Departments of Medicine and Cell Biology, University of Virginia, Charlottesville, VA 22908, USA
* Author for correspondence (e-mail: rnd2v{at}virginia.edu)
Accepted 21 April 2005
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Summary |
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Key words: Transcriptional regulation, Prolactin, Nuclear co-repressor, SMRT, Nuclear structure, Green fluorescent protein, Fluorescence microscopy, Pit-1
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Introduction |
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Many observations indicate that transcription factors and co-regulatory proteins localize to particular subnuclear sites (Day et al., 1999; Downes et al., 2000
; Enwright et al., 2003
; Kim et al., 1996
; Lamond and Earnshaw, 1998
; Misteli, 2001b
; Pombo et al., 1998
; Schaufele et al., 2001
; van Wijnen et al., 1993
; Zeng et al., 1998
). The recent characterization of many small spherical nuclear bodies including Cajal bodies (Gall et al., 1999
), gems (Hebert and Matera, 2000
) and promyelocytic leukemia (PML) bodies (Maul et al., 2000
) illustrate the highly ordered intranuclear environment. In addition to biochemical and immunofluorescence methods, the spectral variants of the fluorescent proteins (FPs) have been used as genetically encoded markers to study these nuclear subcompartments in living cells (Patterson et al., 2001
; van Roessel and Brand, 2002
). The dynamic partitioning of these different subcompartments without intervening membranes provides evidence for self-organization of the proteins that form these structures (Misteli, 2001b
). Moreover, the chromatin in the interphase nucleus is similarly organized into distinct domains, including chromosomal territories, interchromatin spaces and centromeric heterochromatin (Lamond and Earnshaw, 1998
). Recent evidence indicates that genes are transcribed at specific intranuclear sites (Cook, 1999
) and there are examples of genes that become spatially positioned in different subnuclear regions depending on their activation state (Andrulis et al., 1998
; Belmont et al., 1999
; Brown et al., 1999
; Brown et al., 1997
). This area of intense research is revealing how these remarkably organized and structurally complex microenvironments contribute to the regulation of gene expression.
Several studies have shown that SMRT and NCoR are concentrated with their HDAC partners in matrix-associated deacetylase (MAD) bodies (Dhordain et al., 1997; Downes et al., 2000
; Li et al., 2000
; Nagy et al., 1997
; Ordentlich et al., 1999
; Soderstrom et al., 1997
; Wu et al., 2001
). A pharmacological HDAC inhibitor disrupted these subnuclear bodies, suggesting that they are maintained by specific functional interactions (Downes et al., 2000
). This concept is supported by the observation that the spherical co-repressor focal bodies are spatially distinct from several other subnuclear protein compartments (Ariumi et al., 2003
; Downes et al., 2000
; Wu et al., 2001
). Using a quantitative imaging approach, we demonstrated that MAD-body formation by NCoR was directly related to expression level (Voss et al., 2004
), indicating a highly regulated mechanism controlling focal-body formation or maintenance. This regulated enrichment of co-repressor complexes in specific subnuclear compartments might contribute to the control of gene expression by at least two different mechanisms: (1) compartments might concentrate active co-repressor complexes at sites of gene regulation; (2) compartments might assemble or store co-repressor complexes away from sites of active transcription.
In this study, we use a combination of biochemistry, immunofluorescence, live-cell microscopy and quantitative image analysis to investigate the mechanisms controlling the subnuclear organization and function of co-repressor complexes. The data presented here suggest that spherical intranuclear foci serve as co-repressor reservoirs that are in balance with the available interacting partner proteins. As Pit-1 interacts with the co-repressors to regulate transcription, it also disperses the co-repressor complexes into diffuse nucleoplasmic compartments containing high concentrations of chromatin. Importantly, this redistribution of the co-repressor requires a functional Pit-1 DNA-binding domain. These data support the hypothesis that interactions with limiting amounts of transcription factors control co-repressor positioning and function, and form an important mechanism by which transcription factors direct changes in cell-specific gene expression.
