Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA 01655-0106, USA
* Present address: Department of Biochemistry, School of Medicine, Kyungpook National University, 101 Dong-In Jung-Gu, Daegu 700-422, Korea
Author for correspondence (e-mail: gary.stein{at}umassmed.edu)
Accepted May 21, 2001
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SUMMARY |
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Key words: Osteoblasts, Transcriptional control, Nuclear matrix, Osteocalcin, Nuclear localization
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INTRODUCTION |
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Localizing regulatory proteins to specialized sites within the nucleus involves nuclear import, in situ interactions with chromatin and transcriptional co-regulators, and subnuclear targeting. Mechanisms controlling nuclear import and interactions of transcription factors with DNA and co-regulators are well defined (Gorlich and Kutay, 1999; Gorlich and Mattaj, 1996; Lemon and Tjian, 2000). However, molecular determinants that target regulatory factors to specific subnuclear domains involved in transcription remain to be identified.
Modifications in multiple parameters of nuclear architecture occur during progression of osteoblast differentiation, as reflected by subnuclear compartmentalization of gene regulatory proteins (Bidwell et al., 1993; Dworetzky et al., 1990) and alterations in chromatin structure of the bone-specific osteocalcin gene (Montecino et al., 1994; Montecino et al., 1996b; Montecino et al., 1996a; Montecino et al., 1999b; Montecino et al., 1999a). Bone-restricted expression of the osteocalcin gene provides a paradigm for defining the mechanisms underlying tissue-specific transcription in the context of nuclear and chromatin architecture. Two osteocalcin gene regulatory factors, nuclear matrix protein 1 (NMP1) and NMP2, have been identified as YY1 and Runx2/Cbfa1, respectively (Guo et al., 1995; Merriman et al., 1995). Both factors are intimately associated with control of steroid-hormone-responsive and tissue-specific transcription (Guo et al., 1997; Javed et al., 1999), suggesting functional coupling between cell signaling and nuclear architecture.
The Runx/Cbfa proteins (e.g. Runx1/AML-1, Runx2/Cbfa1) play vital roles in cellular differentiation and development. Gene ablation studies have revealed that Runx1 is required for definitive hematopoiesis whereas Runx2 is essential for bone formation (Komori et al., 1997; Otto et al., 1997; Wang et al., 1996). Runx proteins interact with the core DNA sequence (RACCRCW) through the highly conserved runt homology domain (Meyers et al., 1993; Ogawa et al., 1993). These proteins exert their effects in part by modifying chromatin organization of tissue-specific gene promoters (Gutierrez et al., 2000; Javed et al., 1999; Montecino et al., 1996b). In addition, the activities of Runx factors are modulated by interactions with coregulatory proteins (e.g. Groucho/TLE, Yes-associated protein (YAP), Smad) that contribute to the integration of cell-signaling pathways in response to physiological cues (Aronson et al., 1997; Hanai et al., 1999; Javed et al., 2000; Levanon et al., 1998; McLarren et al., 2000; Yagi et al., 1999; Zhang et al., 2000a). Runx proteins reside in discrete subnuclear foci and colocalize with transcriptional regulators which activate or repress Runx-dependent genes (Javed et al., 2000; Prince et al., 2001; Zeng et al., 1997; Zeng et al., 1998).
The identification of Runx2/Cbfa1 as a nuclear-matrix-associated tissue-specific transcription factor (Banerjee et al., 1997; Bidwell et al., 1993; Ducy et al., 1997; Merriman et al., 1995) provided the initial insight into the linkage between subnuclear compartmentalization and osteoblast differentiation. Therefore, one important question is how Runx2 is directed to specific subnuclear domains and whether intranuclear localization contributes to regulation of bone-specific genes. In the present study, we have defined a 38 amino acid segment in the C-terminus of Runx2 that is responsible for subnuclear targeting of the transcription factor. Moreover, this nuclear-matrix-targeting signal (NMTS) is sufficient to direct a heterologous protein to the nuclear-matrix-associated foci containing Runx2. Furthermore, subnuclear targeting of Runx2 contributes to transcriptional regulation of the bone-specific osteocalcin gene.
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MATERIALS AND METHODS |
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Plasmid constructs
An epitope tag version of pcDNA 3.1 (pHA) was generated by inserting the HA-tag in the HindIII-KpnI sites to replace the His-Xpress tag of pcDNA 3.1 (Invitrogen, Carlsbad, CA). HA-tag Runx2 was constructed by cloning a cDNA encoding Runx2 in the BamHI-XbaI sites of pHA to generate pHA-Runx2 (1-528). pHA-Runx2 (1-516) was generated by digesting pHA-Runx2 (1-528) with EcoRI and XbaI followed by fill-in reaction with DNA polymerase I Klenow fragment and blunt end ligation. pHA-Runx2 (1-376) was generated by the addition of a stop codon by PCR-based site-directed mutagenesis. The Runx2 mutant 397-434 was constructed by PCR-based nest-deletion mutagenesis. Internal deletions of Runx2, that is,
400-467 and
433-467, were generated by digesting pHA-Runx2 with SmaI and BspmI-SmaI, respectively. To generate Gal4 DNA-binding domain (DBD) (1-147) fused with Runx2 (397-434), the aforementioned fragments of Runx2 were amplified with EcoRV-PstI sites incorporated into the primers and the digested PCR products were inserted in frame downstream of the Gal4 DBD. The reading frames of deletion mutants were confirmed by sequencing.
