FASEB J. Avanti Polar Lipids
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.08-113639.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.08-113639v1
23/2/546    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Graham, J. D.
Right arrow Articles by Clarke, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Graham, J. D.
Right arrow Articles by Clarke, C. L.
(The FASEB Journal. 2009;23:546-556.)
© 2009 FASEB

Nuclear matrix binding is critical for progesterone receptor movement into nuclear foci

J. Dinny Graham*, Adrienne R. Hanson{dagger}, Amanda J. Croft*, Archa H. Fox{dagger} and Christine L. Clarke*,1

* Westmead Institute for Cancer Research, University of Sydney at the Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales, Australia; and

{dagger} Western Australian Institute for Medical Research, Centre for Medical Research, University of Western Australia, Perth, Western Australia, Australia

1 Correspondence: Westmead Institute for Cancer Research, Westmead Millennium Institute, Darcy Rd., Westmead, NSW 2145, Australia. E-mail: christine_clarke{at}wmi.usyd.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ovarian hormone progesterone is essential for normal breast development, and progesterone analogues are implicated in increasing breast cancer risk. The progesterone receptor (PR) is a transcription factor that, when ligand activated, moves rapidly into nuclear foci associated with transcriptional activity. However, the role of intranuclear trafficking signals in the focal location of PR is unknown. We have identified a mutation in PR that ablates its binding to the nuclear matrix and prevents PR movement into nuclear foci. Nuclear matrix binding mutants lack transcriptional activity and inhibit dimerization, demonstrating the critical role of matrix binding for PR dynamics and activity. DNA binding of PR is required for fidelity of location in foci, as DNA binding domain (DBD) mutants form aberrant foci with reduced mobility and altered tethering to the nucleus. Mutations in either the nuclear matrix targeting sequence or DBD domains were dominant in preventing wild-type receptor from moving to appropriate nuclear locations, demonstrating that both partner proteins in a PR dimer must have intact intranuclear trafficking signals for correct receptor positioning within the nucleus. This study has demonstrated that positioning of PR in foci within the nucleus is critically regulated by intranuclear trafficking signals, which play a key role in transcriptional activity and are relevant to its action in normal and malignant breast cells.—Graham, J. D., Hanson, A. R., Croft, A. C., Fox, A. H., Clarke, C. L. Nuclear matrix binding is critical for progesterone receptor movement into nuclear foci.


Key Words: intranuclear trafficking • breast cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE OVARIAN HORMONE PROGESTERONE is a critical regulator of normal female reproductive function and plays key roles in normal mammary gland development and function (1) . Progesterone is also implicated in breast cancer, and large-scale trials have shown that women exposed to progesterone analogues in hormone replacement therapy are at increased risk of developing breast cancer (2 , 3) . Progesterone effects are mediated by the nuclear progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. Human PR is expressed as two proteins, PRA and PRB (4) , and in normal human target tissues both PRA and PRB are coexpressed at equivalent levels in all PR-positive epithelial cells (5 , 6) .

PR is a ligand-activated transcription factor, and ligand binding causes the redistribution of PR within the nucleus, from being homogeneously distributed throughout the nucleus to being located in focal aggregates (7) . In PR-positive cells, PRA and PRB both form foci, and these foci contain PRA and PRB hetero- and homodimers, as demonstrated directly by fluorescence resonance energy transfer (FRET) of fluorescently tagged PRA and PRB (8) . Endogenous PR foci are colocalized with sites of nascent mRNA expression, and inhibition of transcription blocks formation of PR foci, supporting the view that PR foci are associated with transcriptional activity (8) .

In human tissues, PR foci have clear physiological relevance. In endometrial epithelial cells, PR is evenly distributed throughout the nucleus in the proliferative phase of the menstrual cycle but is detected only in nuclear foci in the luteal phase of the cycle (7) , when serum progesterone levels are high, and at a time when progesterone exerts its physiological actions in that tissue. Similar observations were made in the normal human breast (8) , demonstrating that progesterone regulates the movement of PR into nuclear foci in normal human tissues that are targets for progesterone action. PR foci are also detected in breast and endometrial cancers, but in these tissues PR foci are aberrant. In endometrial cancers, PR foci are significantly larger than PR foci in normal endometrium and also show reduced reliance on ligand for their formation (7) . In breast cancers, PR foci are found in around 50% of tumors and are equally found in premenopausal and postmenopausal patients (8) , suggesting reduced reliance on circulating progesterone, as seen in endometrial cancers. PR foci in cancers are independently associated with poorer clinical features of the tumor, emphasizing their importance in pathophysiology as well as in normal physiology (7) .

Although movement of PR into foci appears to be a critical component of its transcriptional activity in normal target cells and is a feature of PR signaling that is disrupted in cancer and associated with poor prognosis features (7 , 8) , the determinants of PR movement into foci are not known. Intranuclear movement is known to be regulated in part by intranuclear trafficking signals, which regulate dimerization, binding to the nuclear matrix, and binding to DNA (9) . For nuclear receptors, sequences involved in dimerization play a role in receptor intranuclear trafficking, as does the DNA binding domain (DBD), comprising two zinc fingers, which is responsible for association of ligand-activated receptors with response elements in target genes (10 , 11) . Nuclear receptors also bind to the nuclear matrix (12 13 14) , the proposed riboprotein scaffold suggested to be responsible for organization of nuclear architecture and tethering of actively transcribing chromatin. A specific nuclear matrix targeting signal (NMTS) has not been defined in PR. This study aimed to determine the role of intranuclear trafficking signals in formation of PR foci and to determine whether positioning of PR in the nucleus, as regulated by intranuclear trafficking signals, regulates the transcriptional activity of the receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs
The cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) -tagged wild-type PR have been described before (8) . PR DBD and NMTS single and double mutations were created using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The primer sequences used to introduce the desired DBD and NMTS mutations were 5'-tgtccttacctgtgggagcgctaaggtcttctttaagagg-3' and 5'-cagttgattccaccactgatcaacgggggaatgagcattgaaccagatgtgat-3', respectively. The PR dimerization mutant was constructed by deletion of the hinge region of PRA (base positions 1432 to 1567 in PRA) from CFP-PRA. The pA3-PRE2-luc reporter construct contains two progestin response elements upstream of the minimal TATA element of the thymidine kinase promoter and the luciferase cDNA.