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Materials and Methods |
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Transfection of cell lines and reporter-gene assays
For transfection, mouse GHFT1-5 (Lew et al., 1993), mouse 3T3-L1 (ATCC CL-173), rat GH4ZR7 (Elsholtz et al., 1991
) and human HeLa (ATCC CCL-2) cell lines were maintained as monolayer cultures in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cells were harvested and transfected with the indicated plasmid DNA(s) by electroporation. Briefly, approximately 1x106 cells were resuspended in Ca2+/Mg2+-free Dulbecco's PBS and transferred into 0.2 cm gap electroporation cuvettes containing 10 µg of the indicated luc reporter gene and various concentrations of the indicated expression vector DNAs. The total amount of DNA was kept constant using the cytomegalovirus (CMV) green fluorescent protein (CMV-GFP) expression plasmid. This provided a control for the effects of both the co-transfected CMV promoter and the GFP. The transfected cells were transferred to 35 mm dishes and maintained in culture. Extracts were prepared from the cells after 24 hours and Luc activity was determined as described by the manufacturer (Promega). Each experiment was performed a minimum of three times and Luc activity, corrected for total protein, was expressed as the mean±s.e.m. The statistical analysis [analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-hoc test where warranted to compare multiple conditions] was performed using SPSS 11 software, with differences considered significant when P<0.05.
Immunoprecipitation and immunodetection
For immunoprecipitation, cells were transfected with expression vectors encoding combinations of hemagglutinin (HA)-tagged Pit-1, GFP-SMRT or GFP-NCoR, and lysed after 24 hours as previously described for the immunoprecipitation of co-repressor complexes (Downes et al., 2000). The lysates were incubated with agarose beads conjugated with anti-HA antibody (Santa Cruz Biotechnology). The beads were then washed and proteins were eluted with denaturing loading buffer (Invitrogen Life Technologies) and analysed by western blot. Immunodetection was performed using anti-GFP (Molecular Probes) or anti-Pit-1 [131 polyclonal serum (S. Rhodes, Indiana University-Purdue University, Indianapolis, IN)] primary antibody, horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Pierce Biotechnology) and chemiluminescence reagent (Amersham Biosciences).
Live cell microscopy and immunocytochemistry
Cells transfected with the indicated plasmid DNA(s) encoding the FP fusion proteins were transferred to 35 mm dishes that contained a sterile 25 mm circular cover glass and maintained in culture as described above. On the following day, the cover glass with the monolayer of cells was transferred to a medium-filled chamber fitted to the microscope stage (Day et al., 2001). Where indicated, the living cells were stained with Hoechst 33342 (H33342; Molecular Probes) as described previously (Schaufele et al., 2001
). Wide-field microscopy (WFM) was performed with an inverted Olympus IX-70 epifluorescence microscope equipped with a 60x water-immersion 1.2 NA objective and filters sets specific for each fluorophore (Chroma Technology). Grayscale images with no saturated pixels were obtained using a cooled digital interline camera (Orca-200, Hamamatsu). All images were collected at a similar gray-level intensity by controlling the excitation intensity with constant neutral density filtration and by varying the on-camera integration time (0.1-8.0 seconds). Indirect immunocytochemical (ICC) detection of endogenous NCoR and Pit-1 proteins in fixed cells was performed as reported previously (Voss et al., 2005
). Reference images of standard fluorescent beads were acquired to monitor consistency of microscope performance for all quantitative imaging experiments. All image files were processed for presentation using ISee software (Inovision) and Canvas 8.0 software (Deneba)
Integrated image analysis of cell populations
Cell populations producing a fusion between yellow fluorescent protein (YFP) and NCoR (YFP-NCoR) or SMRT (YFP-SMRT), the unfused monomeric red fluorescent protein (mRFP), and the indicated blue fluorescent protein (BFP) fusion protein were co-transfected by electroporation as described above. On the following day, images of the living cells were acquired using an integrated imaging protocol for the unbiased selection of cells based on mRFP expression. The subcellular features defined by the other expressed fusion proteins in the selected cells were measured using automated quantitative image analysis as described earlier (Voss et al., 2004). The area and fluorescence intensity of each automatically selected focal-body region of interest (ROI) was measured and the center of the ROI was used to position a second rectangular ROI that measured the fluorescence intensity of the nucleoplasm surrounding the selected feature. An enrichment factor (EF) was then calculated as the ratio of the intensities in the two regions. All the measurements were exported to text files and linear regression analysis was performed using spreadsheet software (Microsoft Excel) to determine the relationship between the labeled protein expression levels and co-repressor subnuclear organization. SPSS 11 software was used to perform ANOVA and post-hoc tests for the statistical comparison of imaging data from multiple cell populations.
