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Originally published In Press as doi:10.1074/jbc.M414076200 on April 25, 2005

J. Biol. Chem., Vol. 280, Issue 26, 24600-24609, July 1, 2005
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Nuclear Matrix Binding Regulates SATB1-mediated Transcriptional Repression*{boxs}

Jin Seo, Mary M. Lozano, and Jaquelin P. Dudley{ddagger}

From the Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712

Received for publication, December 14, 2004 , and in revised form, April 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor {alpha}, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224–278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse mammary tumor virus (MMTV)1 transcription is controlled by regions largely found within the U3 region of the long terminal repeat (LTR) (1). The MMTV LTR promoter is a paradigm for hormone-regulated transcription (2). Addition of glucocorticoids to virally infected cells results in a 10- to 50-fold increase in transcription that is dependent on transient binding of glucocorticoid receptor (GR) to a hormone-response element (HRE) (3). Ligand-bound GR allows chromatin-remodeling events that then lead to NF1 and Oct1 binding upstream of the LTR transcription start site (4).

Previous work by our group has shown that MMTV transcription is highest in the lactating mammary gland, intermediate in reproductive and lymphoid tissues, and undetectable in brain, heart, liver, and skeletal muscle (5–7). Tissue-specific MMTV expression does not mimic GR distribution. However, MMTV transcription is suppressed in specific tissues by the presence of several negative regulatory elements (NREs) located upstream of the HRE in the viral LTR (6–10). Deletion of one or more NREs leads to increased MMTV expression in non-permissive tissues as well as cell lines derived from semipermissive tissues (6, 11, 12). In addition, MMTV strains that lack the NREs induce primarily T-cell lymphomas rather than breast cancer (8), yet mammotropic strains require replication in B and T cells for transmission to mammary cells (13–18). Because of the limited coding capacity of MMTV and other retroviruses (19), negative regulation of viral transcription must be controlled by cellular proteins.

At least two homeodomain-containing proteins, CCAAT-displacement protein (CDP) (20–24) and special AT-rich binding protein 1 (SATB1) (25–27), have been shown to bind to the MMTV NREs (9, 28–31). CDP overexpression in mammary or fibroblastic cells results in suppression of MMTV LTR-reporter gene expression both in the presence and absence of glucocorticoids (28, 29, 31). Mutations in CDP-binding sites found within the promoter-proximal and promoter-distal NREs elevate MMTV transcription (29). CDP also is differentially expressed during development of the mammary gland, and CDP DNA-binding activity is high in virgin mammary tissue, where MMTV transcription is low. Conversely, MMTV RNA expression is highest in the lactating mammary gland, a tissue in which CDP DNA-binding activity for the MMTV LTR is undetectable (28). Loss of CDP-binding sites within the NREs elevates MMTV transcription in the mammary gland and accelerates tumorigenesis (32). Therefore, CDP is an important regulator of MMTV expression in the mammary gland.

The transcription factor, SATB1, also binds to the MMTV NRE within MAR sequences (9–11) and recognizes a specific sequence context that exhibits a high base-unpairing or -unwinding propensity (9, 25). SATB1 was first isolated as a factor that bound to the nuclear matrix attachment region (MAR) 3' of the immunoglobulin heavy chain intronic enhancer (25), but also has been implicated in the tissue-specific expression of many genes (33–35). SATB1 is most abundant in the thymus (25), a tissue that is semi-permissive for MMTV expression, yet little SATB1 is expressed in the mammary glands, a target tissue for high levels of viral transcription (9, 11, 36). SATB1 appears to be primarily a repressor of gene expression in T cells (9, 10, 37), and mutation of a SATB1-binding site in the promoter-proximal NRE in the MMTV LTR elevates reporter gene activity in the lymphoid tissues of transgenic mice (9, 10). SATB1-null mice are small in size and have disproportionately small thymi and spleens (33). Approximately 9 of 10 genes surveyed by reverse transcription-PCR had increased expression in SATB1-null thymocytes (33), consistent with repressor function.

SATB1 appears to function as a homodimer (38), and the N-terminal dimerization sequence may be involved in signaling through protein-protein interactions (39). Recently, it was shown that SATB1 recruits chromatin remodeling complexes such as nucleosome remodeling and deacetylase complex, chromatin-accessibility complex, and ATP-utilizing chromatin assembly and remodeling factor (40). These chromatin remodeling complexes were suggested to suppress gene expression through histone deacetylation and nucleosome positioning on SATB1-bound sites (40, 41). SATB1 associates with the nuclear matrix (42), an insoluble nuclear structure that is important for many cellular events, including regulation of gene expression and replication (43–46). However, the effect of SATB1 association with the nuclear matrix on transcriptional repression has not been demonstrated.

In this report, we have identified a domain that plays a dominant role in SATB1 binding to the nuclear matrix. Deletion of this nuclear matrix targeting sequence (NMTS) partially relieved SATB1-mediated transcriptional suppression of integrated MMTV proviruses or MMTV LTR-reporter plasmids even in the presence of positive activators, such as ligand-bound GR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructions—The plasmid pMTV-LUC (also called pLC-LUC) contains the C3H MMTV LTR upstream of firefly luciferase (11). Full-length SATB1 cDNA from pMAT (47) was inserted into the EcoRI site of pUHD10-3 (48) to produce pUHD10-3/SATB1. SATB1(1–345) and SATB1(1–494) deletion mutants were constructed by digesting full-length SATB1 cDNA with ScaI or AseI, respectively, followed by treatment with Klenow fragment of DNA polymerase I to fill in the overhangs. Each fragment then was ligated to pUHD10-3 that had been digested with EcoRI and treated with Klenow to blunt the ends. The internal deletion mutant, ID(346–494), was constructed by digestion of full-length SATB1 cDNA with ScaI and AseI, and mung bean nuclease treatment was used to generate blunt ends that were ligated. The deletion mutant was then inserted into pUHD10-3 after digestion with EcoRI and Klenow treatment. The plasmid pUHD10-3/SATB1 was digested with EcoNI followed by mung bean nuclease treatment to prepare ID(274–410). The plasmid pUHD10-3/SATB1 was digested with EcoNI and BamHI, treated with Klenow polymerase to blunt the ends, and religated to generate SATB1(1–274). The plasmids SATB1(1–224), SATB1(55–764), SATB1(168–764), SATB1(275–764), and SATB1(510–764) were constructed using PCR with the following primer pairs: SATB199EGFP and SA870DO, SA631UP and SA2497DO, SA700UP and SA2497DO, SA1021AUG and SA2497DO, and SA1723+ and SA2497DO, respectively. The PCR products were digested with EcoRI and inserted into the EcoRI site of pUHD10-3. The NMTS deletion mutant, ID(224–278), was constructed using recombinant PCR (11). Briefly, two fragments were separately amplified from the SATB1 cDNA with SA196+ and SA867– and SA1033+ and SA2497DO. The NMTS was deleted in a second PCR step with SA196+ and SA2497DO primers using the first PCR products as a template. To make pGFP-NMTS and pLexA-NMTS, the NMTS of SATB1 was amplified by PCR with Sagfp865+ and SA1032– and Sagal865+ and SA1032–, respectively. To prepare pGFP-SATB1, pGFP-SATB1(1–55), and pGFP-SATB1(1–224), fragments were amplified by PCR using primer pairs SATB199EGFP and SA2497DO, SATB199EGFP and SA363DO, and SATB199EGFP and SA870DO, respectively. The products were cloned into the EcoR1 site of pEGFPC1 or pLexA (both from Clontech). Full-length SATB1 cDNA was PCR-amplified with SA196+ and SA2497DO using pMAT as a template and inserted into the EcoRI site of MigR1 (49) to produce the retroviral expression vector, MigR1-SATB1. The {Delta}NMTS and {Delta}DBD cDNAs were obtained from ID(224–278) and ID(346–474) for cloning into the EcoRI site of MigR1 to construct MigR1-{Delta}NMTS and MigR1-{Delta}DBD. High fidelity, Pfu Turbo DNA polymerase (Stratagene) was used for all PCRs as recommended by the manufacturer. Platinum Pfx DNA polymerase (Invitrogen) was used for the second round of recombinant PCR. The final clones were verified by sequencing and restriction enzyme digestions. All restriction enzymes and Klenow DNA polymerase were obtained from NEB.