Cell culture and transfections
U-2 OS osteosarcoma cells were maintained in phenol red-free Dulbecco modified Eagle medium (DMEM) with 10% fetal calf serum (FCS, Invitrogen, Mount Waverley, VIC, Australia). In experiments where cells were depleted of ATP, U-2 OS cells were transferred into glucose-free DMEM (Invitrogen), containing 6 mM 2-deoxyglucose (Sigma-Aldrich, Castle Hill, NSW, Australia), 10 mM sodium azide, and 10% FCS. MCF-7M11 breast cancer cells were grown in phenol red-free DMEM containing 10% fetal calf serum and insulin (0.28 IU/ml; Novo Nordisk, Baulkham Hills, NSW, Australia). Cells were grown on chamber slides (imaging experiments) and in 10 cm dishes (PR expression and transcriptional assays). Constructs were introduced into cells by transfection using Fugene (Roche, Castle Hill, NSW, Australia) at a ratio of 6:1, DNA:Fugene, and following the manufacturer’s protocol. Cells were treated with ORG2058 (10 nM) or vehicle, as indicated. Luciferase activity was measured in cell lysates using the Promega Bright-Glo reporter system (Promega, Annandale, NSW, Australia). Each PR and reporter combination was transfected from a single master mix, and luciferase activities were normalized to the protein concentrations of the lysates.

Protein extraction and immunoblot analysis
Cells were harvested and washed with cold PBS, and cell pellets were collected by centrifugation. Whole-cell extracts were prepared by lysis in RIPA buffer for 30 min at 4°C, and PR proteins were detected in supernatants by immunoblotting as described previously (8) .

Live imaging of focus formation and FRET
Real-time imaging of CFP- and YFP-tagged PR focus formation was performed using a Zeiss Axiovert 200M inverted fluorescent microscope (Carl Zeiss, Oberkochen, Germany), at 37°C, 5% CO2, using an x630 oil-immersion objective. Only low- to medium-intensity nuclei were imaged to avoid artifacts of overexpression. For fixed time points, cells were treated with 10 nM ORG2058 or vehicle for the times indicated (1 h for FRET), washed with cold PBS, and fixed with fresh formaldehyde solution (3.7% in PBS) for 1 h at 4°C. PR dimer formation was measured using FRET as described previously (8) . By this method, CFP and YFP intensities should be within 0.1- to 10-fold of each other. This was not possible in the case of PR construct pairs in which one PR forms foci and the other remains evenly distributed in the nucleus. In this instance, the intensity of the foci formed is in >10-fold excess over the nonfocal PR, and as a result, a low-level bleedthrough to the FRET channel is unavoidable but was minimal compared with true FRET signals. PR protein localization was scored in fixed cells by systematic image analysis of multiple fields captured at x400 using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA). Localization was scored as exclusively nuclear, nuclear and cytoplasmic, or exclusively cytoplasmic.

Confocal imaging and fluorescence recovery after photobleaching (FRAP)
Confocal imaging of YFP-tagged PR was performed using an Olympus Fluoview FV1000 confocal microscope and FV10-ASW 1.7 image analysis software (Olympus, Tokyo, Japan). As in live imaging, only low- to medium-intensity nuclei were imaged. A single Z section was imaged using an x630 aqueous-immersion objective and 473 nm excitation diode laser. An elliptical region of interest was imaged 3 times by continuous scanning followed by a bleach pulse of 40 frames at 100% laser power. The region of bleaching was identical for all samples. Fluorescence recovery was recorded by continuous scanning for a subsequent 37 frames to 78.5 s after bleaching. Fluorescence intensities were obtained for the regions of interest, and pre- and postbleach values were normalized between 1 and 0. Mean normalized intensities were plotted for each treatment group, and recovery half-times were estimated from the curves generated. Nuclei (12–15) were analyzed for the evaluation of each recovery half-time.

Permeabilization and chromatin extraction
Cells growing on chamber slides were washed with cold PBS, followed by incubation in cytoskeleton buffer (15) for 3 min at 4°C. Cells were then incubated with digestion buffer (cytoskeleton buffer with NaCl reduced to 50 nM) in the presence or absence of 200 U/ml DNaseI (Invitrogen), 30 min at room temperature. After digestion, ammonium sulfate was added to the digest to a final concentration of 0.25 M, followed by fixation. Removal of DNA was confirmed by staining with DAPI. Retained PR was visualized by fluorescent microscopy, and the level of focus formation was quantitated using Image-Pro Plus software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PR foci form rapidly
Tagged PRA and PRB (CFP-PRA and YFP-PRB) were largely distributed in the nucleoplasm when unliganded, with a small amount of cytoplasmic signal that became more distinctly nuclear when treated with ligand (Fig. 1B ). Ligand treatment caused movement of both PRA and PRB into foci (Fig. 1A ), which were exclusively nuclear. Tagged PRB was active, as demonstrated by its ability to stimulate transcription of a reporter gene, although to a lesser extent than untagged receptor, whereas YFP-PRA had very low transcriptional activity (Supplemental Fig. 1C). The dynamics of PR movement into foci were determined by live-cell image analysis of cells transfected with fluorescently tagged PRA, PRB, or both PR isoforms. In vehicle-treated cells, PR was evenly distributed throughout the nucleus but moved into foci detectable within 20–40 s of exposure to the synthetic PR ligand ORG2058 (PRA is shown in Fig. 2A ). PR foci continued to become more prominent up to 120 s after treatment, and the rate of focus formation was similar for PRA and PRB homodimers and PRA-PRB heterodimers (not shown).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		Figure 1. PRA and PRB are distributed into foci in response to progestin treatment. A) U-2 OS cells transfected with CFP-PRA or YFP-PRB, as indicated, were treated 1 h with 10 nM ORG2058 (ORG) or vehicle (V). PR was visualized in fixed cells by fluorescent microscopy at x630. B) PR distribution was scored in ORG-treated (PRA, n=314; PRB, n=570) and vehicle-treated (PRA, n=312; PRB, n=494) cells.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Figure 2. Wild-type PR moves rapidly in and out of foci in response to ligand. A) YFP-PRA was transiently expressed in U-2 OS cells, and focus formation was imaged in real time as described in Materials and Methods. Cells were treated with 10 nM ORG2058 or vehicle, and images were captured at 20 s intervals. A subset of the captured images is shown. B) Formation of foci in cells transfected with YFP-PRB was documented up to 20 min after ORG2058 treatment. C) Mobility of PR foci and unliganded PR was characterized by FRAP, and recovery half-time was estimated: PRA V T1/2 = 3.0 s, PRA ORG T1/2 = 11.0 s, PRB V T1/2 = 2.3 s, PRB ORG T1/2 = 12.1 s.