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Results |
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By contrast, images acquired of cells that co-expressed mRFP-SMRT and GFP-NCoR showed that these related co-repressor proteins occupied the same focal bodies (Fig. 2B and overlay). Earlier studies demonstrated that the MAD bodies formed by NCoR and SMRT also contain their HDAC partners, which function as part of the co-repressor complex to modify chromatin and repress transcription (Dhordain et al., 1997; Downes et al., 2000
; Li et al., 2000
; Nagy et al., 1997
; Ordentlich et al., 1999
; Soderstrom et al., 1997
; Wu et al., 2001
). We then examined the subcellular localization of HDAC5 to determine whether it was co-localized with NCoR. Some HDACs, including HDAC5, are known to shuttle between the cytoplasm and nucleus (McKinsey et al., 2000
), and, when expressed in living GHFT1-5 cells, CFP-HDAC5 was distributed throughout the cell (Fig. 2C). However, when co-expressed with YFP-NCoR, there was redistribution of HDAC5 to the intranuclear foci occupied by NCoR (Fig. 2D and overlay). Equivalent results were obtained when CFP-HDAC5 was co-expressed with YFP-SMRT, but there was no association of the CFP-HDAC5 with the focal bodies formed by the co-expressed PML-RFP (data not shown). These living-cell observations are consistent with previous ICC studies showing that MAD bodies contain other members of the co-repressor complex and suggest a mechanism whereby specific protein interactions organize the co-repressor protein complex within specialized intranuclear compartments. To explore the dynamics of co-repressor foci, fluorescence recovery after photobleaching (FRAP) experiments were performed on GHFT1-5 cells producing YFP-SMRT. Following the photobleaching of YFP-SMRT in selected foci, the fluorescence intensity recovered to 70% in approximately 5 minutes (Fig. 2E,right). These results clearly demonstrate the dynamic exchange of co-repressor protein between the foci and nucleoplasm.
Co-repressor proteins functionally interact with Pit-1 to repress transcription
We next investigated the inhibition of Pit-1-dependent gene transcription by the co-repressors. Pit-1 functions during development to establish the pituitary somatolactotrope cell lineage, and is required for the transcription of both the growth hormone (GH)-encoding and PRL-encoding genes in these cells (Elsholtz et al., 1991). The pituitary somatolactotrope cell line GH4ZR7 expresses high levels of endogenous Pit-1 protein, which strongly activated the transfected rat PRL-promoter-fused luc reporter gene (-2.5 rPRL luc; Fig. 3A). Co-transfection with increasing amounts of an expression vector encoding SMRT resulted in a dose-dependent decrease in Luc activity, with greater than 80% inhibition of -2.5 rPRL Luc activity at the maximum amount of SMRT expression vector tested. Importantly, similar activity was observed for increasing amounts of expression vector encoding GFP-SMRT, demonstrating that the GFP fusion did not interfere with the PRL gene inhibitory activity of SMRT.
