP-SRC1 when it is associated with the PML body (before its recruitment by the agonist-activated AR) and after its translocation to subnuclear speckles. Figure 10Go shows the representative FRAP pattern in two cells, one with (Fig. 10AGo) and the other without (Fig. 10BGo) androgen (10-8 M DHT) treatment. The rate of recovery (t1/2) of the YFP-SRC1 associated with a single PML body was 30 sec as compared with 12 sec when this coactivator translocated to subnuclear speckles by the activated AR (Fig. 10CGo). Because a longer t1/2 generally indicates a more stable association, this observation suggests an active recruitment of SRC-1 by AR from the PML bodies to the nuclear speckles, rather than translocation based only on the kinetics of equilibrium between these two subnuclear sites. It should also be noted that the exchange kinetics of YFP-SRC1 when it is associated with the filamentous structure is almost the same as that of CFP-AR (Fig. 10Go, A and D). Thus, it appears that at this site the SRC-1/AR heteromeric complex moves in and out together as a single unit.



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Figure 9. Determination of the Relative Intranuclear Mobility of the Agonist and Antagonist-Bound AR by FRAP

A, Cell showing rapid mobility of the free YFP within the nuclear compartment. The boxed area indicates the attempted photobleached region. An overall decline of nuclear fluorescence during photobleaching indicates rapid equilibration of the free YFP within the nuclear compartment. B, Photobleaching and recovery in a cell transfected with CFP-AR and treated with the antagonist casodex (10-6 M). Photobleaching around the boxed area shown in the left frame and a complete recovery after 90 sec (right frame) is seen. C, Slow recovery of the photobleached area in cells transfected with CFP-AR and treated with 10-8 M DHT. D, Quantitative comparison of the recovery rate in casodex (square)- and DHT (diamond)-treated cells. Each curve represents measurements from 9 cells at different time points ± SD.

 


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Figure 10. Relative Recovery Rates of Photobleached YFP-SRC1 Associated with the PML Body and Subnuclear Speckles

A, FRAP of YFP-SRC1 associated with subnuclear speckles. Cells were cotransfected with CFP-AR and YFP-SRC1 and subsequently treated with DHT (10-8 M). The boxed area (4.5 µm x 0.8 µm) was subjected to photobleaching and recovery as visualized through the YFP filter. B, FRAP of YFP-SRC1 associated with the PML body. Cells were cotransfected as in panel A but not treated with androgen. A single PML body-associated SRC-1 foci was photobleached (1.0-µm-diameter circle) and allowed to recover over time. C, Relative recovery rates of YFP-SRC1 when associated either at subnuclear speckles (diamonds) or in the PML body (squares). Data represent averages of nine cells ± SD.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormonal regulation of gene expression by nuclear receptors is a multistep process that utilizes at least three distinct pathways. One of these pathways is typified by the thyroid hormone receptor where the unliganded receptor is associated with the chromatin and provides a repressor function. Ligand binding causes reversal of this repressor role of the receptor with the exchange of corepressor with coactivator proteins (27). The second mechanism, exemplified by the estrogen receptor (ER) and peroxisome proliferator-activated receptor, in which the unliganded receptor also exists as a nuclear protein and only after ligand activation, migrates to the chromatin site for promotion of target gene expression (13, 28). Androgen and glucocorticoid receptors utilize a third pathway, where the inactive receptor is housed in the cytoplasmic compartment as a complex with heat shock proteins. Upon ligand activation, AR translocates into the nucleus and moves through distinct subnuclear compartments before interacting with the target gene (6, 7). Such movements may have important regulatory implications not only for stepwise interaction with various coregulatory proteins but also for integration of cell function through a regulatory network involving common factors such as CBP, GRIP-1, and SRC-1.

The PML body appears to play an important coordinating role for multiple regulatory pathways. It has already been implicated in the regulation of cell proliferation, cellular senescence, and apoptosis (15, 18, 19, 22, 29, 30, 31, 32, 33, 34). Our finding, along with the other two examples, i.e. CBP and GRIP-1 (21, 35, 36), expands the role of the PML body to include regulation of steroid hormone action. The basic structural integrity of the PML body is dependent on the SUMO-ylated form of the PML, a RING finger protein (23). Proteins containing RING fingers possess an inherent tendency for self-association (20). Fibroblasts derived from PML-/- mice do not contain PML bodies. However, transfection of these cells with an expression vector for the wild-type PML protein can restore their ability to form PML bodies. On the other hand, ectopic expression of a mutant PML protein, where the three SUMO-ylation sites are altered, fails to do so (30, 31). From these results it appears that the SUMO-ylated PML protein provides the structural framework for the formation of PML bodies, a subnuclear unit that harbors a number of regulatory factors. Additionally, most of the proteins associated with the PML body also undergo reversible SUMO-ylation. In fact, it has already been suggested that the SUMO-ylation serves as a mechanism of recruitment for various PML-associated proteins (17, 30). An examination of the amino acid sequence of SRC-1 indicates a number of potential SUMO-ylation sites. Mutation of the lysine-to-arginine codon within only one of these SUMO-ylation sites of the SRC-1 cDNA prevents the association of SRC-1 with the PML body (Rivera, O., and A. K. Roy, manuscript in preparation).