PR foci were not observed to move in any discernable way within the nucleus and persisted in discrete nuclear locations for up to 20 min of the live-cell imaging experiment (PRB shown in Fig. 2B ) and thereafter for several hours, as shown in fixed cells (not shown). Within foci, however, PR was highly mobile. FRAP of PR foci showed that PR foci recovered rapidly after bleaching (Fig. 2C ). Both PRA and PRB were mobile within foci, with recovery half-times of 11 and 12.1 s, respectively. The mobility of liganded receptor was slower than that for the unliganded receptor, where the recovery half-times were 3 s for PRA and 2.3 s for PRB (Fig. 2C ).

Dimerization is required for PR focus formation
The rapid movement of PR within the nucleus raised the possibility that intranuclear trafficking signals within PR, including dimerization signals (16 , 17) , NMTSs (18 , 19) , and DBDs (11) may be required for PR movement into foci. Dimerization is a key requirement for PR binding to chromatin: to disrupt PR dimerization, the short hinge region between the DNA-binding and hormone-binding domains was deleted from PRA (Supplemental Fig. 1A ). When transfected into U-2 OS cells, this PR mutant was localized to the cytoplasm in the absence of ligand (Fig. 3A ) as expected, since the PR hinge region, along with the second DNA-binding zinc finger, constitutes a nuclear localization signal (20) . After 1 h exposure to ligand, nuclear localization was modestly enhanced (Fig. 3B ). However, the dimerization mutant was evenly distributed in the nucleus, and no foci were formed (Fig. 3A ). This finding demonstrated that, although ligand may lead to partial restoration of nuclear localization, the inability of the hinge mutant to form homodimers prevents the formation of foci. We noted that the dimerization mutant was particularly sensitive to aggregation and displayed a peri-nuclear localization that was particularly evident in vehicle-treated cells (Fig. 3A ). This condition was likely due to the deletion of the dimerization and nuclear localization signals, both of which influence PR conformation. Confocal and FRAP imaging of apparently focal aggregates of this mutant demonstrated that they were predominantly extranuclear and were immobile (Supplemental Fig. 2 and data not shown).


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		Figure 3. PRA dimerization mutant localization and focus formation are aberrant. A) U-2 OS cells transiently expressing the PRA dimerization mutant (PRA-{Delta}hinge) were treated with 10 nM ORG2058 or vehicle for 1 h, as indicated, and PR localization was visualized by fluorescent microscopy at x630. B) PRA dimerization mutant localization was scored in ORG2058-treated (n=150) and vehicle-treated cells (n=174). C) CFP-PRA wild-type and dimerization mutant were cotransfected with YFP-PRB into U-2 OS cells and treated 1 h with 10 nM ORG2058 or vehicle as indicated. FRET analysis was performed on cells expressing both receptors at equivalent levels.

Although the hinge mutant lacks the dimerization domain, it could form dimers with wild-type PR. Coexpression of wild-type and hinge mutant PR and subsequent ligand treatment resulted in a pattern of focus formation that was similar to wild-type alone (Fig. 3C ). These foci contained wild-type-hinge mutant dimers, as evidenced by FRET signals at these foci (Fig. 3C ), demonstrating that although the hinge mutant is unable to form mutant homodimers, the intact N-terminal sequences known to participate in PR dimer formation (17 , 21) were sufficient to allow rescue of the dimerization mutant by wild-type PR.

PR binding to nuclear matrix is required for focus formation
To disrupt nuclear matrix binding, we targeted two adjacent leucine residues at positions 690 and 691 with respect to the start of PRB (Supplemental Fig. 1A ). These residues are highly conserved among steroid receptors (22) and are essential for nuclear matrix targeting of the glucocorticoid receptor (19) . Mutation of these residues abolished nuclear matrix binding of PR in permeabilized, DNase digested cells (Fig. 4 ), demonstrating the critical requirement for these residues for nuclear matrix binding of PR. The PRA and PRB NMTS mutants were localized to both the nucleus and cytoplasm in vehicle-treated cells (Fig. 5A, B ) with a relative nucleus-cytoplasm distribution that was distinctly different from wild-type receptor (compare Fig. 1A, B with Fig. 5A, B ), suggesting that the retention of NMTS mutant PR in the nucleus was compromised. The PRA NMTS mutant was more nuclear overall than PRB NMTS. With ligand treatment the localization of the PR NMTS mutants became more distinctly nuclear, demonstrating the capacity of NMTS mutant PRs to bind ligand and relocate in response to this, although some fluorescence was still detectable in the cytoplasm (Fig. 5B ).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Figure 4. Characterization of a nuclear matrix targeting sequence in PRB. U-2 OS cells were transiently transfected with wild-type and NMTS mutant PRB. After transfection, the medium was removed and replaced with ATP-depletion medium, as described in Materials and Methods. Cells were permeabilized and incubated in digestion buffer in the presence or absence of DNaseI, as indicated. PR was visualized at x630, and images were recorded at matched exposure times for all conditions. Transfected, nonpermeabilized cells are shown for comparison.