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To determine whether the co-repressor activity observed in pituitary somatolactotrope cells was dependent upon Pit-1, PRL promoter activity was reconstituted in a heterologous cell line. Non-pituitary HeLa cells do not express crucial regulators of the PRL-encoding gene, including Pit-1, allowing dissection of Pit-1-dependent PRL promoter activation (Bradford et al., 1997; Ingraham et al., 1988
; Nelsen et al., 1993
; Walter et al., 1985
). The expression of GFP-SMRT did not inhibit the RSV promoter in the HeLa cells (Fig. 3D), indicating that SMRT was not a global inhibitor of promoter activity in these cells. Linking the 1P Pit-1 RE to the RSV promoter (1P RSV luc) conferred responsiveness to Pit-1 when co-expressed in the HeLa cells (Fig. 3D). Western blotting showed that the expression of SMRT did not inhibit the expression of the RSV-Pit-1 plasmid (Fig. 3D, inset). Although the magnitude of the Pit-1-response was much less than that observed in GH4ZR7 cells (Fig. 3B), which probably reflects the high basal activity of the RSV promoter, the co-expression of GFP-SMRT led to a significant inhibition of the Pit-1-dependent reporter-gene activity (Fig. 3D). The rat PRL-encoding gene proximal promoter (-306 rPRL) contains four Pit-1 binding sites (Ingraham et al., 1988
) and has very low basal activity in HeLa cells, but could be induced more than 600-fold by co-transfection with the RSV Pit-1 expression plasmid (Fig. 3E). The co-expression of GFP-SMRT with Pit-1 resulted in more than 90% inhibition of the Pit-1-dependent promoter activity, and a similar level of inhibition occurred with the co-expression of GFP-NCoR (Fig. 3F). Together, these reporter gene experiments indicated that both SMRT and NCoR inhibited Pit-1-dependent gene expression.
These results implied a functional interaction between Pit-1 and the co-repressor protein complex, and earlier studies indicated a direct physical interaction between NCoR and Pit-1 (Xu et al., 1998). We used co-immunoprecipitation (co-IP) to assess potential direct interactions between the FP-labeled co-repressor proteins and Pit-1 (Fig. 3G). Lysates were prepared from cells that co-expressed HA-Pit-1 and either GFP-NCoR or GFP-SMRT. The HA-Pit-1 expression in the cell lysates was confirmed by western blot analysis (data not shown). Following IP with an antibody specific for the HA epitope tag, western blotting with a GFP-specific antibody was used to detect co-IPed GFP fusion proteins. The results showed that GFP-NCoR, but not GFP-SMRT, was co-IPed with HA-Pit-1, despite similar levels of both proteins in the input lysate (Fig. 3G). This indicated stable interactions between GFP-NCoR and Pit-1, consistent with that previously reported for untagged NCoR (Xu et al., 1998
). However, under the same conditions, an interaction with SMRT could not be detected. Given the equivalent activity of SMRT and NCoR in vivo, this was an unexpected result and might indicate a weaker physical association between SMRT and Pit-1 under the conditions used for the co-IP assay.
NCoR and Pit-1 co-localize in mouse pituitary somatotrope progenitor cells
We next examined whether NCoR co-localized with Pit-1 in mouse pituitary cells, which endogenously express both proteins. The mouse pituitary GHFT1-5 cell line was derived by targeted transformation of embryonic pituitary cells and has characteristics of the progenitor for the GH-secreting pituitary somatotrope cell lineage (Lew et al., 1993). The GHFT1-5 cells express Pit-1 at a much lower level than differentiated PRL and GH-secreting cell lines, including GH4ZR7 cells (Schaufele et al., 2001
). ICC staining was used to compare the subnuclear distribution of the endogenous NCoR and Pit-1 in fixed GHFT1-5 cells, and the results demonstrated that the endogenous NCoR and Pit-1 were both distributed in a nuclear reticular pattern that contained very small focal structures (Fig. 4). The profile analysis shows a very high degree of co-localization of the immunostained proteins (Fig. 4). The specificity of staining was confirmed by analysis of cells stained with both secondary antibodies alone, each individual primary antibody with the opposite secondary antibody, or each primary with both secondary antibodies.
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Quantitative cell-population analysis confirms that Pit-1 reorganizes NCoR
The subnuclear organization of FP-NCoR in transfected cells can be heterogeneous (Voss et al., 2004) and the ability of Pit-1 to reorganize the co-expressed FP-NCoR was also variable. This is exemplified by the images of YFP-NCoR in randomly selected cells that co-express BFP-Pit-1 (Fig. 6A). Although YFP-NCoR was clearly dispersed in some cells (Fig. 6A, top), other cells had NCoR in a dispersed pattern with some small foci (Fig. 6A, middle) or in larger more distinct foci (Fig. 6A, bottom). The BFP-Pit-1 and YFP-NCoR were strongly co-localized to the dispersed compartments in these cells (Fig. 6A, profile). This heterogeneity in the subnuclear organization of NCoR creates a problem for image analysis, in which the interpretation of protein distribution in representative high-resolution microscopy images is subjective and might not accurately reflect the cell population.