Sequences of PCR primers mentioned above are as follows: SATB199EGFP (5'-CGG AAT TCA CTG AGT ATG GAT CAT TTG AAC-3'); SA363DO (5'-GGC GAA TTC TTA CAC TCC CTG CAT CTT TCC-3'); SA870DO (5'-GGC GAA TTC TTA GTA CGT GCT GTT CAC AAT G-3'); SA631UP (5'-GCG AAT TCC TGA GTA TGG TGC CTT TAA AAC ACT CG-3'); SA2497DO (5'-CGG AAT TCT CTC TCA GTC TTT CAA GTC G-3'); SA700UP (5'-GCG AAT TCC TGA GTA TGA TTC AGT TAC ACA GTT GC-3'); SA1021AUG (5'-GCG AAT TCC TGA GTA TGC CAG GAA ACA CAG CTG AG-3'); SA196+ (5'-CGG AAT TCA GTA TGG ATC ATT TGA ACG AG-3'); SA867–(5'-CTG CTC AGC CGT GCT GTT CAC AAT GGA G-3'); SA1033+ (5'-AAC AGC ACG GCT GAG CAG CCT CCA TCC-3'); Sagfp865+ (5'-CGG AAT TCT ACG TAC TAT GCA AAT GTC-3'); SA1032–(5'-CTG AAT TCT GTG TTT CCT GGG ACA GG-3'); Sagal865+ (5'-CGG AAT TCA CGT ACT ATG CAA ATG TC-3'); SA1723+ (5'-CGG AAT TCA GTA TGG CTA AAG TGT CCC AAG CAC C-3').

Cell Culture and Transfections—The cell lines MCF-7/Tet-off (Clontech), XC, and 293T were grown at 37 °C in 7.5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics (29, 50, 51). Growth conditions for HC11 (28) and Jurkat cells (9) have been described before. MCF-7/Tet-off cells were grown in media supplemented with 100 µg/ml G418 to maintain the transactivator plasmid as recommended by the supplier. Cells were plated at a density of 7.5 x 105 cells (XC) or 4 x 106 cells (MCF-7/Tet-off) per 60-mm plate for 24 h prior to transfection. The pUHD10-3/SATB1 or SATB1 mutant (6 µg) and pUHD15–1/hygro (4 µg) constructs or only pUHD10-3/SATB1 construct (6 µg) were introduced into XC or MCF-7/Tet-off cells, respectively, using DMRIE-C reagent (Invitrogen) according to the manufacturer's protocol. The transfected cells were incubated for 24–48 h before analysis. XC cells were infected with purified MMTV virions (C3H strain) to produce XC/MMTV cells. The HC11/MTV-LUC and XC/MTV-LUC cells were generated by transfection of HC11 mouse mammary cells or XC rat fibroblasts, respectively, with pTR174 containing the hygromycin-resistance cassette and pMTV-LUC (pLC-LUC). The Jurkat/MTV-LUC cells were obtained by transfection of Jurkat cells with pMTV-LUC modified by insertion of a hygromycin-resistance cassette in the vector backbone (pMTV-LUC/Hyg). Stably transfected cells were selected in the presence of 250 µg/ml hygromycin B for ~3 weeks. Resistant colonies were pooled and used for analysis. Jurkat/MTV-LUC cells were electroporated with 600 nM SATB1-specific small interfering RNAs (siRNAs) (Dharmacon, SMART pool siRNA) or a non-targeting siRNA (Dharmacon, siCONTROL Non-Targeting siRNA #1). Mock treatment consisted of electroporation in the absence of siRNA. Cells were incubated for 60 h in RPMI medium containing 2% fetal bovine serum prior to preparation of protein extracts for Western blotting and luciferase assays.

Subcellular Fractionation—Subcellular fractions were prepared according to the method of de Belle et al. (42) with slight modifications. MCF-7/Tet-off cells transfected with full-length or mutant SATB1 expression constructs were treated with 0.5% Triton X-100 in CSK buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 1 mM EGTA) supplemented with 1 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mixture (Sigma, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, bestatin, E-64, leupeptin, pepstatin A, and 1,10-phenanthroline) on ice. The supernatant was recovered after centrifugation and referred to as the soluble fraction. The insoluble fraction was suspended in CSK buffer and treated with 20 units of DNase I (Roche Applied Science) for 15 min at 37 °C, and ammonium sulfate was added to a final concentration of 0.25 M. The soluble material was referred to as the chromatin fraction, and the insoluble fraction was washed with 2 M NaCl. The remainder (nuclear matrix fraction) was dissolved in 8 M urea and 10 mM Tris-HCl, pH 8.0. The same proportion of each fraction was analyzed on denaturing polyacrylamide gels.