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Figure 5. PR NMTS mutant localization and focus formation are aberrant. A) U-2 OS cells, transfected with CFP-PRA NMTS and YFP-PRB NMTS mutants, were treated for 1 h with 10 nM ORG2058 or vehicle, as indicated, and PR expression was visualized at x630. B) PR NMTS mutant distribution was scored in ORG-treated (PRA NMTS, n=249; PRB NMTS, n=540) and vehicle-treated cells (PRA NMTS, n=305; PRB NMTS, n=577). C) Mobility of wild-type and NMTS mutant PRB in the presence of ORG2058 or vehicle was characterized by FRAP. Recovery half-time: PRB V T1/2 = 2.3 s, PRB ORG T1/2 = 12.1 s, PRB NMTS V T1/2 = 2.5 s, PRB NMTS ORG T1/2 = 3.2 s.

NMTS mutants were mobile, as shown by rapid FRAP recovery times similar to the recovery times observed for unliganded wild-type receptors (Fig. 5C ). However, while ligand-binding decreased the mobility of wild-type receptors (Figs. 2C and 5C ), liganded and unliganded NMTS mutants were equivalently mobile (Fig. 5C ). Although the limitations of comparing recovery times of diffusely vs. focally distributed proteins are acknowledged, FRAP of the NMTS mutant demonstrates that the inability to bind the nuclear matrix prevents the reduction in mobility of the liganded mutant receptor that is observed for wild-type receptor. Mutation of the NMTS prevented the movement of PR into foci on exposure to ligand. One hour after ligand treatment wild-type PR redistributed into foci (Fig. 1A ), but no foci were observed in PRA or PRB NMTS mutant transfected cells (Fig. 5A ).

DNA binding of PR is required for normal focus formation
To determine the role of DNA binding in PR subnuclear localization, a cysteine to alanine mutation was introduced at residue 587 in the first DNA-binding zinc finger (Supplemental Fig. 1A). This mutation is known to abolish zinc incorporation (23) , resulting in loss of the first zinc finger in PR. DBD mutant PRs were expressed at similar level and size as wild-type PR (Supplemental Fig. 1B) but lacked transcriptional activity (Supplemental Fig. 1C). The PRA-DBD and PRB-DBD mutants showed both nuclear and cytoplasmic distribution in the absence of ligand, and this became more predominantly nuclear in the presence of ligand (Fig. 6B, C ). Treatment with ligand caused a striking redistribution of the receptor into foci. Compared to the foci observed in wild-type PR (Fig. 1A ), DBD mutant foci were more numerous and distributed throughout the nucleus (Fig. 6A ). The proportion of PR that moved into foci was higher for the DBD mutant than for wild-type PR. Whereas liganded wild-type receptor was distributed both evenly throughout the nucleus and in foci (Fig. 1A ), the liganded DBD mutants, particularly the PRB-DBD mutant, were almost exclusively located in foci (Fig. 6A ). The capacity of the DBD-mutant to move into foci was dependent on matrix binding, as mutation of both DBD and NMTS signals (Supplemental Fig. 1A) ablated the capacity of DBD-mutant PR to move into foci (Fig. 6D ). Line scan analysis of confocal images demonstrated that the basal intensity of nonfocally distributed PR was markedly lower for cells expressing the DBD mutant than for those expressing the wild-type receptor (Fig. 6E ), demonstrating little nonfocal DBD mutant PR distributed in the nucleus.


Figure 6
View larger version (30K):
[in this window]
[in a new window]

  		Figure 6. Disruption of DNA binding alters PR localization and focus characteristics. A) U-2 OS cells transfected with CFP-PRA DBD and YFP-PRB DBD mutants were treated for 1 h with 10 nM ORG2058 or vehicle, as indicated. Cells were fixed, and PR expression was visualized at x630. B) PR DBD mutant distribution was scored in ORG-treated (PRA, n=272; PRB, n=326) and vehicle-treated cells (PRA, n=397; PRB, n=246). C) U-2 OS cells were transiently transfected with YFP-PRB DBD mutant and treated with 10 nM ORG2058 or vehicle. Cells were then fixed at the indicated times, and YFP-PRB DBD mutant distribution was visualized by confocal microscopy at x630. D) CFP-PRA DBDNMTS and YFP-PRB DBDNMTS mutants transfected into U-2 OS cells were treated 1 h with ORG2058 or vehicle, as indicated. Cells were fixed, and PR expression was visualized at x630. E) U-2 OS cells transfected with YFP-PRB and YFP-PRB DBD were exposed to 10 nM ORG2058 for 90 min, then fixed. A single confocal Z section was imaged, and intensity was estimated across the nucleus by line scan.

The dynamics of focus formation were different for DBD mutant PR. Whereas wild-type PR was detectable in foci within seconds of ligand exposure and became maximal by 2–5 min after treatment (Fig. 2 ), DNA mutant foci were absent from most nuclei 5 min after ligand treatment and did not reach maximal detection until after 30 min of ligand exposure (Fig. 6C ). FRAP analysis further highlighted differences in the dynamics of wild-type and DBD mutant PR foci. Whereas wild-type foci recovered to prebleach levels (when corrected for sample fade; not shown) after bleaching, DBD mutant foci were immobile and showed little recovery within the experimental period (Fig. 7 ).


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Figure 7. Mutation of PR DNA binding alters focus mobility. A) U-2 OS cells expressing wild-type YFP-PRB were treated with 10 nM ORG2058 (top panels) or vehicle (bottom panels). PR was visualized by confocal microscopy at x630, and an elliptical region of interest was bleached by exposure to maximum laser power for 40 continuous cycles. Images were recorded immediately before and after bleaching and for a series of continuous scan cycles up to 78 s postbleaching. B) U-2 OS cells expressing YFP-PRB DBD mutant were treated with 10 nM ORG2058 (top panels) or vehicle (bottom panels). Fluorescence before and after photobleaching was measured as in A. C) Mean normalized intensity of bleached regions was quantified in wild-type and DBD mutant PR. Recovery half-time: PRB V T1/2 = 2.3 s, PRB ORG T1/2 = 12.1 s, PRB DBD V T1/2 = 2.8 s, PRB DBD ORG T1/2 = 5.5 s.