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Pit-1 also disperses the SMRT/HDAC co-repressor complex to a chromatin-enriched subnuclear pattern
NCoR and SMRT occupied the same focal bodies when co-expressed (Fig. 2B) and reporter-gene analysis indicated that both SMRT and NCoR inhibited Pit-1-dependent gene expression (Fig. 3E,F). However, biochemical analysis failed to detect an interaction between Pit-1 and SMRT (Fig. 3G). Because this could reflect a basic difference in Pit-1 interactions with the SMRT co-repressor complex, we next determined whether Pit-1 also reorganized SMRT in the nuclei of living cells. When they were co-expressed, BFP-Pit-1 was reorganized GFP-SMRT but not the PML bodies into a more dispersed distribution in which SMRT was localized with Pit-1 (Fig. 7A,B). The results in Fig. 7C further show that, when RFP-SMRT was recruited by GFP-Pit-1, both proteins were co-localized with the H33342-stained chromatin (Fig. 7C and profile). In addition, when Pit-1 reorganized SMRT, there was concomitant reorganization of the co-expressed HDAC-5 (Fig. 7D). This repositioning of multiple members of the co-repressor complex provides further support for the hypothesis that functional interactions with transcription factors direct the subnuclear organization and activity of co-repressor complexes.
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Discussion |
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Many proteins involved in transcriptional regulation and RNA-processing localize to specific compartments within the nucleus (for reviews, see Cook, 1999; Isogai and Tjian, 2003
; Lamond and Spector, 2003
; Spector, 2001
). These self-organizing subnuclear compartments are maintained through protein-protein interactions, promoting coordinated multistep processes at specialized sites within the nucleus (Misteli, 2001a
). For instance, extensive analysis has shown specific protein interactions are required for the formation of PML and Cajal bodies in distinct regions of the nucleus (Hebert and Matera, 2000
; Maul et al., 2000
). Although they are morphologically similar to these well-characterized intranuclear structures, the focal bodies formed by the co-repressor proteins were distinct from PML bodies, Daxx bodies and RNA splicing factor compartments (Wu et al., 2001
). Furthermore, we have shown that the focal bodies formed by SMRT co-repressor and those formed by the coactivator glucocorticoid-receptor-interacting protein (GRIP1/TIF2) are also different (Voss et al., 2005
). By contrast, the related co-repressor proteins SMRT and NCoR concentrate in the same intranuclear compartments (Fig. 2). Additionally, the Ataxin-1 protein, which functions as a transcriptional co-repressor and interacts physically with SMRT, was also found to co-localize with SMRT in the same subnuclear foci (Tsai et al., 2004
). These co-repressor proteins function through their direct physical association with the HDACs, and earlier studies showed the recruitment of HDAC-1, HDAC-3 and HDAC-5 by SMRT and recruitment of HDAC-1 by NCoR (Downes et al., 2000
; Soderstrom et al., 1997
; Wu et al., 2001
). We have demonstrated here that the co-producion of YFP-NCoR led to the recruitment of CFP-HDAC-5, but not the unfused CFP (Fig. 2), to the intranuclear focal bodies. These observations indicate that specific protein interactions target the co-repressors and their protein partners to specialized subnuclear compartments.