In earlier studies, several groups have used fluorescently labeled SRC-1 and GRIP-1 to address various issues related to nuclear receptor function. Some of these reports suggested uniform intranuclear distribution of the labeled coactivator (37, 39), whereas one report describes both focal and smooth intranuclear distribution of the coactivator (21). Most of these reports present only single-cell images, and some with less than optimal resolution. However, our results are consistent with those of Baumann et al. (21), who showed both focal and smooth intranuclear distribution of GRIP-1. It may be noted that both our studies and those of Baumann et al. were conducted with unsynchronized cell populations. Formation and disassembly of PML bodies, as well as mobilization and utilization of coactivators, are cell cycle-dependent processes. SRC-1 is used as a coactivator by many transcription factors for normal cell function. Thus, during the stages of the cell cycle when these transcription factors are active, almost all of the SRC-1 is expected to be associated with the chromatin and, therefore, diffusely distributed. On the other hand, during the stages of the cell cycle when these transcription factors are less active or inactive, SRC-1 may be sequestered in the PML bodies. The significance of PML body association of SRC-1 and its coactivator function at various stages of the cell cycle remains an interesting issue.

It was reported previously that SUMO conjugation of AR inhibits its transactivation function (11). Although the enzymatic mechanism for SUMO-ylation is similar to ubiquitination, the E3 SUMO ligases have not yet been well characterized. However, PIASy (protein inhibitor of activated signal transducer and activator of transcription) appears to be a strong candidate enzyme (40, 41). Furthermore, overexpression of PIASy causes inhibition of a number of transcriptional regulators including the AR (41, 42). In rare situations (<0.1% of transfected cells) we have found that both AR and SRC-1 are associated with the PML bodies (data not presented). These observations raise the possibility that AR may transiently enter into the PML body and interact with SRC-1 to retrieve the coactivator.

It is possible that transient SUMO-ylation of AR introduces it to the PML body and simultaneous de-SUMO-ylation of AR and SRC-1 may allow the formation of AR/SRC-1 complex leading to their removal from the PML body and uptake by other subnuclear compartments for subsequent steps involved in the activation of target genes. Since submission of this manuscript, Kotaja et al. (43, 44) published two interesting articles providing more substantial correction between SUMO conjugation and AR/coactivator function. These authors demonstrate that PIAS family members can function as SUMO-1-tethering proteins for a number of transcriptional regulators and that mutational inactivation of potential SUMO conjugation sites of GRIP-1 decreases its ability to enhance AR-mediated transcriptional activation.

FRAP analysis of the unconjugated YFP reveals that the proteins that are not associated with any subnuclear structures are highly mobile. Moreover, transcriptionally incompetent AR translocated into the nuclear compartment under the influence of the pure antagonist casodex maintains a relatively high mobility. These observations indicate that acquisition of transcriptional competency of AR is correlated to its association with specific subnuclear structures. In addition to PML bodies, association of both the SRC-1 and AR to an interconnected filamentous nuclear structure appears to be another requisite step in the eventual signaling function of AR. At this point the exact nature of this filamentous nuclear structure is uncertain. Because heterochromatin and euchromatin structures within the living cell nucleus are differentially distributed (7), the uniform nuclear distribution of the interlacing filamentous structure may represent a nonchromatin compartment, possibly nuclear matrix (45). However, the concept of nuclear matrix still remains controversial (46), and an exact definition of this subnuclear compartment will require further investigation.

FRAP analysis of the ER in living cells after agonist and antagonist treatment has been reported previously (13). These studies have shown that the unliganded ER within the nuclear compartment is highly mobile and the receptor mobility is constrained after agonist/antagonist binding. Unlike ER, the unliganded AR is localized within the cytoplasmic compartment; therefore it is difficult to compare the intracellular dynamics of these two receptors in their unliganded states. However, a reduced mobility of both of these receptor after ligand binding and equalization of the receptor and the coactivator movement after agonist uptake are consistent with a common intranuclear pathway for both AR and ER function. It has also been reported that binding of ER to a pure antagonist (ICI 182,780) immobilizes the receptor within a subnuclear compartment (13). This finding differs from our observation of the highly mobile state of the AR when bound to a pure antagonist, i.e. casodex. Although authors of these studies suggested that binding of ER to the pure antagonist immobilizes the receptor to the nuclear matrix, the possibility that antagonist-bound ER is sequestered into the proteasomal compartment has not been ruled out.

Lastly, our results for the first time reveal the surface characteristics of the PML body and the nuclear speckles (a likely candidate for the nuclear matrix) in living cells. Based on electron microscopic evaluation, it has been suggested that PML bodies are doughnut-shaped intranuclear structures (30). However, our results show that in living cells the PML bodies are round to elliptical in shape. The doughnut-shaped structure indicated by the electron microscopic evaluation is possibly due to the changes introduced during fixation and drying and may indeed suggest that the outer shell of the PML body is denser than its inner core. Such a fluid-rich inner core may be functionally relevant for the dynamic movement of regulatory proteins in and out of the PML bodies, necessary for the coordinated control of cell proliferation, apoptosis, and hormone action.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
The chimeric CFP-AR was constructed by inserting the human AR cDNA into the EcoRI site of the 3'-end of pECFP-C1 (CLONTECH Laboratories, Inc., Palo Alto, CA). The chimeric YFP-SRC1 and CFP-SRC1 were generated by inserting the human SRC-1e cDNA into the 3'-end of pEYFP-C1 (CLONTECH Laboratories, Inc.) at the BglII and SalI sites. CFP-Sp100 was created by inserting the Sp100 cDNA (GenBank no. 4507164) into the 3'-end of pECFP-C1 at the EcoRI site. The pCMV-AR vector (a gift from Frank French and Elizabeth Wilson) contains the human AR cDNA in a cytomegalovirus (CMV) expression plasmid. The plasmid pCMV-AR{Delta}LBD was generated by insertion of a mutant AR cDNA sequence containing a deleted ligand-binding domain (truncation of the AR cDNA at the 1986-bp position) into the CMV expression vector. The pARR2-Luc reporter construct is based on the rat probasin promoter. The androgen response region (ARR) of this promoter corresponds to bp -244 to -96 and was inserted immediately upstream of the sequence corresponding to bp -286 to +28 of the same promoter. This tandem arrangement was inserted into the pGL3-Basic vector (Promega Corp., Madison, WI). Nucleotide sequences of all DNA constructs were authenticated by manual sequencing.

Cell Culture and Transfection
COS-1 and HEK293 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s Modified Media supplemented with 9% fetal bovine serum. All transfections used Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. For luciferase reporter assays, cells were seeded in 24-well plates at 50 x 103 cells per well in MEM supplemented with 5% charcoal-stripped serum and grown overnight before transfection. Transfected DNA consisted of 200 ng of the pARR2-Luc promoter-reporter plasmid, 300 ng of pCFP-AR, and 100 ng of pYFP-SRC1. Twenty-four hours after transfection, hormone was added to the media and cells were collected after an additional 24 h. Cell extracts were assayed for protein concentration (Bradford protocol) and luciferase activity (assay kit, Promega Corp.). All values were normalized to equivalent protein concentration.

Cells for confocal microscopy were seeded in Nunc Lab-Tek no. 1 borosilicate four-well chambered slides (Fisher Scientific, Pittsburgh, PA) in MEM supplemented with 5% charcoal-stripped serum at 30 x 103 cells per well and grown overnight before transfection with 100 ng of the appropriate chimeric plasmid. The cells for experiments shown in Fig. 7CGo were transfected with 75 ng of pYFP-SRC1 and 375 ng of either the wild-type human AR, pCMV-AR, or its truncated counterpart, pCMV-AR{Delta}LBD, which lacks the ligand-binding domain. All images were collected at least 28 h after transfection to allow sufficient expression of the chimeric fluorescent protein. Before imaging, media were supplemented with HEPES to a final concentration of 25 mM.

Fluorescence Imaging and FRAP Analysis
Fluorescence imaging of live cells, FRAP experiments, and 3-D imaging were conducted on a LSM 510 laser confocal microscope (Carl Zeiss, Thornwood, NY) equipped with a 63 x 1.4 NA oil immersion objective, a BP 470–500 filter, a LP 530 filter, a LP 560 filter (Chroma Technology Corp, Brattleboro, VT), and a temperature-controlled stage. All FRAP experiments were conducted at a stage setting of 37 C. To conduct FRAP, a single z-section was imaged before bleach, immediately after bleach (~0.5 sec later), and at subsequent time intervals. For CFP-AR and nucleomatrix-distributed YFP-SRC1, bleaching was performed on a 4.5 x 0.8 µm boxed region at wavelengths of 458 and 514 nm, respectively. The intensity was adjusted to 90% maximum power for 25 iterations for approximately 3 sec. To compensate for z-directional movement of the foci-distributed YFP-SRC1 over time, the FRAP experiments in this case were performed under the same conditions except that an individual focus was targeted for bleaching. After bleaching, a z-dimensional section 3 µm thick was collected at each time point by taking four optical z-sections at 0.75-µm intervals. To compensate for xy-directional movement of YFP-SRC1 foci over time, the region of interest was manually directed to the position of the foci and measured at each time point. Raw images were collected with LSM software (Carl Zeiss), whereas fluorescence intensity measurements of regions of interest were performed with Metamorph software (Universal Imaging Corp., West Chester, PA) and analyzed with Excel (Microsoft Corp.). Fluorescence signals for different fluorophores were normalized by adjusting the intensity in each image so that the brightest pixels were just below saturation. Using this procedure, the average pixel intensity was approximately equalized for each fluorophore.

The 3-D surface-rendered images presented in Fig. 8Go were generated with LSM software (Carl Zeiss) by reconstructing 30–40 optical z-sections throughout the cell. Each scan interval was 0.2 µm, and the total number of scans depended on the total thickness of the cell. Scanning through a single PML body took 2–4 sec, whereas scanning through the whole nucleus took 40–50 sec. To minimize distortion due to the random movement of the PML bodies (YFP-SRC1 foci), optical sections for 3-D imaging were taken at 22 C rather than 37 C. The lower temperature reduced PML body movement by 75% thus minimizing distortion of the 3-D reconstruction. Images for all FRAP experiments were collected every 10 sec including the time required to scan the image. The extent of bleaching after laser exposure varied from experiment to experiment, and the recovery was always incomplete possibly due to the replacement of the bleached fluorophore-containing proteins and the corresponding endogenously derived nonfluorescent protein. Therefore, graphical representations of FRAP data are shown as a ratio of the measured fluorescence intensity divided by the original fluorescence intensity at a given time point. For statistical comparison of CFP-AR in the presence of DHT or casodex, the values were normalized by setting the bleached fluorescence value to 0 and the maximal recovery value to 1. The same method was used to compare the recovery rate of the two distribution patterns of YFP-SRC1 (foci or nucleomatrix). Normalizing the values makes the fluorescence intensity measurement at each time point directly comparable and is necessary because the intensity after bleaching is not always the same. One-way ANOVA was employed to compare the treatments at each time point. At P < 0.01, the recovery rate of CFP-AR treated with DHT differed significantly from casodex treatment up to 60 sec. At P < 0.01, the recovery rate of the YFP-SRC1 foci differed significantly from the YFP-SRC1 nucleomatrix distribution up to 150 sec.

Immunofluorescent Identification of PML Bodies
Primary antibody to PML protein used for this study was made against human protein. Therefore, immunolabeling experiments were performed in human kidney-derived (HEK293) cells. HEK293 cells were transfected in Nunc Lab-Tek No. 1 borosilicate four-well chambered slides as described above for COS-1 cells. Twenty-eight hours after transfection the cells were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and postfixed in ice-cold methanol for 5 min. After fixation, cells were incubated overnight with rhodamine-conjugated antibody specific for human PML (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The antibody was diluted 1:100 in PBS with 4% BSA and 0.1% Tween-20. After washing three times with PBS for 10 min, each of the cells was imaged as described above. For immunostaining of endogenous SRC-1, untransfected HEK293 cells were fixed and permeabilized in a single step using ice-cold methanol for 10 min. Primary antibody against SRC-1 was diluted 1:500 in PBS with 4% BSA and 0.1% Tween-20 and incubated overnight. The cells were washed three times in PBS for 10 min each. Secondary rhodamine-conjugated antibody was diluted 1:1000 and incubated for 1 h followed by three washes in PBS for 10 min each.


    ACKNOWLEDGMENTS
 
We thank Dr. Michael Stallcup for the human SRC-1e plasmid. Valuable technical assistance from Mr. Gilbert Torralva and help from Dr. Thomas Oh in clone identification are gratefully acknowledged.


    FOOTNOTES
 
This work was supported by NIH Grants RO1-DK-14744, R37AG-10486, T32-AG-00165, and a MERIT Review grant from Veterans Affairs. B.C. is a senior career scientist of the Veterans Affairs. A.K.R. is a MERIT awardee from the National Institute on Aging.

Abbreviations: AR, Androgen receptor; ARR, androgen response region; CBP, cAMP response element binding protein; CFP, cyan fluorescent protein; CMV, cytomegalvirus; 3-D, three dimensional; DHT, dihydrotestosterone; ER, estrogen receptor; FRAP, fluorescence recovery after photobleaching; GRIP, glucocorticoid receptor-interacting protein; LBD, ligand-binding domain; PIAS, protein inhibitor of activated signal transducer and activator of transcription; PML, promyelocytic leukemia; SRC-1, steroid receptor coactivator 1; SUMO, small ubiquitin-like modifier; YFP, yellow fluorescent protein.

Received for publication May 2, 2002. Accepted for publication October 22, 2002.


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 DISCUSSION
 MATERIALS AND METHODS
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