The lack of mobility of DBD mutant PRs suggested that these foci may be differently tethered to the nucleus than wild-type foci, which was confirmed using permeabilization studies. If cells are permeabilized, PR is normally lost from the nucleus by ATP-dependent mechanisms, and PR can only be retained in the nucleus of permeabilized cells if ATP is depleted (14) . Comparison of nuclear retention of wild-type and DBD mutant PR foci showed that, while the wild-type receptor was lost in permeabilized cells, DBD mutant PR foci were not (Fig. 8 ). ATP depletion prevented the loss of wild-type foci, as expected, but interestingly did not prevent the loss of the unliganded DBD mutant (Fig. 8 ). Taken together, the permeabilization studies show that DBD mutant PR is tethered to the nucleus by ATP-independent mechanisms that differ from those regulating the location of wild-type PR foci.


Figure 8
View larger version (34K):
[in this window]
[in a new window]

  		Figure 8. Mutation of PR DNA binding results in altered ATP dependence. U-2 OS cells were transfected with wild-type or DBD mutant YFP-PRB. Cells were treated 30 min with 10 nM ORG2058 or vehicle, then medium was removed and replaced with fresh ATP-depleted or complete medium as indicated. Cells were treated an additional 1 h with 10 nM ORG2058 or vehicle, then fixed immediately or permeabilized then fixed. YFP-PR was visualized by confocal microscopy at x630.

Nuclear matrix and DNA binding are both required for PR focus formation
The findings that nuclear matrix and DNA binding are key determinants of PR foci raised the question of whether intranuclear trafficking signals are required in both the PR proteins in a PR dimer, in order for foci to form. To explore this, NMTS or DBD mutants were cotransfected with wild-type PR, and the ability of wild-type and mutant proteins to dimerize and form foci was evaluated using FRET. While wild-type PRA and PRB dimerized to form PR heterodimers in foci, which represented the highest concentrations of heterodimers in the nucleus as evidenced by FRET (Fig. 9 A), NMTS mutant receptors prevented the movement of wild-type receptor into foci. This finding was evidenced by the lack of wild-type foci in cells where wild-type and NMTS mutant receptors were at similar high levels; in such cells the wild-type-mutant heterodimer would be the predominant molecular species (Fig. 9B , top ORG-treated panels). The wild-type receptor was competent to form foci in these experiments, as shown by the presence of wild-type, but not mutant, foci in cells where wild-type receptor was in excess over the mutant, where the wild-type homodimer would be the predominant molecular species (Fig. 9B , bottom ORG-treated panels). The absence of true FRET signals in cells with wild-type receptor foci confirmed that there was no dimerization of wild-type and mutant receptors (Fig. 9B ). It should be noted that the minimal signal in the FRET panel in Fig. 9B represents low-level CFP bleedthrough to the FRET channel, as described in Materials and Methods, which occurs when the intensity of the CFP focal signal is greater than 10-fold in excess over the nonfocal PR in the YFP channel.


Figure 9
View larger version (48K):
[in this window]
[in a new window]

  		Figure 9. Characterization of PR wild-type and mutant heterodimerization by FRET. U-2 OS cells were cotransfected with the indicated combinations of CFP and YFP PR wild-type and mutant constructs, treated 1 h with ORG2058 or vehicle, and fixed. CFP, YFP, and CFP to YFP FRET were imaged as described in Materials and Methods. A) CFP-PRA and YFP-PRB. B) CFP-PRA and YFP-PRB-NMTS. C) CFP-PRA and YFP-PRB-DBD. D) CFP-PRA-{Delta}hinge and YFP-PRB-DBD.

When the DBD mutant PR was coexpressed with wild-type PR, wild-type-mutant dimers were formed, as evidenced by FRET signals in cells cotransfected with CFP-wild-type PR and YFP-DBD mutant PR (Fig. 9C ). Interestingly, wild-type-DBD mutant PR dimers adopted the pattern of focus formation observed for the DBD mutant alone (compare the pattern of PRA foci in Fig. 9A, C ), suggesting that the DBD mutant PR was dominant in sequestering wild-type PR into foci. The DBD mutant could also heterodimerize with and move the hinge mutant into foci, as evidenced by FRET signals in cells coexpressing DBD and hinge mutants (Fig. 9D ).

Taken together, these results show that mutations in the NMTS and DBD intranuclear trafficking signals in one dimer partner protein prevent normal movement of the wild-type PR partner into normal foci and highlight the fact that for PR foci to form, intact NMTS and DBD intranuclear trafficking signals must be present in both PR proteins in a PR dimer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The location of PR in nuclear foci is characteristic of the endogenous receptor in human tissues and is associated with exposure to circulating progesterone (7) . We have previously shown in cell lines that ligand is required for PR movement into foci and that PR foci are associated with transcriptional activity (8) . This study has used live cell imaging, FRAP, and FRET of wild-type and mutant PRs to determine the role of intranuclear trafficking signals in movement of PR into nuclear foci. The findings are that PR dimerization, binding to nuclear matrix, and binding to DNA are all required for normal movement of PR into foci. In addition, the study has shown that both PR partners in a PR dimer must have intact intranuclear trafficking signals in order for PR to move into foci.

Nuclear receptors commonly move into foci within the nucleus, and transfection of tagged receptors has shown that estrogen receptor {alpha} (ER{alpha}) (24) , androgen receptor (AR) (25) , glucocorticoid receptor (GR) (26) , and mineralocorticoid receptor (MR) (27) form foci when exposed to ligand (24 , 25 , 27 , 28) . Formation of PR foci is associated with transcriptional activity, as shown by the reliance for PR focus formation on recruitment of the coregulator SRC-1, colocation of PR foci with components of the transcription machinery, and colocation of PR foci with nascent RNA (8) .