The proteins within these nuclear compartments are continuously exchanged with proteins in surrounding nuclear regions, providing a mechanism for dynamic regulation (Lamond and Spector, 2003; Misteli, 2001b
). Using FRAP, we have demonstrated the exchange of FP-labeled co-repressor protein between focal bodies and the surrounding nucleoplasm (Fig. 2). Given that the size of the focal bodies is very sensitive to changes in co-repressor protein expression level, these results suggest that the relative concentrations of co-repressor complexes in different regions of the nucleus and the concentrations and distributions of other interacting proteins control the flux of co-repressor protein through this subnuclear compartment. In this regard, the co-repressors interact with many different classes of DNA-binding transcription factors (Bailey et al., 1999
; Dhordain et al., 1997
; Hong et al., 1997
; Hu et al., 2001
; Jimenez-Lara and Aranda, 1999
; Kakizawa et al., 2001
; Lavinsky et al., 1998
; Lee et al., 2000
) and these transcription factors compete for limiting quantities of co-repressor proteins (Shibata et al., 1997
; Zhang et al., 1998
). The activity of the pituitary-gland-specific transcription factor Pit-1 is regulated through its interactions with both coactivator and co-repressor proteins (Scully et al., 2000
; Xu et al., 1998
), and the precise balance of co-regulatory proteins is thought to play a key role in the regulation of gene expression (Glass and Rosenfeld, 2000
; Sohn et al., 2003
). In a previous study, we demonstrated that a GFP-Pit-1 fusion protein that had proper DNA-binding specificity was localized to distinct subnuclear compartments, where it interacted with other transcription factors to induce target gene expression (Day, 1998
; Enwright et al., 2003
).
Although best characterized for its role as an activator, Pit-1 also functions as an inhibitor of pituitary-hormone genes through an association with the nuclear co-repressor NCoR (Xu et al., 1998). We have showed here that tipping the balance in favor of the co-repressors resulted in the inhibition of Pit-1-dependent gene expression (Fig. 3). We confirmed that GFP-NCoR physically interacts with Pit-1 and functions to repress Pit-1-dependent transcriptional activity (Fig. 3). We found that SMRT also inhibited Pit-1-dependent transcription and retained this inhibitory activity when fused to GFP. These results are consistent with the overlapping activities of NCoR and SMRT that have been described for many DNA-binding factors (Gelmetti et al., 1998
; Hu et al., 2001
; Lavinsky et al., 1998
; Yamamoto et al., 2001
). Because both SMRT and NCoR are present in PRL-producing pituitary cells (Misiti et al., 1998
), it seems likely that both function as important physiological regulators of Pit-1-dependent gene activity. Unlike NCoR, however, a strong interaction between SMRT and Pit-1 was not detected by co-IP experiments (Fig. 3). This could indicate different affinities of SMRT and NCoR for Pit-1 or could mean that in vitro binding conditions were not favorable for the interaction of SMRT with Pit-1. Together, the results support direct interactions of Pit-1 with the co-repressor protein, leading to the inhibition of Pit-1-dependent transcription.
We tested the hypothesis that Pit-1 could function to control co-repressor subnuclear positioning in living cells. We observed that, in mouse pituitary GHFT1-5 cells, endogenous NCoR and Pit-1 were co-localized within the nuclear compartment (Fig. 4). When GFP-NCoR was co-expressed with BFP-Pit-1, there was a striking reorganization of GFP-NCoR into the diffuse nuclear distribution of Pit-1 (Fig. 5). By contrast, the nuclear bodies formed by PML protein were unaffected by the co-produced Pit-1 protein. To quantify this, we used an integrated image analysis of populations of randomly selected cells from the transfected population (Voss et al., 2004; Voss et al., 2005
). The co-expression of Pit-1 in the cell population resulted in statistically significant decreases in both YFP-SMRT focus size and the relative protein concentration in the foci compared with the surrounding nucleoplasm (Fig. 6). In cells in which the co-repressor concentration was high, focal bodies were still formed in the presence of Pit-1 but Pit-1 was never observed to localize to the co-repressor foci. This implies that most Pit-1/co-repressor interactions occur outside the spherical focal bodies. Interestingly, our linear regression models revealed that Pit-1 expression caused a graded dispersal of YFP-SMRT foci in the cell population instead of the `all or none' dispersal implied by previous qualitative imaging studies of co-repressors and nuclear receptors (Tazawa et al., 2003
; Wu et al., 2001
). These are the first statistical data supporting the hypothesis that DNA-binding factors position co-repressor complexes in specific subnuclear domains, moving co-repressor protein out of spherical foci and into more widely distributed nuclear compartments.
The similar reorganization of HDAC-5 in cells that co-expressed SMRT and Pit-1 suggested that the co-repressor complex was reorganized by Pit-1 to the widely distributed compartments it occupied (Fig. 7). However, we could not distinguish a direct interaction between Pit-1 and the co-repressors from an indirect association of these proteins through common protein partners. Interestingly, several qualitative imaging studies have reported similar effects of DNA-binding nuclear receptors on co-repressor subnuclear organization. The non-ligand-bound retinoic-acid receptor (RAR
) physically interacts with SMRT and recruits SMRT into a diffuse nuclear pattern during repression of target gene promoters (Wu et al., 2001
). In the presence of ligand, SMRT dissociates from the RAR
, allowing coactivator complexes to bind the receptor and to stimulate target genes (Glass and Rosenfeld, 2000
; Wu et al., 2001
). Similar results were reported for receptor-interacting protein 140 (RIP140), another co-repressor protein that is redistributed from spherical foci to a diffuse pattern through physical interactions with the non-ligand-bound glucocorticoid receptor (GR). This dispersal of RIP140 also correlated with repression (Tazawa et al., 2003
). In combination with these results, our imaging and functional studies indicate that transcriptional repression correlates with the dispersal of co-repressor complexes by DNA-binding factors in living cells.
Several lines of evidence suggest that the DNA-binding activity of Pit-1 is necessary for the recruitment of the co-repressor complexes. When expressed alone, Pit-1 distributed in a web-like pattern that was co-localized with H33342-stained euchromatin throughout the nucleus (Fig. 5A). In addition, the dispersed Pit-1/SMRT compartments are positioned in regions of H33342-stained euchromatin. Our earlier studies showed disruption of the Pit-1 DNA-binding activity prevented its co-localization with the chromatin stain (Enwright et al., 2003) (data not shown). This suggested that interactions with the chromatin control the position of Pit-1 in the nucleus. Here, we examined the naturally occurring point mutant Pit-1W261C, which is defective in DNA binding and failed to co-localize with the stained chromatin. Using quantitative image-analysis, we demonstrated that this Pit-1 point mutant failed to reorganize the focal bodies containing SMRT (Fig. 8). Instead, the DNA-binding-defective Pit-1 became co-localized with SMRT to the focal bodies. This indicated that, although the mutant Pit-1 retained its ability to interact with the co-repressor complex, DNA-binding activity was necessary to establish the final positioning of the Pit-1/co-repressor complexes in the cell nucleus. Similar results have been reported for qualitative imaging studies of the RIP140 co-repressor and a GR DNA-binding-domain mutant (Tazawa et al., 2003
).
In summary, these observations provide striking evidence that DNA-binding factors alter the organization of their interacting co-repressor proteins and that this redistribution contributes to transcriptional regulation. The results indicated that dynamic protein interactions lead to the assembly of proteins in common subnuclear compartments and models that characterize these sites as aggregates of misfolded protein do not adequately account for this dynamic behavior. Our studies support the view that, in the presence of limiting amounts of DNA-binding transcription factors such as Pit-1, any excess of the co-repressor protein is organized in focal bodies. These foci are dynamic and exchange co-repressor complexes with the surrounding nucleoplasm. When Pit-1 is in excess, co-repressor protein is recruited to the nuclear sites occupied by Pit-1. The co-localization of these proteins was independent of the Pit-1 DNA-binding activity, but the dominant organizer activity of Pit-1 required the DNA-binding activity, suggesting either direct association with chromatin or through interactions with chromatin-associated factors. This dominant organizer activity overrides the co-repressor default targeting signal, resulting in dispersal of the co-repressor and HDAC to regions containing chromatin. We propose that active transcriptional regulation occurs in the widely distributed subnuclear compartments that contain high concentrations of Pit-1, SMRT/NCoR, HDAC and chromatin. Other nuclear proteins have subnuclear distributions that are similar to the patterns observed here for the co-repressor proteins, and may be similarly regulated.
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Acknowledgments |
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References |
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