Fluorescence Microscopy—HC11 mouse mammary cells were transfected with plasmids encoding GFP alone or GFP fused to wild-type or mutant SATB1 proteins. After 24–48 h, cells grown on glass coverslips were fixed with 4% paraformaldehyde, stained with 300 nM 4',6-diamidino-2-phenylindole, and washed extensively in phosphate-buffered saline. Coverslips were mounted in Vectashield (Vector Laboratories) and observed by fluorescence microscopy.

Western Blotting and Antibodies—Immunoblotting was performed according to standard procedures. Protein samples were mixed with 6x Laemmli buffer (0.35 M Tris-HCl, pH 6.8, 10% SDS, 36% glycerol, 0.6 M dithiothreitol, and 0.012% bromphenol blue), boiled, and separated on denaturing polyacrylamide gels (percentage varied from 8 to 15%) containing 0.1% SDS prior to transfer onto nitrocellulose (Schleicher & Schuell). Nonspecific binding to the membrane was blocked with TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk prior to incubation with primary antibodies (mouse monoclonal SATB1 (BD Biosciences), polyclonal goat anti-SATB1 (Santa Cruz) or polyclonal rabbit anti-SATB1 (34), GFP (Clontech), LexA (Clontech), anti-capsid (CA or p27) (generously provided by Dr. Tatyana Golovkina, The Jackson Laboratory) (52), cytochrome c (Santa Cruz Biotechnology), histone H1 (Santa Cruz Biotechnology), and lamin B (Santa Cruz Biotechnology)) for 1 h. Subsequently, the nitrocellulose membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase (Jackson Immunoresearch) for 30 min. Enhanced chemiluminescence reagents (PerkinElmer Life Sciences) were used for antibody detection. For quantitation, Western blots were incubated with GFP-specific or lamin B-specific antibodies, washed, and incubated with Alexa Fluor 680-conjugated rabbit immunoglobulin-specific (Molecular Probes) or IRDye800-conjugated goat immunoglobulin-specific (Rockland) antibodies. Band intensities were determined using infrared fluorescence and an Odyssey imaging system (LI-COR Biotechnology). Blocking buffer (LI-COR) was used for pretreatment of blots and antibody dilution.

Electrophoretic Mobility Shift Assays—Gel mobility shift assays were performed essentially as described by Dickinson et al. (25) and Liu et al. (9). Nuclear extracts were prepared by NE-PER nuclear and cytoplasmic extraction reagent (Pierce) supplemented with phenylmethanesulfonyl fluoride and protease inhibitor mixture (Sigma). The nuclear extracts (0.5 µg) were incubated with a reaction mixture (10 mM HEPES, pH 7.9, 50 mM KCl, 2.5 mM MgCl2, 4% glycerol, 0.1 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, and 0.1 mg/ml poly(dIdC) (Amersham Biosciences)) on ice for 10 min before end-labeled DNA probe (~2.5 fmol) was added. The binding reaction then proceeded on ice for an additional 20 min. For antibody ablation experiments, antibodies diluted 5-fold in phosphate-buffered saline were added to the nuclear extract and incubated on ice for 30 min prior to addition of the radiolabeled DNA probe. Aliquots of each sample were subjected to electrophoresis on 4% non-denaturing polyacrylamide gels containing 8% glycerol and 90 mM Tris borate buffer, pH 8.3, containing 2 mM EDTA. Gels were dried and subjected to autoradiography.

RNA Extractions and RNase Protection Assay—RNA was extracted using the guanidinium thiocyanate method (53). RNase protection assays were performed essentially as described by Yang and Dudley (54). MMTV(C3H) riboprobe template was constructed by inserting the MMTV LTR sequence from +843 to +1086 into the pGEMT-Easy vector (Promega). The mouse Gapd template (Ambion) was used as a control for RNA amount and integrity.

Pseudotyped Virus Production and Titering—On the day before transfection, 293T cells (8 x 106) were plated on a 100-mm dish. Retroviral vector MigR1, pHIT60 (gag-pol expression vector) (55) and pHITG (VSV-G expression vector) (55, 56) were co-transfected at a 2:1:1 ratio using SuperFect (Qiagen) according to the manufacturer's instructions. Cells were incubated with the transfection mix overnight, and fresh medium supplemented with 10 mM n-butyric acid was added for 5 h to increase protein expression. The media with n-butyric acid was removed, and fresh medium was added. Supernatant was harvested after 24 h followed by centrifugation to remove cell debris. The viral supernatant was concentrated by centrifugation for 100 min in a SW55Ti rotor at 20,000 rpm (38,000 x g), and the pellet was resuspended in a 0.08 volume of the original media. Virus was aliquoted and frozen at –70 °C until use. The efficiency of infection after transduction was determined using the number of GFP-positive cells obtained by fluorescence-activated cell sorting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mapping of SATB1 Domains Involved in DNA Binding to the MMTV NRE—SATB1 has been reported to have three DNA-binding domains, including two Cut repeat domains (CR1 and CR2) and an atypical homeodomain (26, 57, 58) (Fig. 1A). The major DNA-binding domain (also called the nuclear matrix association region or MAR domain) has been localized to amino acids 346–495, which spans CR1 and a small portion of CR2 (26) (Fig. 1A). The SATB1 N terminus contains a dimerization domain (amino acids 90–204) (38) as well as a region involved in recruitment of histone deacetylase 1 (40).

To determine whether nuclear matrix targeting of SATB1 is important for transcriptional repression of the MMTV LTR, we constructed a series of truncation/deletion mutants in an overexpression vector (Fig. 1A). SATB1 expression plasmids or the empty vector were transfected into MCF-7 human mammary carcinoma cells, which have little endogenous SATB1 expression. After incubation for 48 h, Western blot analysis was used to assess expression of wild-type and mutant proteins (Fig. 1, B and C). Two different SATB1 antibodies were used to detect the SATB1 mutants, because the polyclonal antibody (Fig. 1B) detects the N-terminal 10 amino acids, and the monoclonal antibody (Fig. 1C) detects the C-terminal region between amino acids 550 and 667. All mutant proteins were expressed at similar levels.

To test the DNA-binding ability of these mutants, we prepared nuclear extracts from MCF-7 cells that had been transfected with the SATB1 overexpression plasmids. Nuclear extracts then were used in electrophoretic mobility shift assays using the promoter-proximal NRE4 probe (9), which has four repeats of the strong SATB1-binding sites between –287 and –265 located in the MAR region of the MMTV LTR (9) (Fig. 2A). Jurkat nuclear extract was included as a positive control because these cells were shown to express high levels of endogenous SATB1 (Fig. 2B). The mobilities of endogenous and transfected SATB1 were similar. An internal deletion mutant, ID(224–278), had approximately wild-type DNA-binding activity (Fig. 2A, compare lanes 3 and 8). A SATB1 mutant with a deletion of the first 54 amino acids (55–764) had similar binding ability for the MMTV NRE (Fig. 2A, lane 11). The reasons for the different mobilities of the complexes are unknown, but could be due to differences in interactions with other proteins and/or protein conformation.