The rapidity of PR movement into foci supports the role of foci in transcription. PR movement into foci takes place within seconds, and PRA or PRB homodimers and PRA-PRB heterodimers move into foci at the same rate. Notwithstanding the rapidity of PR movement into foci, once foci are formed, they do not move detectably within the nucleus. Persistence of PR foci at the same location is consistent with residence of PR at sites of transcription, which is now accepted to occur in specific nuclear locations where RNA polymerase enzymes, transcriptional cofactors, and other components of the transcriptional machinery assemble at high concentrations (29 30 31 32) . The binding of nuclear receptors to specific DNA sequences associated with transcriptional regulation of target genes has been extensively described. However, the link between sites of active transcription within chromatin and the positioning of nuclear receptors has received less attention. The demonstration that ligand binding causes PR movement into foci that are associated with transcriptional activity (8) and that are tethered at specific nuclear locations (present study) provide support for the importance of positioning of PR in specification of its transcriptional activity. Moreover, the findings link the ligand-dependent redistribution of PR into nuclear foci, observable by live imaging of intact cells, to chromatin-associated functions of PR that are measurable by more direct molecular methods.

Within foci, PR dynamics are very rapid, consistent with their cyclical occupancy of response elements. This finding is consistent with the fact that nuclear receptors are known to cycle rapidly on and off their target genes, and this exchange, and the ordered recruitment of comodulators to sites of hormone-regulated transcription, is required for effective gene expression (12 , 33 34 35 36) . PR dynamics are slower once the ligand is bound than in the unliganded state, in line with findings in other nuclear receptors (37 , 38) . This lower mobility of ligand-bound nuclear receptors has been attributed to their tethering to the nuclear matrix, along with coregulators and other protein components of the transcriptional machinery, to facilitate transcriptional activity (12 , 38) . Association with the nuclear matrix is thought to facilitate recruitment by nuclear receptors of proteins involved in altering chromatin structure, such as SWI/SNF-related proteins, which in turn causes dissociation of histones H2A and H2B from the nearby chromatin and a more open DNA structure allowing transcriptional activation (39 40 41) . The ligand-dependent recruitment of specific histone demethylases is also required to oppose the actions of histone methyl transferases, which act as inhibitory gatekeepers and prevent unliganded activation of target genes (42) . Nuclear matrix tethering may be critical for the fidelity of this highly ordered series of cofactor associations, and the failure of the NMTS mutant to show lower mobility on ligand binding is consistent with the relevance of this process for PR.

The nuclear matrix, which has been described as a riboprotein scaffold, is thought to be responsible for the functional and physical compartmentalization of the nucleus into discrete domains (43 , 44) , including maintenance of higher-order chromatin structure. Although it is acknowledged that controversy exists regarding whether the nuclear matrix is a distinct entity in vivo, or whether the methods used to isolate or reveal the nuclear matrix contribute to its characteristic properties (45) , it is clear that there is tethering of specific proteins to nuclear structures that are frequently given the overarching description of nuclear matrix. Nuclear receptors associate with the nuclear matrix, and nuclear matrix target signals have been described in most steroid receptors (18) . This study has identified a sequence motif responsible for the binding of PR to the nuclear matrix, and this is analogous to a similar motif in GR. Mutation of this sequence prevents the binding of PR to the matrix and also allows the receptor to redistribute to nuclear and cytoplasmic locations, in contrast with the predominantly nuclear location of the wild-type receptor, demonstrating that nuclear matrix binding contributes to the predominant residence of PR in the nuclear compartment.

PR binding to the nuclear matrix is essential for focus formation, as PR mutants that lack the ability to bind to the nuclear matrix also fail to form foci. Mutation of the nuclear matrix binding capacity of PR ablates its transcriptional activity, highlighting the critical role of matrix binding for PR activity. The mechanism of this effect is unknown, but could involve sequestration by the mutant PR, which retains coregulator binding sites, of factors essential for full transcriptional activity of the wild-type receptor. Normal matrix attachment is also critical for PR dimerization because when one partner PR protein lacks matrix-binding capacity, PR proteins fail to form dimers, despite intact dimerization domains being present, and fail to move into foci. The importance of nuclear matrix binding has been shown for GR, AR, and ER (14 , 46 , 47) , and the capacity of PR to bind to the nuclear matrix was first described some time ago (13) . However, prior to this study the matrix attachment signal for PR was not described, nor was the role of matrix attachment in PR dimer formation, movement into foci, and transcriptional activity known.

DNA binding is required for PR to move into normal foci, as mutants lacking DNA-binding capacity move into aberrant foci, and these mutant PRs are transcriptionally inactive. DBD mutant PRs are mobile unless engaged in foci, and DBD mutant PR foci form considerably more slowly than wild-type foci. Once in foci, mutant PRs have very low mobility, which suggests that DBD mutant foci form immobile aggregates, from which PR does not emerge once sequestered. This finding is also supported by the evidence that most DBD mutant PR is engaged in foci, with little nonfocal DBD mutant PR distributed in the nucleus. This finding contrasts with that of the wild-type PR. Wild-type PR can be distributed both in focal and nonfocal locations within the same nucleus. The DBD mutant PR foci have reduced reliance on ATP-dependent mechanisms for nuclear retention, supporting the view that they are immobile and emphasizing the importance of chromatin tethering for normal PR focus formation.

This study has demonstrated that positioning of PR in the nucleus requires intact intranuclear trafficking signals in both protein partners of the PR dimer (Fig. 10 ). When intranuclear trafficking mutants of PR were coexpressed with the wild-type receptor, they were dominant over wild-type in movement of PR into foci. Our findings suggest a model in which both nuclear matrix association and DNA binding are critical for normal PR positioning. In response to hormones, the wild-type receptor recruits transcriptional cofactors and associates with target loci. The NMTS mutant does not associate with the nuclear matrix and is dispersed throughout the nucleus. Although the NMTS mutant might sequester transcriptional cofactors, the lack of nuclear matrix association contributes to a failure to relocate to target loci in the presence of hormones. Ablation of DNA binding blocks redistribution to target loci, and hormone exposure results in formation of aberrant PR aggregates.


Figure 10
View larger version (15K):
[in this window]
[in a new window]

  		Figure 10. Proposed model of ligand-dependent PR subnuclear distribution. Intact nuclear trafficking signals are required in both partners of the PR dimer for normal PR transcriptional function. Disrupted nuclear matrix or DNA binding signals on either PR protein lead to aberrant PR localization and failure to associate with target genes. Nuclear localization signals allow appropriate subnuclear positioning and priming for transcriptional activation.