Nuclear extracts and electrophoretic mobility shift assay
Preparation of nuclear extracts from cells expressing proteins of interest and the protocol for electrophoretic mobility shift assays have been described (Javed et al., 1999). The wild-type Runx consensus 5' CGAGTATTGTGGTTAATACG 3' (Meyers et al., 1993) was synthesized using a Beckman synthesizer 2000. The Runx binding site is indicated in bold.
In situ immunofluorescence microscopy
ROS 17/2.8 or HeLa cells grown on gelatin-coated coverslips were transfected with 0.5 µg cytomegalovirus (CMV)-driven wild-type Runx2, its deletion mutants or with expression constructs for Gal4 DBD fused to the NMTS of Runx2. Cells were extracted for in situ nuclear matrix preparations and were analyzed by digital microscopy essentially as described (Javed et al., 2000). HA-tag full-length or deletion mutants of Runx2 were detected by a mouse monoclonal antibody against HA-tag at a dilution of 1:3000 (Zeng et al., 1997). Expression of the Gal4 DBD fusion constructs was detected by a mouse monoclonal antibody against Gal4 DBD (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a dilution of 1:1000. We used either Alexa 488 anti-rabbit or Alexa 568 anti-mouse secondary antibodies (Molecular Probes, Eugene, OR). To detect endogenous Runx2 in ROS 17/2.8 cells, the rabbit polyclonal antibody (Javed et al., 2001) was used at a dilution of 1:200. Mouse monoclonal antibodies were used to detect SC35, RNA processing speckles (1:500) (Spector et al., 1991), promyelocytic leukemia (PML) bodies (1:1000; Santa Cruz Biotechnology Inc.), coilin (1:100 (Smith et al., 1995)) and nucleolin (1:300; provided by P.-K. Chan, Baylor College of Medicine, TX).
Biochemical fractionation and western blotting
Cells were biochemically fractionated using a protocol previously described (Merriman et al., 1995), with several modifications. HeLa cells grown on 100 mm plates were transfected with 10 µg of full-length Runx2 or deletion constructs and subcellular fractions were prepared 24 hours after transfection. For whole-cell lysate, cells were harvested in 300 µl direct lysis buffer (2% SDS, 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 0.002% bromophenol blue, 10 mM DTT and 1x CompleteTM protease inhibitors (Roche, Indianapolis, IN)). Samples were immediately boiled for 5 minutes and stored at -70°C until used. For subcellular fractionation, cells were collected in ice-cold 1x PBS containing 1x CompleteTM protease inhibitors. Cell pellets were resuspended in 300 µl CSK buffer (100 mM NaCl, 0.3 M sucrose, 10 mM Pipes, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, pH 6.8) for 10 minutes on ice. Extracted cells were then subjected to centrifugation to collect nuclei. The supernatant containing cytosolic proteins was frozen in liquid nitrogen and stored in aliquots at -70°C. Nuclei were extracted with 300 µl digestion buffer (50 mM NaCl, 0.3 M sucrose, 10 mM Pipes, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, pH 6.8) containing 400 U DNaseI (Roche) for 30 minutes at room temperature. DNaseI activity was stopped by adding 2 M ammonium sulfate to a final concentration of 250 mM for 5 minutes at room temperature. Extracted nuclei were centrifuged to separate soluble nuclear proteins (chromatin fraction) and insoluble nuclear-matrix-intermediate filament fraction (NM-IF). The NM-IF fraction was then boiled in 300 µl of direct lysis buffer for 5 minutes. The same volume percentage of each fraction was analyzed by 10% SDS-PAGE. Separated proteins were transferred to a PVDF membrane (Millipore) and processed for western blotting as described elsewhere (Ausubel et al., 1997). Antibodies were purchased from Santa Cruz and used in the following dilutions: monoclonal antibody against HA tag (SC-7392; 1:3000), goat polyclonal antibody against lamin B (SC-6217; 1:1000), and the corresponding horse-radish peroxidase (HRP)-conjugated secondary antibodies (SC-2042; 1:2000). Immunoreactivity was assessed using an ECL chemiluminescence kit (Amersham-Pharmacia, Piscataway, NJ).
Reporter gene activity assays
Rat osteosarcoma (ROS 17/2.8) or HeLa cells grown on six-well plates were transfected with 0.1 µg of expression vector for Runx2 or its deletion mutants along with 1 µg of -208 osteocalcin-CAT (OC-CAT), spanning one Runx-binding site (site C) (Banerjee et al., 1996; Javed et al., 1999) of the rat osteocalcin promoter. Rous sarcoma virus-luciferase (RSV-Luc) was included as an internal control for transfection efficiency. Cells were harvested 24 hours after transfection and were processed for CAT (chloramphenicol acetyl transferase) assays as described (Javed et al., 1999). All quantitation was done on the Storm PhosphorImager using ImageQuant software (ABI/Molecular Dynamics, Sunnyvale, CA). The luciferase activity was assessed with Luciferase Assay Kit (Promega, Madison, WI) from the same cell lysate. CAT values were normalized with respect to the luciferase values. The graphs are representative of three independent experiments (n=6 in each experiment) with different DNA preparations.
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RESULTS |
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We assessed whether subnuclear targeting of the Gal4 DBD is functionally linked to transcriptional control of a Gal4-responsive promoter. We transfected the Gal4 DBD or the Gal4-NMTS into HeLa cells containing a genomically integrated luciferase gene driven by Gal4 binding sites. As shown in Fig. 8C, the Gal4-NMTS protein mediates 2.5-fold activation relative to Gal4 DBD alone. Thus, our data indicate that the NMTS supports transcriptional activation of a Gal4-responsive promoter.
The NMTS of Runx2 contributes to activation of the bone-specific osteocalcin gene
We addressed the biological consequences of NMTS-mediated intranuclear targeting of Runx2 on osteoblast-specific gene expression. We compared the transcriptional activities of wild-type Runx2 and mutants lacking the NMTS by monitoring their ability to enhance promoter activity of the rat osteocalcin gene (Fig. 9). Wild-type Runx2 (1-528) activates osteocalcin gene transcription 7-8 fold. This response is reduced to 1.5-2 fold for the Runx2 (1-376) mutant, which lacks the entire C-terminus spanning the NMTS. This result demonstrates that the entire C-terminus of Runx2 is required for optimal physiological activity of the protein. Runx2 mutants missing only the NMTS (397-434) or 68 amino acids including the NMTS (
400-467) exhibit dramatically reduced transactivation potential similar to the Runx2 mutant (1-376) compared to wild-type Runx2 (1-528). Runx2 mutants in which other segments of the C-terminus were deleted (i.e. Runx2 (
433-467) or Runx2 (1-516)) show only moderately reduced transactivation potential (i.e. from 7-8 fold to 4-5 fold). Interestingly, all three mutants displaying severely reduced transcriptional activity are expressed at comparable levels, are imported into the nucleus (Figs 4, 5) and have normal DNA binding activity (Fig. 3), yet exhibit compromised subnuclear targeting (Figs 4-6). These data strongly suggest that subnuclear targeting of Runx2 is obligatory for optimal transcriptional activity of the protein.
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DISCUSSION |
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Components of nuclear architecture may influence the spatial organization of nucleic acids and regulatory proteins. Two types of interactions have been reported: stable interactions through nuclear-matrix-associated sequences designated MARs (Bode et al., 2000) and dynamic interactions involving transcription factors and tissue-specific nuclear matrix proteins (Bidwell et al., 1993; Dworetzky et al., 1992; van Wijnen et al., 1993). Distinct transcription factors exhibit characteristic subnuclear distributions. These regulatory proteins interact with DNA in a sequence-specific manner and associate with the components of nuclear architecture at distinct subnuclear sites. Such specific intranuclear domains can provide an environment that increases the probability of achieving a threshold for protein-DNA and protein-protein interactions to organize the regulatory machinery required for tissue-specific transcription.
The Runx family of transcription factors provides a paradigm to understand the link between transcriptional regulation and nuclear architecture. Gene ablation studies suggest that Runx2 is a principal regulator of bone formation in vivo (Komori et al., 1997; Otto et al., 1997). Recently, a nonsense mutation has been reported in exon 7, which results in expression of Runx2 lacking the NMTS (Zhang et al., 2000b). This natural mutation causes the autosomal dominant human disease the cleidocranial dysplasia (CCD), which is characterized by severe craniofacial abnormalities. The C-terminus of Runx1 containing the NMTS is a frequent target of chromosomal translocations in leukemia (Lutterbach et al., 2000; Rowley, 1998), which is consistent with physiological implications for intranuclear trafficking. Loss of the NMTS and aberrant subnuclear targeting of Runx1 has been implicated in the pathology of leukemia. Compromised subnuclear targeting of Runx2 may also contribute to the development of the CCD phenotype. Additional characterization of mechanisms that control localization of Runx proteins at subnuclear sites should contribute to understanding further the development of hematopoietic and skeletal disorders.
Our data suggest that optimal transactivation of the bone-restricted osteocalcin gene by Runx2 is dependent on subnuclear localization and/or intranuclear targeting of the protein. Interestingly, the NMTS of Runx2 we have identified in this study is contained within a larger region that has a transactivation function (Kanno et al., 1998; Thirunavukkarasu et al., 1998). Moreover, this region is responsible for the interaction of Runx2 with several proteins and integrates regulatory cues related to transforming growth factor ß (TGF-ß)/bone morphogenetic protein 2 (BMP2) and Yes/Crk/Src signaling pathways (Hanai et al., 1999; Yagi et al., 1999; Zhang et al., 2000a). We propose that extracellular signaling and protein-protein interactions involving the C-terminus of Runx2 and bone-specific transcriptional control may converge at subnuclear foci to optimize physiological responses.
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ACKNOWLEDGMENTS |
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