The N-terminal deletion mutants, SATB1(168–764), -(275–764), and -(510–764) (Fig. 2A, lanes 12–14), did not bind the MMTV NRE, consistent with previous data that the dimerization domain is required for SATB1 DNA binding (38). SATB1(1–494), which contains both the MAR and dimerization domains, showed only one complex with the NRE4 probe (Fig. 2A, lane 7), suggesting that the homeodomain and the C-terminal domain may provide additional protein-protein interactions. Internal deletions ID(346–494) and ID(274–410) missing the MAR domain had no detectable DNA binding to the MMTV NRE probe (Fig. 2A, lanes 9 and 10). As expected, the C-terminal truncation mutants (1–224, 1–274, and 1–345) (lanes 4–6), which also lack the MAR domain, CR2, and the homeodomain, did not bind DNA. The ability of SATB1-specific antibodies to abolish binding in electrophoretic mobility shift assays confirmed the identities of the protein-DNA complexes observed in Jurkat T cells and mammary cells (Fig. 2B). Similar results were observed when the rat fibroblast cell line, XC, was used for transfection and nuclear extract preparation (see Supplement Fig. S1), indicating that the DNA-binding activity of SATB1 was not cell type-specific.

Mapping of an NMTS—SATB1 was first identified as a MAR-binding protein (25) that associates with the bases of chromatin loops (42). A number of transcription factors bind to the nuclear matrix (59–63), and SATB1 has been associated with the nuclear matrix in Jurkat cells (42). However, the requirement for SATB1 binding to the nuclear matrix in transcriptional regulation has not been addressed. The fractionation method of de Belle et al. (42) was used to determine the intranuclear localization of various SATB1 mutants. Initially, we tested MCF-7 cells to verify the efficiency of the fractionation method. As anticipated, lamin B, histone H1, and cytochrome c (a mitochondrial protein) were found in the nuclear matrix, chromatin, and soluble fractions, respectively (Fig. 3A). The soluble fraction was defined as proteins that are soluble in 0.5% Triton X-100, and this fraction contains soluble nuclear as well as cytosolic proteins.



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  		FIG. 1.
Expression of SATB1 and SATB1 mutants. A, schematic diagram of SATB1 domain structure. Positions of the dimerization domain, the proposed nuclear matrix targeting sequence (NMTS), the MAR-binding domain, the homeodomain, and two Cut-like repeats (CR1 and CR2) are indicated. Dotted lines indicate deleted sequences. The numbers in the SATB1 mutant constructs indicate the amino acids present in the construct, whereas the numbers in the internal deletion (ID) mutants indicate the amino acids deleted from the mutants. B and C, protein expression of wild-type and SATB1 mutants. MCF-7 human mammary cells transfected with wild-type or mutant SATB1 expression vectors were incubated for 24–48 h prior to analysis of protein extracts by Western blotting. The position of mutant proteins detected by the polyclonal antibodies is indicated. SATB1-specific goat polyclonal antibodies (B) or mouse monoclonal antibodies (C) were used to detect the mutant proteins, depending on the deleted regions. The polyclonal goat antibody recognizes the SATB1 N terminus, and the monoclonal mouse antibody recognizes the C-terminal region.

 
To determine the domain needed for SATB1 localization to the nuclear matrix, we transfected human mammary MCF-7 cells with wild-type or mutant SATB1 expression vectors, and the cells were fractionated (Fig. 3B). An equal proportion of each fraction was loaded on denaturing polyacrylamide gels to allow semi-quantitative comparisons of protein localization to the nuclear matrix. Our results confirmed that the wild-type protein was present in the nuclear matrix, but SATB1 also was present in the chromatin and soluble fractions. Interestingly, C-terminal deletion to amino acid 494, SATB1(1–494), redistributed SATB1 so that the protein was localized exclusively to the chromatin and nuclear matrix fractions. Deletion of C-terminal sequences to amino acid 274 (SATB1(1–274)) decreased association with the nuclear matrix. Further deletion to amino acid 224, SATB1(1–224) abolished nuclear matrix localization (Fig. 3B). The mutant SATB1(1–224) was localized to the nucleus, but reduced amounts of mutants SATB1(510–764), -(168–764), and -(275–764) were apparent in the nucleus by fluorescent microscopy (Fig. 4 and data not shown). Such results are consistent with a SATB1 nuclear localization signal (NLS) within the N-terminal 224 amino acids and an NMTS between amino acids 224 and 278.

Localization of a SATB1 NLS was confirmed by fluorescent microscopy of cells expressing wild-type and mutant SATB1 proteins fused to GFP (Fig. 4). GFP alone is localized throughout the cell, whereas GFP-SATB1 was strictly nuclear with a slightly punctate appearance. Linkage of the first 55 amino acids of SATB1 to GFP resulted in slightly more nuclear localization compared with GFP alone, but fusion to the N-terminal 224 amino acids had an exclusively nuclear punctate appearance as previously described (64). GFP fusion to SATB1 amino acids 224–278 (GFP-NMTS) showed a similar localization as GFP alone, suggesting that this region lacks an NLS. Because no consensus NLS is localized in the first 224 amino acids of SATB1, these results are consistent with multiple weak NLSs in this region.



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  		FIG. 2.
Both dimerization and MAR domains are necessary to bind to the MMTV NRE. A, electrophoretic mobility shift assays from cells expressing wild-type and mutant SATB1 proteins. Nuclear extracts were prepared from MCF-7 human mammary cells transfected with SATB1 expression plasmids. Extracts then were used in gel shift assays with an NRE4 probe, a four-repeat concatamer of strong SATB1-binding sites in the MMTV NRE. Lane 1 contains the labeled probe in the absence of added nuclear extract, whereas lane 2 shows probe binding to nuclear extracts transfected with the empty vector. Gel shifts with nuclear extracts obtained from transfections with wild-type SATB1 (lane 3) or various mutants (lanes 4–14) are shown. Wild-type SATB1 and three mutants, which contain both the dimerization and the MAR-binding domains, showed DNA binding (lanes 3, 7, 8, and 11). A bracket shows the distribution of various SATB1-specific complexes. B, antibody ablation assays reveal the identity of the retarded bands. Even-numbered lanes indicate assays performed in the presence of preimmune sera, and odd-numbered lanes (except lane 1) indicate assays performed in the presence of SATB1-specific rabbit polyclonal antibody. Gel shifts with nuclear extract from human Jurkat T cells (lanes 12 and 13) were used as a positive control. Lanes 2–11, 14, and 15 contain nuclear extract from transfected MCF-7 cells. Gel shifts shown in lanes 4 and 5 and lanes 14 and 15 were performed using nuclear extracts from different transfections with wild-type SATB1.

 
Additional mutants were examined to confirm the localization of a SATB1 NMTS. As predicted, deletion of the N-terminal 54 amino acids had no effect (SATB1(55–764)), but deletion of the first 274 amino acids (SATB1(275–764)) virtually eliminated nuclear matrix association. Examination of the internal deletion mutants ID(346–494) and ID(274–410) revealed that the deletion of the MAR-binding domain or Cut repeats reduced, but did not abolish, nuclear matrix binding. Because these mutants retain an NLS, SATB1 may contain additional nuclear matrix-binding regions. Deletion of the region between amino acids 224 and 278, ID(224–278), which retains the NLS, significantly reduced SATB1 association with the nuclear matrix (Fig. 3B). These results are consistent with the presence of a SATB1 NMTS in a region outside the DNA-binding domains.



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  		FIG. 3.
Subcellular fractionation of wild-type and mutant SATB1 proteins. A, integrity of fractions obtained from MCF-7 cells. Cells were fractionated as described under "Materials and Methods" to obtain a combined nuclear and cytoplasmic soluble (SF), chromatin (CH), and nuclear matrix (NM) fractions. To verify the integrity of each fraction, lamin B, histone H1, and cytochrome c antibodies were used for Western blotting to determine the distribution of endogenous proteins among the fractions. B, deletion of the NMTS(224–278) decreased nuclear matrix association of SATB1. Various SATB1 mutants were transfected into MCF-7 cells, incubated for 24 h, and fractionated. Equal amounts of each fraction were separated by gel electrophoresis prior to Western blotting and incubated with monoclonal mouse anti-SATB1(internal and N-terminal deletion mutants) or polyclonal rabbit anti-SATB1 antibody (C-terminal deletion mutants).

 
Fusion of the SATB1 NMTS(224–278) to Heterologous Proteins Promotes Nuclear Matrix Association—To assess whether the SATB1 region from 224 to 278 can localize a heterologous protein to the nuclear matrix, C-terminal fusions to GFP or LexA were engineered. Each construct was transfected into MCF-7 cells, which were fractionated to determine whether the heterologous proteins associated with the nuclear matrix. Fusion of GFP to the SATB1 NMTS(224–278) localized a portion of the hybrid protein to the nuclear matrix, whereas GFP was not associated with the nuclear matrix. Similarly, because LexA is a soluble protein that appears equally distributed in the nucleus and the cytoplasm (65), we observed LexA in both the soluble and chromatin fractions (Fig. 5A, lanes 1 and 2). LexA-NMTS, but not LexA, also associates with the nuclear matrix (Fig. 5A, lane 3). These data indicate that SATB1 amino acids between 224 and 278 are sufficient to partially relocalize heterologous proteins to the nuclear matrix.



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  		FIG. 4.
Intracellular localization of wild-type and mutant SATB1 fusion proteins by fluorescence microscopy. HC11 mouse mammary cells were transiently transfected with plasmids encoding GFP or GFP fusions to wild-type or mutant SATB1 proteins. GFP-SATB1 expresses a GFP fusion to full-length SATB1, whereas GFP-SATB1(1–55), GFP-NMTS, and GFP-SATB1(1–224) express GFP fusions to the N-terminal 55 amino acids, amino acids 224–278, and the N-terminal 224 amino acids of SATB1, respectively. After 24–48 h, cells grown on glass coverslips were fixed in paraformaldehyde, stained with 4',6-diamidino-2-phenylindole, and viewed under a fluorescence microscope at two different wavelengths.

 
To quantitate the effect of the NMTS on nuclear matrix association, we performed Western blotting of fractions obtained from cells transfected with plasmids expressing GFP or various GFP-fusion proteins (Fig. 5B). The amount of these proteins in different fractions was determined using the Odyssey imaging system and compared with that of a known nuclear matrix protein, lamin B. At least 90% of lamin B was detected in the nuclear matrix in multiple sample preparations, whereas only 3% of GFP was in this fraction. However, 42% of GFP-NMTS containing SATB1 amino acids from 224 to 278 associated with the nuclear matrix, a 14-fold increase. GFP-SATB1(1–55) containing the first 55 amino acids of SATB1 showed no increased association with the nuclear matrix compared with GFP alone, whereas GFP-SATB1(1–224) showed a 4-fold increase in association with the nuclear matrix compared with GFP. The latter observation may result from increased GFP localization to the nucleus due to the NLS or to weak NMTS activity of these sequences or both. Nevertheless, these results confirm localization of a SATB1 NMTS to a region between amino acids 224 and 278.

The SATB1 NMTS(224–278) Contributes to Transcriptional Repression of Integrated MMTV Promoters—Initially, transient transfections were performed to determine the effect of NMTS(224–278) deletion on SATB1-mediated repression of an MMTV LTR-reporter gene. SATB1 repression was observed, but most SATB1 mutants, including those lacking the MAR-binding domain, gave similar reporter gene expression, perhaps due to squelching (data not shown). SATB1 has been reported to organize chromatin on the nuclear matrix and recruit chromatin remodeling complexes to regulate gene expression (40, 41, 66). To confirm the ability of wild-type SATB1 to repress transcription from the integrated MMTV promoter, human Jurkat T cells were stably transfected with an MMTV LTR-luciferase reporter plasmid. Stable transfectants then were transiently transfected with a control siRNA or an siRNA specific for SATB1 prior to preparation of lysates for Western blotting and luciferase assays (Fig. 6). Western blots indicated that the control siRNA had little effect on SATB1 levels compared with the mock-transfected cells, whereas the SATB1-specific siRNA had a major effect (Fig. 6A). Luciferase levels in cells transfected with the SATB1-specific siRNA were significantly elevated (p < 0.01) compared with those of cells treated with the control siRNA (Fig. 6B). These results indicated that wild-type SATB1 suppresses transcription from the integrated MMTV LTR. However, the high levels of endogenous SATB1 in T-cell lines interfered with analysis of various mutants. Therefore, we assessed the effect of SATB1 mutants on exogenous MMTV proviruses integrated in rat cells, which lack both endogenous MMTV proviruses and high SATB1 levels that might complicate the results.



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  		FIG. 5.
SATB1 NMTS(224–278) targets heterologous proteins to the nuclear matrix. A, biochemical fractionation of MCF-7 mammary cells expressing SATB1 NMTS-fusion proteins. GFP-NMTS, LexA-NMTS, GFP, and LexA constructs were transfected into MCF-7 cells, incubated for 24–48 h, and fractionated. The proteins were detected by Western blotting with rabbit GFP-specific or goat LexA-specific antibody. SF, soluble fraction; CH, chromatin fraction; NM, nuclear matrix fraction. B, quantitation of nuclear matrix association of GFP-SATB1 fusion proteins. Plasmids encoding GFP or GFP-SATB1 fusion proteins were transfected into MCF-7 cells, incubated for 24–48 h, and fractionated. Fractions were analyzed by Western blotting and incubation with GFP or lamin B-specific antibodies. Alexa Fluor 680-conjugated rabbit immunoglobulin-specific or IRDye800-conjugated goat immunoglobulin-specific antibodies were used to detect bands. Band intensities were measured using the Odyssey imaging system. The total amount of fluorescence in the soluble, chromatin, and nuclear matrix fractions was set to 100. The small numbers below each figure indicate the percentage of fluorescence of either GFP or lamin B present in each fraction. The whole cell lysate (WL) represents the amount of protein in one-quarter of the unfractionated transfected cells after sonication.

 
Information on SATB1 domain structure is incomplete, primarily due to the lack of a good tissue culture system where SATB1 can be expressed at high levels. A retroviral vector, MigR1, was used to express wild-type or mutant proteins in cells that lack significant SATB1 expression. MigR1 allows production of a bicistronic transcript that expresses GFP using an internal ribosomal entry site downstream of the cloning site. MMTV-infected XC cells (XC/MMTV) were super-infected with MigR1 viruses, expanded, and induced with dexamethasone (DEX) for 24 h. Analysis of GFP expression revealed that at least 95% of the cells expressed the cloned proteins (data not shown). Western blotting was used to test the effect of wild-type and {Delta}NMTS (also called ID(224–278); Fig. 1A) on MMTV Gag protein levels (Fig. 7A). MigR1-SATB1 significantly suppressed Gag expression compared with the MigR1 vector control, and deletion of the SATB1 NMTS relieved this suppression (compare lanes 2–4, upper panel). Western blotting with SATB1 antibody showed that the expression levels of SATB1 and {Delta}NMTS were comparable (Fig. 7A, middle panel), and actin expression verified similar protein loading in each lane (lower panel).



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  		FIG. 6.
Reduction of SATB1 levels in Jurkat T cells leads to increased expression from integrated MMTV LTRs. Jurkat cells stably transfected with the MTV-LUC reporter plasmid were electroporated with SATB1-specific siRNAs or non-targeting siRNAs. Mock treatment consisted of electroporation without added siRNAs. Cells were incubated for 60 h prior to preparation of protein extracts. A, SATB1-specific siRNAs reduce SATB1 expression. Western blots were incubated with SATB1-specific mouse monoclonal antibodies (upper panel) or with actin-specific antibodies (lower panel). Scion software was used to quantitate SATB1 levels relative to actin. B, SATB1-specific siRNAs increase expression from integrated MMTV LTR-reporter genes. Luciferase levels in cells treated with the non-targeting siRNA were assigned a value of 100, and luciferase activity in the other samples was normalized to this value. Values are shown as the means of triplicate determinations ± S.D. The luciferase activities of cells treated with control and SATB1-specific siRNAs are statistically different by the Student's t test (p < 0.01), whereas the luciferase activities of mock-treated and control siRNA-treated cells are not (p > 0.39).

 
Because Western blotting is only semi-quantitative, ribonuclease protection assays were performed to compare the level of MMTV (C3H) expression in different virally transduced XC/MMTV cells. SATB1 overexpression reduced MMTV transcription ~4- to 5-fold after DEX induction (Fig. 7B, compare lanes 4 and 5). However, the suppression by {Delta}NMTS was 2.7-fold less than that observed with wild-type SATB1 overexpression (lane 6). RNA from non-transduced XC cells was used to distinguish nonspecific and specific bands (lane 3). To accurately compare the expression levels, MMTV expression was normalized to Gapd RNA levels as measured by a phosphorimaging device. Relative levels of MMTV expression in MigR1 (vector), MigR1-SATB1, and MigR1-{Delta}NMTS cells were 100, 23, and 62, respectively (Fig. 7B).

A similar experiment was performed using rat XC cells that had stably integrated copies of an MMTV LTR-luciferase reporter gene (XC/MTV-LUC). Stably transfected cells were transduced with MigR1 or MigR1 expressing wild-type SATB1, {Delta}NMTS, or {Delta}DBD (also called ID(346–494); Fig. 1A). Western blotting confirmed that similar levels of each protein were expressed in the transduced cells (Fig. 7C). Cells were then assayed for reporter gene activity (Fig. 7D). Wild-type SATB1 reduced reporter gene expression ~2.5-fold, whereas deletion of either the NMTS or the MAR-binding domain significantly relieved this suppression. Thus, the NMTS was important for SATB1-mediated repression when the native target was used (integrated proviruses) or when integrated MMTV LTR-reporter genes were used.

A second cell line also was used to determine the cell-type specific effect of the NMTS on SATB1-mediated repression of MMTV expression. HC11 mouse mammary cells, which lack significant SATB1 expression, were stably transfected with an MMTV-luciferase reporter gene. Transfected cells were then transduced with MigR1, MigR1-SATB1, MigR1-{Delta}DBD, or MigRI-{Delta}NMTS infectious particles. Comparable expression of full-length SATB1 and the {Delta}NMTS and {Delta}DBD mutants was confirmed by Western blot analysis (Fig. 8A). Reporter gene activity then was measured in cells grown in the presence or absence of glucocorticoids (Fig. 8B). SATB1 suppressed MMTV expression ~2-fold in each case. Similar to results obtained using the XC cells, SATB1-mediated suppression was relieved by NMTS deletion. As expected, deletion of the MAR-binding domain (MigR1-{Delta}DBD) completely abolished SATB1-mediated repression of the MMTV LTR, and the luciferase activity was similar to that obtained for vector-transduced cells (Fig. 8B). Therefore, the NMTS(224–278) contributes to repression of MMTV expression by SATB1 in both the presence and absence of glucocorticoids.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MMTV expression is positively regulated by the interaction of ligand-bound steroid hormone receptors with multiple sites within the hormone response element (HRE) in the U3 region of the LTR (67). MMTV expression also is negatively regulated by NREs containing MARs located upstream of the HRE (8, 9, 11). Our previous experiments indicate that the effects of the NREs are due in part to binding of two homeoprotein transcription factors, CDP and SATB1, which have independent differentiation and cell type-specific distributions (9, 28).

SATB1 originally was identified as a thymus-specific transcription factor that bound to DNA associated with the nuclear matrix (i.e. MARs) (25). Because SATB1 associates with the nuclear matrix (42), a series of SATB1 deletion mutations were constructed to delineate sequences that contribute to nuclear matrix localization (Fig. 1A). Using biochemical fractionation of transiently transfected cells, an NMTS was mapped to the 55 amino acids between the dimerization and MAR domains (residues 224–278) (Fig. 3B). An internal deletion mutant of SATB1 lacking an NMTS ({Delta}NMTS or ID(224–278)) significantly reduced nuclear matrix association. However, because this deletion did not completely abolish the association, SATB1 may have more than one domain that specifies matrix binding. The presence of multiple NMTSs has been observed in steroid hormone receptors, ETO, the AML1/ETO fusion, and ying-yang 1 (68). Our data are consistent with the idea that different factors may function as receptors for localization of NMTS-containing proteins to specific regions of the nuclear matrix. Separate subdomains within the nuclear matrix may explain the lack of a consensus NMTS (61) (see Supplement Fig. S2 for alignment of SATB1 NMTS(224–278) with those from Runx family members (69)). The most conserved amino acids contain PXP, but this sequence is outside the critical region for Runx NMTS function. Mutagenesis of the SATB1 NMTS(224–278) will be necessary to determine required amino acids.



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  		FIG. 7.
The NMTS is required for optimal SATB1-mediated MMTV suppression in rat fibroblast cells. A, deletion of the NMTS relieves SATB1 suppression of MMTV Gag protein expression. MMTV-infected XC cells (XC/MMTV) cells were incubated for 4–5 days after infection with retroviruses expressing SATB1 prior to addition of 10–6 M DEX for 24 h. Transduction efficiency was >95% as determined by fluorescence-activated cell sorting analysis. MMTV Gag protein was measured by Western blot analysis using monoclonal antibodies to the viral capsid protein (upper panel). Overexpression of SATB1 and {Delta}NMTS was confirmed using SATB1-specific monoclonal antibody (middle panel). Incubation with actin-specific antibody was used as a control for protein loading (lower panel). B, ribonuclease protection assay using RNA extracted from rat XC cells expressing MMTV and either wild-type SATB1 or {Delta}NMTS. XC/MMTV cells were infected with MigR1 (Vector), MigR1-SATB1, or MigR1-{Delta}NMTS virions. After treatment as described above, total RNA was extracted prior to ribonuclease protection assays. MMTV expression was quantitated using a phosphorimaging device and normalized relative to Gapdh RNA levels. The positions of full-length probes and smaller protected bands are indicated. Faint bands near the MMTV-specific bands (asterisks) represent incompletely digested probe, because these also appear using RNA extracted from XC cells. C, expression of wild-type and SATB1 mutants in XC rat cells containing stably integrated MMTV LTR-reporter constructs (XC/MTV-LUC). The upper panel shows Western blotting using the monoclonal SATB1-specific antibody. Blots also were incubated with actin-specific antibody (lower panel). D, luciferase activity of XC/MTV-LUC cells after transduction with MigR1 virus or MigR1 virions expressing wild-type or mutant SATB1 proteins. The luciferase activity of the same amount of protein from each set of SATB1-transduced cells was normalized to that of cells transduced with the MigRI control virus (assigned a relative value of 100). The luciferase activity of extracts from MigR1-SATB1-transduced cells was significantly different (p < 0.01; two-tailed Student's t test) from that obtained with MigR1-transduced cells. Results from MigR1-{Delta}NMTS and MigR1-{Delta}DBD-transduced cells were also significantly different (p < 0.01) from results with MigR1-SATB1-transduced cells.

 



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  		FIG. 8.
Deletion of the NMTS(224–278) relieves SATB1-mediated repression of MMTV in mammary cells. A, overexpression of SATB1 proteins in HC11 mammary cells. SATB1, {Delta}NMTS, and {Delta}DBD were ectopically expressed in mouse mammary cells using the retroviral MigR1 vector. Transduction efficiency was ~90% as determined by fluorescence-activated cell sorting analysis. A monoclonal SATB1-specific antibody showed expression of wild-type SATB1 and the deletion mutants, {Delta}NMTS and {Delta}DBD. Incubation of the blot with actin-specific antibody confirmed similar protein loading. B, deletion of the NMTS relieves SATB1-mediated suppression of integrated MMTV LTR-reporter genes. The HC11/MTV-LUC cells were transduced with MigR1, MigR1-SATB1, MigR1-{Delta}NMTS, or MigR1-{Delta}DBD infectious particles. The transduced cells were incubated in the presence or absence of 10–6 M DEX for 24 h prior to assays for luciferase activity. Luciferase values were calculated as described in Fig. 7. Data are expressed as an average of three independent luciferase assays from the same transduction, and error bars represent the standard deviations of the means. The luciferase activity of extracts from MigR1-SATB1-transduced cells was significantly different (p < 0.01; two-tailed Student's t test) from that obtained with control virus-transduced cells (either in the presence or absence of hormone). Results from MigR1-{Delta}NMTS and MigR1-{Delta}DBD-transduced cells (with or without hormone) also were significantly different (p < 0.01) from those obtained with comparably treated MigR1-SATB1-transduced cells. The entire experiment was repeated with similar results.

 
The presence of an NMTS within the SATB1 N terminus was confirmed by tethering amino acids 224–278 to either LexA or GFP, which do not normally bind the nuclear matrix. Both GFP-NMTS and LexA-NMTS partially relocalized to the nuclear matrix (Fig. 5A). Interestingly, the SATB1 NMTS also partially redirected GFP to the chromatin fraction. This result may indicate that the 55 amino acids designated as NMTS(224–278) might be further delimited to give either nuclear matrix or chromatin association. In addition, the SATB1 mutant (1–494) containing the dimerization domain, the NMTS(224–278), and the MAR-binding domain localized entirely to the chromatin and nuclear matrix fractions, whereas SATB1(1–224) containing only the dimerization domain was exclusive to the soluble nuclear fraction (Figs. 3B and 4) (26, 38, 57). SATB1 localization to the chromatin and nuclear matrix fractions decreased after deletion of all or part of the MAR domain in various mutants (Fig. 3B). Although localization in the soluble fraction may indicate the lack of specific intranuclear trafficking signals, it also is possible that the presence of both the MAR and NMTS(224–278) domains increases the affinity for both the nuclear matrix and chromatin. Previous data have shown that GR association with the nuclear matrix is increased by the presence of the GR DNA-binding domain (60). Therefore, the NMTS(224–278) and C-terminal sequences, including the DNA-binding domains, may regulate chromatin and nuclear matrix association.

The nuclear matrix, a subnuclear domain, plays an important role in transcriptional regulation (70, 71). Therefore, we also determined whether nuclear matrix binding affects SATB1-mediated repression of MMTV transcription. Structure/function studies have been particularly difficult because of problems associated with SATB1 overexpression in cell culture (37),2 and the greatest effects of SATB1 have been observed using integrated genes in transgenic and knock-out mice (9, 33). To overcome these problems, we have analyzed integrated MMTV proviruses or transgenes in cell lines infected with retroviral vectors overexpressing wild-type or mutant SATB1 proteins. Use of retroviral vectors allows overexpression in >90% of the cell population while allowing analysis of integrated gene expression within a timeframe that may prevent extensive selection during cell growth. Cells with low levels of SATB1 or loss of SATB1-signaling pathways may have a growth advantage.

Deletion of the NMTS relieved SATB1-mediated repression of transcription from an integrated MMTV provirus or reporter plasmid (Figs. 7 and 8), but there was no detectable effect using transiently transfected plasmid containing the MMTV LTR (data not shown). Our observations are reminiscent of the results of Kohwi-Shigematsu et al. (37) in which SATB1 suppressed the expression of stably transfected, but not transiently transfected, MAR constructs. Although reduced suppression by {Delta}NMTS (ID(224–278)) may be due to conformational changes within the protein, this mutant maintained several functional properties, including nuclear import and DNA binding (Fig. 2 and data not shown). Partial relief of suppression after NMTS deletion might result from the retention of other nuclear matrix association sequences (see above). Repression by SATB1 and the effect of NMTS(224–278) also were independent of the cell type examined, because similar results were obtained using mouse mammary epithelial or rat fibroblast cells. Previous data have shown that MAR binding to the nuclear matrix is independent of cell type (9). However, more recent evidence suggests that the nuclear matrix from normal cells has a cell type and differentiation-dependent composition that is modified in cancer cells (reviewed in Zink et al. (72)), but our assays may not detect these differences (73).

Nuclear matrix binding clearly has functional implications for transcription. Fractionation studies show that transcriptionally active DNA is tightly associated with the nuclear matrix, whereas inactive loci are not. For example, the ovalbumin gene in the chicken oviduct is associated with the nuclear matrix when the gene is stimulated by steroid hormones. However, when the hormone is withdrawn, the gene is detached from the nuclear matrix (74). Genes expressed by integrated polyoma or avian sarcoma viruses in transformed cells also were associated with the nuclear matrix, but subclones that lack the transformed phenotype and express no detectable viral transcripts show little association with the nuclear matrix (75). Mutations of Pit-1, a transcriptional activator of prolactin, altered nuclear matrix localization and led to dwarfism in mice (76). Knock-in mice that express an NMTS-deleted form of Runx2 have abnormal bone formation and die during embryonic development (71). The Runx2 NMTS autonomously exhibits transactivation in a mammalian one-hybrid system (69), whereas deletion of the Runx2 NMTS abrogated the transactivation effect on the bone-specific promoters, osteocalcin, and transforming growth factor {beta} R1 (69, 71, 77). An NMTS-deletion mutant of Hic-5/ARA55, a GR coactivator, maintained its GR-binding ability in yeast cells, but did not fully activate a glucocorticoid-responsive promoter (62). NMTS deletion from transcription regulators also affects repression of certain promoters. RGS12TS-S, a regulator of G protein signaling, inhibits transcription in situ and in a mammalian one-hybrid assay, and NMTS deletion relieved this suppression (61). Removal of the Runx2 NMTS abrogated repression of the bone sialoprotein promoter (71). Therefore, the nuclear matrix affects gene activation as well as repression (71).

SATB1 suppressed integrated reporter gene expression in the presence and absence of ligand-bound GR (Fig. 8). GR functions by recruitment of coactivators that acetylate histones and chromatin remodeling complexes (78), and the subsequent chromatin reorganization allows promoter-proximal NF1 and Oct1 binding and assembly of the basal transcription machinery (79, 80). Both GR and SATB1 are bound to the nuclear matrix (42, 60), where there may be competition between recruitment of histone acetyltransferases and histone deacetylases. Such competition may explain the partial effect of SATB1 overexpression on hormone-induced MMTV expression. NMTS deletion would decrease SATB1-mediated histone deacetylase recruitment, which may impede interaction with chromatin-accessibility complex, ATP-utilizing chromatin assembly and remodeling factor, and nucleosome remodeling and deacetylase complex chromatin-remodeling complexes (40). SATB1 is bound to MARs at the bases of chromatin loops (42) and may regulate effects of cis-acting regulatory elements in cellular DNA by acting as an insulator (81). For MMTV, which contains MARs at either end of the genome in the LTR (9), insulation from surrounding cellular regulatory control after genome insertion may provide repression of viral transcription in T cells and a selective advantage for virus survival.


   FOOTNOTES
 
* This work was supported by Grants CA34780 and CA77760 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

{ddagger} To whom correspondence should be addressed: One University Station, A5000, 24th St. and Speedway, ESB 226, Austin, TX 78712-0162. Tel.: 512-471-8415; Fax: 512-471-7088; E-mail: jdudley{at}uts.cc.utexas.edu.

1 The abbreviations used are: MMTV, mouse mammary tumor virus; LTR, long terminal repeat; SATB1, special AT-rich binding protein 1; CDP, CCAAT-displacement protein; NRE, negative regulatory element; GFP, green fluorescent protein; GR, glucocorticoid receptor; NMTS, nuclear matrix targeting sequence; MAR, nuclear matrix-associated region; NLS, nuclear localization signal; DEX, dexamethasone; ID, internal deletion; HRE, hormone responsive element; DBD, DNA-binding domain; siRNA, small interfering RNA. Back

2 J. Seo, M. M. Lozano, and J. P. Dudley, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank members of the Dudley laboratory, Jon Huibregtse, David Johnson, and Phil Tucker for critical evaluation of the manuscript.



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 MATERIALS AND METHODS
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 DISCUSSION
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