This study demonstrates a nuclear matrix binding domain of PR and shows the critical reliance on nuclear matrix binding for PR dimerization, movement into foci, and transcriptional activity. Sequences responsible for DNA binding have also been demonstrated to be essential for PR movement into foci, and inability to bind DNA results in immobile PR foci that tether to the nucleus differently to foci formed by wild-type PR. The intranuclear trafficking signals of PR are both required for dimer formation and foci, suggesting their critical role for positioning of PR within the nucleus and transcriptional activity. The findings of this study now provide the basis for investigation of the composition and function of the aberrant PR foci identified in human cancers.


   ACKNOWLEDGMENTS
 
These studies were supported by grants from the National Health and Medical Research Council of Australia and the National Breast Cancer Foundation.

Received for publication June 23, 2008. Accepted for publication September 25, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Conneely, O. M., Mulac-Jericevic, B., Lydon, J. P. (2003) Progesterone-dependent regulation of female reproductive activity by two distinct progesterone receptor isoforms. Steroids 68,771-778[CrossRef][Medline]
  2. Rossouw, J. E., Anderson, G. L., Prentice, R. L., LaCroix, A. Z., Kooperberg, C., Stefanick, M. L., Jackson, R. D., Beresford, S. A., Howard, B. V., Johnson, K. C., Kotchen, J. M., Ockene, J., . Writing Group for the Women’s Health Initiative, I (2002) Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288,321-333[Abstract/Free Full Text]
  3. Santen, R. J. (2003) Risk of breast cancer with progestins: critical assessment of current data. Steroids 68,953-964[CrossRef][Medline]
  4. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H., Chambon, P. (1990) Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J. 9,1603-1614[Medline]
  5. Mote, P. A., Balleine, R. L., McGowan, E. M., Clarke, C. L. (1999) Colocalisation of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle. J. Clin. Endocrinol. Metabol. 84,2963-2971[Abstract/Free Full Text]
  6. Mote, P. A., Bartow, S., Tran, N., Clarke, C. L. (2002) Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis. Breast Cancer Res. Treat. 72,163-172[CrossRef][Medline]
  7. Arnett-Mansfield, R. L., DeFazio, A., Mote, P. A., Clarke, C. L. (2004) Subnuclear distribution of progesterone receptors A and B in normal and malignant endometrium. J. Clin. Endocrinol. Metabol. 89,1429-1442[Abstract/Free Full Text]
  8. Arnett-Mansfield, R. L., Graham, J. D., Hanson, A. R., Mote, P. A., Gompel, A., Scurr, L. L., Gava, N., de Fazio, A., Clarke, C. L. (2007) Focal subnuclear distribution of progesterone receptor is ligand dependent and associated with transcriptional activity. Mol. Endocrinol. 21,14-29[Abstract/Free Full Text]
  9. Zaidi, S. K., Young, D. W., Javed, A., Pratap, J., Montecino, M., van Wijnen, A., Lian, J. B., Stein, J. L., Stein, G. S. (2007) Nuclear microenvironments in biological control and cancer Nat. Rev. Cancer 7,454-463
  10. Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., Yamamoto, K. R. (1988) The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature 334,543-546[CrossRef][Medline]
  11. Gronemeyer, H. (1991) Transcription activation by estrogen and progesterone receptors. Ann. Rev. Genet. 25,89-123[CrossRef][Medline]
  12. Elbi, C., Walker, D. A., Romero, G., Sullivan, W. P., Toft, D. O., Hager, G. L., DeFranco, D. B. (2004) Molecular chaperones function as steroid receptor nuclear mobility factors. Proc. Natl. Acad. Sci. U. S. A. 101,2876-2881[Abstract/Free Full Text]
  13. Schuchard, M., Subramaniam, M., Ruesink, T., Spelsberg, T. C. (1991) Nuclear matrix localisation and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor. Biochemistry 30,9516-9522
  14. Tang, Y., DeFranco, D. B. (1996) ATP-dependent release of glucocorticoid receptors from the nuclear matrix. Mol. Cell. Biol. 16,1989-2001[Abstract/Free Full Text]
  15. He, D., Nickerson, J. E., Penman, S. (1990) Core filaments of the nuclear matrix. J. Cell Biol. 110,569-580[Abstract/Free Full Text]
  16. DeMarzo, A. M., Beck, C. A., Onate, S. A., Edwards, D. P. (1991) Dimerisation of mammalian progesterone receptors occurs in the absence of DNA and is related to the release of the 90-kDa heat shock protein. Proc. Natl. Acad. Sci. U. S. A. 88,72-76[Abstract/Free Full Text]
  17. Tetel, M. J., Jung, S., Carbajo, P., Ladtkow, T., Skafar, D. F., Edwards, D. P. (1997) Hinge and amino-terminal sequences contribute to solution dimerisation of human progesterone receptor. Mol. Endocrinol. 11,1114-1128[Abstract/Free Full Text]
  18. DeFranco, D. B., Guerrero, J. (2000) Nuclear matrix targeting of steroid receptors: specific signal sequences and acceptor proteins. Crit. Rev. Eukaryot. Gene Expr. 10,39-44[Medline]
  19. Tang, Y., Getzenberg, R. H., Vietmeier, B. N., Stallcup, M. R., Eggert, M., Renkawitz, R., DeFranco, D. B. (1998) The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal. Mol. Endocrinol. 12,1420-1431[Abstract/Free Full Text]
  20. Guiochon-Mantel, A., Lescop, P., Christin-Maitre, S., Loosfelt, H., Perrot-Applanat, M., Milgrom, E. (1991) Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J. 10,3851-3859[Medline]
  21. Tetel, M. J., Giangrande, P. H., Leonhardt, S. A., McDonnell, D. P., Edwards, D. P. (1999) Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo. Mol. Endocrinol. 13,910-924[Abstract/Free Full Text]
  22. Milhon, J., Lee, S., Kohli, K., Chen, D., Hong, H., Stallcup, M. R. (1997) Identification of amino acids in the tau 2-region of the mouse glucocorticoid receptor that contribute to hormone binding and transcriptional activation. Mol. Endocrinol. 11,1795-1805[Abstract/Free Full Text]
  23. Takimoto, G. S., Tasset, D. M., Eppert, A. C., Horwitz, K. B. (1992) Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299. Proc. Natl. Acad. Sci. U. S. A. 89,3050-3054[Abstract/Free Full Text]
  24. Htun, H., Holth, L. T., Walker, D., Davie, J. R., Hager, G. L. (1999) Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol. Biol. Cell 10,471-486[Abstract/Free Full Text]
  25. Tyagi, R. K., Lavrovsky, Y., Ahn, S. C., Song, C. S., Chatterjee, B., Roy, A. K. (2000) Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol. Endocrinol. 14,1162-1174[Abstract/Free Full Text]
  26. Van Steensel, B., Brink, M., van der Meulen, K., van Binnendijk, E. P., Wansink, D. G., de Jong, L., de Kloet, E. R., van Driel, R. (1995) Localisation of the glucocorticoid receptor in discrete clusters in the cell nucleus. J. Cell. Sci. 108,3003-3011[Abstract]
  27. Fejes-Toth, G., Pearce, D., Naray-Fejes-Toth, A. (1998) Subcellular localisation of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc. Natl. Acad. Sci. U. S. A. 95,2973-2978[Abstract/Free Full Text]
  28. Htun, H., Barsony, J., Renyi, I., Gould, D. L., Hager, G. L. (1996) Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc. Natl. Acad. Sci. U. S. A. 93,4845-4850[Abstract/Free Full Text]
  29. Cook, P. R. (2002) Predicting three-dimensional genome structure from transcriptional activity. Nat. Genet. 32,347-352[CrossRef][Medline]
  30. Fraser, P., Bickmore, W. (2007) Nuclear organization of the genome and the potential for gene regulation. Nature 447,413-417[CrossRef][Medline]
  31. Martin, S., Pombo, A. (2003) Transcription factories: quantitative studies of nanostructures in the mammalian nucleus. Chromosome Res. 11,461-470[CrossRef][Medline]
  32. Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W., Fraser, P. (2004) Active genes dynamically colocalize to shared sites of ongoing transcription. Nat. Genet. 36,1065-1071[CrossRef][Medline]
  33. DeFranco, D. B. (2002) Navigating steroid hormone receptors through the nuclear compartment. Mol. Endocrinol. 16,1449-1455[Abstract/Free Full Text]
  34. Hager, G. L., Elbi, C., Becker, M. (2002) Protein dynamics in the nuclear compartment. Curr. Opin. Genet. Dev. 12,137-141[CrossRef][Medline]
  35. Li, X., Lonard, D. M., O'Malley, B. W. (2004) A contemporary understanding of progesterone receptor function. Mech. Ageing Dev. 125,669-678[CrossRef][Medline]
  36. McKenna, N. J., O'Malley, B. W. (2002) Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108,465-474[CrossRef][Medline]
  37. Marcelli, M., Stenoien, D. L., Szafran, A. T., Simeoni, S., Agoulnik, I. U., Weigel, N. L., Moran, T., Mikic, I., Price, J. H., Mancini, M. A. (2006) Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. J. Cell. Biochem. 98,770-788[CrossRef][Medline]
  38. Stenoien, D. L., Patel, K., Mancini, M. G., Dutertre, M., Smith, C. L., O'Malley, B. W., Mancini, M. A. (2001) FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent. Nat. Cell Biol. 3,15-23[CrossRef][Medline]
  39. Nagaich, A. K., Walker, D. A., Wolford, R., Hager, G. L. (2004) Rapid periodic binding and displacement of the glucocorticoid receptor during chromatin remodeling. Mol. Cell 14,163-174[CrossRef][Medline]
  40. Nye, A. C., Rajendran, R. R., Stenoien, D. L., Mancini, M. A., Katzenellenbogen, B. S., Belmont, A. S. (2002) Alteration of large-scale chromatin structure by estrogen receptor. Mol. Cell. Biol. 22,3437-3449[Abstract/Free Full Text]
  41. Vicent, G. P., Nacht, A. S., Smith, C. L., Peterson, C. L., Dimitrov, S., Beato, M. (2004) DNA instructed displacement of histones H2A and H2B at an inducible promoter. Mol. Cell 16,439-452[CrossRef][Medline]
  42. Garcia-Bassets, I., Kwon, Y.-S., Telese, F., Prefontaine, G. G., Hutt, K. R., Cheng, C. S., Ju, B.-G., Ohgi, K. A., Wang, J., Escoubet-Lozach, L., Rose, D. W., Glass, C. K., Fu, X.-D., Rosenfeld, M. G. (2007) Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell 128,505-518[CrossRef][Medline]
  43. Zink, D., Fischer, A. H., Nickerson, J. A. (2004) Nuclear structure in cancer cells. Nat. Rev. Cancer 4,677-687[CrossRef][Medline]
  44. Zaidi, S. K., Young, D. W., Choi, J. Y., Pratap, J., Javed, A., Montecino, M., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. (2004) Intranuclear trafficking: organization and assembly of regulatory machinery for combinatorial biological control. J. Biol. Chem. 279,43363-43366[Abstract/Free Full Text]
  45. Pederson, T. (2000) Half a century of "the nuclear matrix". Mol. Biol. Cell 11,799-805[Abstract/Free Full Text]
  46. Barrack, E. R. (1983) The nuclear matrix of the prostate contains acceptor sites for androgen receptors. Endocrinology 113,430-432[Abstract/Free Full Text]
  47. Metzger, D. A., Korach, K. S. (1990) Cell-free interaction of the estrogen receptor with mouse uterine nuclear matrix: evidence of saturability, specificity, and resistance to KCl extraction. Endocrinology 126,2190-2195[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.08-113639v1
23/2/546    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Graham, J. D.
Right arrow Articles by Clarke, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Graham, J. D.
Right arrow Articles by Clarke, C. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS