Characterization of Homo- and Heterodimerization of Cardiac Csx/Nkx2.5 Homeoprotein*

Hideko KasaharaDagger §, Anny Usheva, Tomomi UeyamaDagger , Hiroki AokiDagger ||, Nobuo Horikoshi**, and Seigo IzumoDagger DaggerDagger

From the Dagger  Cardiovascular Division and the  Division of Endocrinology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, the ** Section of Cancer Biology, Radiation Oncology Center, Washington University School of Medicine, St. Louis, Missouri 63108, and the || Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube 755-8505, Japan

Received for publication, June 8, 2000, and in revised form, October 11, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Csx/Nkx2.5 is an evolutionarily conserved homeodomain (HD)-containing transcription factor that is essential for early cardiac development. We found that the HD of Csx/Nkx2.5 binds as a monomer as well as a dimer to its DNA binding sites in the promoter of the atrial natriuretic factor (ANF) gene, an in vivo target gene of Csx/Nkx2.5. Csx/Nkx2.5 physically interacts with each other in vitro as well as in cells, and the HD is critical for homodimerization. Lys193 and Arg194, located at the COOH-terminal end of HD, are essential for dimerization. Lys193 is also required for a specific interaction with the zinc finger transcription factor GATA4. Csx/Nkx2.5 can heterodimerize with other NK2 homeodomain proteins, Nkx2.3 and Nkx2.6/Tix, with different affinities. A single missense mutation, Ile183 to Pro in the HD of Csx/Nkx2.5, preserved homodimerization function, but totally abolished DNA binding. Ile183 right-arrow Pro mutant acts in an inhibitory manner on wild type Csx/Nkx2.5 transcriptional activity through the ANF promoter in 10T1/2 cells. However, Ile183 right-arrow Pro mutant does not inhibit wild type Csx/Nkx2.5 function on the ANF promoter in cultured neonatal cardiac myocytes, possibly due to failure of dimerization in the presence of the target DNA. These results suggest that complex protein-protein interactions of Csx/Nkx2.5 play a role in its transcriptional regulatory function.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The homeodomain (HD)1-containing transcription factors, characterized by their 60-amino acid DNA binding domain, play critical roles in developmental patterning and differentiation. The HD forms three alpha -helices and contacts the major groove of DNA through the third helix (1). Contrary to the highly specific biological functions of individual homeobox genes, in vitro DNA binding studies have demonstrated that most HD proteins bind to similar short consensus sequences containing the TAAT motif (1, 2). This apparent discrepancy may result from target gene's specificity for each HD protein in vivo being achieved by multiple mechanisms, such as interaction with other factors (3), small differences in DNA binding affinities to target sites (4), translational regulation of homeobox gene expression (5, 6), subcellular localization (7), or protein phosphorylation (8, 9).

Homo- or heterodimerization of transcription factors has been proposed to regulate transcriptional activity of many transcription factors. Combinatorial use of a limited number of transcription factors allows the regulation of a larger number of biological processes, increasing both the diversity as well as the specificity of control. However, a very limited number of studies have addressed the homo- and heterodimerization of HD-containing transcription factors among more than 400 members of HD proteins from yeast to human. Homodimerization ability has been demonstrated for Oct1 (10), Paired (11), Cdx2 (12), Even-skipped (13), Mix.1 (14), and Pit1 (15). Heterodimerization was shown for HNF1alpha -HNF1beta (16), Oct1-Oct2 (15), Mix.1-Siamois (14), MCM1-MATalpha 2 (17, 18), and Extradenticle-Ultrabithorax (19). The monomer of HD proteins is sufficient to interact with DNA, and the DNA-bound monomer recruits other partners to the complex (11, 20). Most likely, the monomer HD proteins regulate through the monomeric DNA binding site, whereas homo- or heterodimerized HD proteins regulate through the dimeric sites. These differential interactions would provide more precise gene regulation at each developmental stage (21).

The biological significance of dimerization of paired-like class HD proteins has been demonstrated in Xenopus Mix.1, which regulates dorsal-ventral patterning (14). To date, the significance of homodimerization of HD-containing transcription factors has not been well established in mammals. Interestingly, 10 heterozygous mutations of human CSX/NKX2.5 were recently identified in patients with congenital heart disease. These patients show progressive atrioventricular conduction defects, left ventricular dysfunction, atrial septal defect, ventricular septal defect, and tetralogy of Fallot (22, 23). Four mutations are single missense mutations in the HD that result in markedly reduced DNA binding (24), raising the possibility that if Csx/Nkx2.5 forms homodimers, the mutants with DNA binding defects may dominantly inhibit CSX/NKX2.5 function in human cardiac development and maturation. In Xenopus, injections of mRNA encoding non-DNA binding mutants of Xenopus XNkx2.3 and XNkx2.5 suppressed normal heart formation and resulted in a small heart or no heart formation in the most severe cases (25). This in vivo evidence suggests that a non-DNA binding mutant of Csx/Nkx2.5 may act in a dominant inhibitory manner on wild type Csx/Nkx2.5 through protein-protein interaction. Therefore, it is critical to examine whether Csx/Nkx2.5 proteins homo- or heterodimerize to regulate their transcriptional activity.

The NK2 class of HD proteins, first described in Drosophila (26), is highly conserved from nematode to human and is characterized by a unique Tyr residue at position 54 in the third helix of the HD. The most frequently observed HD binding motif is T/AAAT, but the NK2 class HD binds to the unique T/CAAG motif. The guanine nucleotide at the fourth position is distinct from all other HD·DNA complexes that usually have thymidine at this position, and 54Tyr is responsible for the unique DNA recognition (27-30).

Several NK2 class HD proteins are expressed in the heart. In Xenopus heart, XNkx2.3 and XNkx2.5 are coexpressed in precardiac mesoderm as well as in the underlying anterior endoderm from the gastrulation stage (stage 13) through adult stage (31, 32), whereas XNkx2.9 expression is restricted to the cardiogenic region of the embryo prior to differentiation, but transcript levels decrease rapidly in the heart (33). In other species, cNkx2.3, cNkx2.5, and cNkx2.8 are coexpressed in chick heart (34-36); and nkx2.3, nkx2.5, and nkx2.7 are coexpressed in zebrafish heart with distinct, but overlapping, spatio-temporal patterns (28, 37). In contrast, Csx/Nkx2.5 is the predominant NK2 class HD protein expressed in mouse cardiac myocytes from 7.5 days postcoital to adulthood (38-40), and another NK2 HD gene, Nkx2.6/Tix expression is restricted to the sinus venosus, dorsal pericardium, and outflow tract from 8 to 10 days postcoital (41).

The ANF promoter has been proposed to be a direct target of Csx/Nkx2.5 (42-46). Csx/Nkx2.5 binds at -87 and -242 bp sites, and transactivates the ANF gene synergistically with the zinc finger transcription factor GATA4 (42-45). In this study, we demonstrate the homodimerization of Csx/Nkx2.5 at ANF -242 site and determine the critical amino acid residues for homodimerization as well as heterodimerization with GATA4. In addition, we generated a single missense mutation, Ile183 to Pro in the HD of Csx/Nkx2.5, which preserved homodimerization function, but totally abolished DNA binding. Ile183 right-arrow Pro mutant acted in an inhibitory manner on wild type Csx/Nkx2.5 transcriptional activity through the ANF promoter depending on the cellular context.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construct-- FLAG epitope (GAC TAC AAA GAC GAT GAC GAC AAG) or HA epitope (TAC CCA TAC GAT GTT CCA GAT TAC GCT) was tagged at the amino terminus of Csx/Nkx2.5 (40). pcDNA3-Csx/Nkx2.5 were digested with SacII-NotI, PflI-NotI, KpnI (partial digestion)-NotI, or AccI-NotI, Klenow-treated, and then religated to produce pcDNA3-Csx/Nkx2.5-(1-230), -(1-199), -(1-159), and -(1-149). PCR amplifications were performed using a forward primer 5'-CCAAGTGCTCCTGCTTC-3' with the following reverse primers: 5'-GGAATTCCTACTGTCGCTTGCACTTGTAGC-3' for 1-196, 5'-GGAATTCCTATCGCTTGCACTTGTAGCGA-3' for 1-195, 5'-GGAATTCCTACTTGCACTTGTAGCGACGGT-3' for 1-194, 5'-GGAATTCCTAGCACTTGTAGCGACGGTTCT-3' for 1-193, 5'-CCAAGCTTCTACTTGTAGCGACGGTTCTGG-3' for 1-191, 5'-CCAAGCTTCTACTGGAACCAGATCTTGACC-3' for 1-186, and 5'-CCAAGCTTCTAGACCTGCGTGGACGTGAGC-3' for 1-181. PCR products digested with BstEII-EcoRI, or Klenow-treated BstEII-digested, were replaced with that of pcDNA3-Csx/Nkx2.5. Mutations at Lys193, Arg194, and Lys193-Arg194 were introduced using the following primers Lys193 (F, 5'-GCTACAAGTGCattCGACAGCGGC-3'; R, 5'-TGCCGCTGTCGaatGCACTTGTAGC-3'); Arg194 (F, 5'-TACAAGTGCAAGatcCAGCGGCAGGAC-3'; R, 5'-GTCCTGCCGCTGgatCTTGCACTTGTA-3'); Lys193-Arg194 (F', 5'-GCTACAAGTGCattgatCAGCGGCAGGAC-3'; R, 5'-GTCCTGCCGCTGatcaatGCACTTGTAGC-3'). Initial PCRs were performed with the combination of each forward (F) primer and C2 reverse (R) primer, as well as each reverse primer and P4 forward primer, then equal amounts of these two PCR products were applied to a second PCR using C2 and P4 primers as described previously (47). BstEII-PflMI-digested PCR fragments were replaced with that of pcDNA3-Csx/Nkx2.5.

Maltose-binding protein (MBP)-HD plasmid was kindly provided by R. Schwartz (48). Using pMAL-malE primer (5'-GGTCGTCAGACTGTCGATGAAGCC-3') and Csx/Nkx2.5 internal primer (5'-ATCTTGACCTGCGTGGACGTGAGC-3'), fragments of MBP-HD plasmid were amplified, digested with SacI-KpnI, then replaced with that of MBP-Csx/Nkx2.5 plasmid (47). To construct MBP-(1-250), SphI-EcoRI (filled) MBP-Csx/Nkx2.5 was replaced with SphI-digested PCR fragment using forward 5'-CCAAGTGCTCCTGCTTC-3' and reverse 5'-CTCTAGACTAGGGTAGGCGTTGTAGCC-3' primers.

MBP-Csx/Nkx2.5 and pcDNA3-Csx/Nkx2.5 were mutated with two primers (5'-CAGGTCAAGccaTGGTTCCAG-3', 5'-CTGGAACCAtggCTTGACCTG-3') to construct MBP-Csx/Nkx2.5(Ile183 right-arrow Pro) and pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro). All the PCR-amplified fragments were sequenced for sequence confirmation.

Electrophoretic Mobility Shift Assay (EMSA)-- MBP fusion proteins were prepared as described previously (47). Briefly, cultured Escherichia coli BL21(DE3) (Novagen) induced with 0.3 mM IPTG were lysed by sonication in lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1% Triton, 2 µg/ml aprotinin, 0.7 µg/ml pepstatin, 0.1 mM PMSF, 1 mM DTT, 10% glycerol), and lysates were incubated with amylose resin (New England Biolabs). Fusion proteins were eluted from the beads with the lysis buffer containing 10 mM maltose.

Two pM end-labeled oligonucleotides, 5'-TCACACCTTTGAAGTGGGGGCCTCTTGAGGCAAATC-3' (-264 to -227), was annealed with 5'-GATTTGCCTCAAGAGGCCCCCACTTCAAAGGTGTGA-3'; 5'-TCACACCTTTGAAGTGGGGGCCT-3' was annealed with 5'-AGGCCCCCACTTCAAAGGTGTGA-3'; 5'-GCCGCCGCAAGTGACAGAATGGGGA-3' (-93 to -65) was annealed with 5'-TCCCCATTCTGTCACTTGCGGCGGGCCA-3' and used for EMSA. 3-Fold serial dilutions of 66 ng of bacterially expressed fusion proteins were incubated with 50,000 cpm of probe, 50 µg of bovine serum albumin, 0.5 µg of poly(dG-dC) in 10 mM HEPES, pH 8.0, 50 mM KCl, 1 mM EGTA, 10% glycerol, 2.5 mM DTT, 7 mM MgCl2 in 15-µl reaction volume for 20 min at room temperature, separated in 5% native polyacrylamide gel with 0.5 × Tris-glycine buffer at 15 mA for ~20 min.

Nuclear extracts of neonatal cardiac myocytes were prepared as follows: cells on 10-cm plates were washed with HBS buffer (25 mM HEPES, pH 7.6, 130 mM NaCl) and soaked in 3 ml of low salt buffer (25 mM HEPES, pH 7.6, 1 mM DTT, 0.1% Triton X-100, 0.5 mM PMSF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate) for 10 min on ice. Cells were scraped with the low salt buffer and lysed by Dounce homogenizer. Nuclei pelleted by centrifugation at 200 × g for 5 min were resuspended in 100 µl of extraction buffer (20 mM HEPES, pH 7.6, 450 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 25% glycerol, 0.1 mM PMSF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate) and incubated for 30 min at 4 °C. After centrifugation at 15,000 × g for 10 min, the supernatant was used as a nuclear extract for EMSA using the same methods described above with 66 mM NaCl in the reaction.

Protein-DNA complexes analyzed by electrophoresis in 5% native polyacrylamide gel were transferred to polyvinylidene difluoride membrane with Tris-glycine-methanol buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membrane was blotted with anti-Csx/Nkx2.5 Ab (40), anti-FLAG Ab (Sigma), or anti-HA Ab (Roche Molecular Biochemicals). EMSA in which proteins were transferred to membranes contains 0.3 µg/µl final concentration of nuclear extract from noninfected cardiac myocytes or 0.044 µg/µl final concentration of nuclear extract from adenoviral infected cardiac myocytes and 0.16 pmol/µl final concentration of nonradiolabeled double-stranded oligonucleotides.

Protein-DNA binding affinity (Kd) was estimated by the protein concentration at which 50% of the DNA probe has become bound (49). Molecular mass of MBP fusion protein was estimated by addition of MBP protein (molecular mass = 42 kDa) and HD (aa 122-212, molecular mass = 10.9 kDa) or full length (molecular mass = 32.8 kDa) or Delta C (aa 1-250, molecular mass = 27.5 kDa) or Delta N (aa 122-318, molecular mass = 21.6 kDa).

Recombinant Adenoviruses-- FLAG- or HA-tagged wild type or FLAG-tagged Ile183 right-arrow Pro mutant was inserted into the shuttle vector pADloxp vector (50), creating pADloxp-Csx/Nkx2.5(FLAG-wild) or pADloxp-Csx/Nkx2.5(HA-wild) or pADloxp-Csx/Nkx2.5(FLAG-IP). One µg of plasmids was cotransfected with 1 µg of Psi 5 viral DNA into Cre8 cells to produce adenoviruses according to the methods reported previously (50). For control, Psi 5 viral DNA expressing no transgene was infected to 293 cells. The viral particle number was determined by plaque assays, and 5-15 multiplicity of infection was used for infection to neonatal rat cardiac myocytes prepared as described previously (51). The expression of wild type or Ile183 right-arrow Pro mutant protein was determined by Western blotting and immunostaining using anti-FLAG mAb (Sigma) or anti-HA mAb (Roche Molecular Biochemicals).

Protein-Protein Interaction-- Bacterially produced MBP-Csx/Nkx2.5, MBP-HD, MBP, glutathione S-transferase (GST)-GATA4 (provided by D. Wilson), and GST protein were made as described previously (47). In vitro transcribed and translated proteins were generated by using TNT-coupled reticulocyte lysate systems (Promega). 1 µl of reticulocyte lysate containing 35S-labeled wild type or mutant Csx/Nkx2.5, Nkx2.3, or Nkx2.6/Tix protein was mixed with fusion proteins in a 400 µl of binding buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, aprotinin (2 µg/ml), pepstatin (0.7 µg/ml), 0.1 mM PMSF, 1 mM DTT, and 1% bovine serum albumin) at 4 °C for 2 h. Beads were washed with binding buffer (without bovine serum albumin) and subjected to SDS-PAGE.

To perform coimmunoprecipitation assay, 293 cells in 100-mm plates were transfected with 9 µg of pcDNA3-FLAG-Csx/Nkx2.5 and/or 9 µg of pcDNA3-HA-Csx/Nkx2.5 using the calcium phosphate method. Total plasmid amount was adjusted with pcDNA3 empty vector to 18 µg. Cells were lysed in the lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, aprotinin (2 µg/ml), pepstatin (0.7 µg/ml), 0.1 mM PMSF, 1 mM DTT) and precleared with normal mouse IgG-bound protein G. Approximately 1 mg of protein in 1 ml of lysis buffer was incubated with 3 µg of anti-FLAG mAb affinity gel (Sigma), washed five times with lysis buffer, and resolved on SDS-PAGE and subjected to Western blotting using peroxidase-conjugated anti-HA Ab (Roche Molecular Biochemicals).

Reporter Gene Assays-- 10T1/2 fibroblast cells cultured in six-well plates were cotransfected with 1.0 µg of ANF(-638)-Luc reporter construct (provided by K. R. Chien), 0.4 µg of Rous sarcoma virus beta -galactosidase (provided by B. Markham), 0.4 µg of pcDNA3-Csx/Nkx2.5 with or without 0.4 µg or 0.8 µg of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) using the calcium phosphate method. 0.4 µg of pAT2-GATA4 expression vector (provided by B. Markham) was cotransfected together with the plasmids listed above. Total plasmid amount was adjusted to 3.0 µg with pcDNA3 vector plasmid. After glycerol shock using 1× HEPES buffer containing 15% glycerol, cells were cultured for another 48 h, lysed with 300 µl of reporter lysis buffer (Promega), and assayed for luciferase activity (Promega) and beta -galactosidase activity.

Rat neonatal cardiac myocytes cultured in six-well plates were cotransfected with 1.8 µg of ANF(-638)-Luc reporter, 0.6 µg of murine sarcoma virus (MSV) beta -galactosidase, 0.75 µg of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) with or without 0.75 µg of pcDNA3-Csx/Nkx2.5(Wild) using 10 µl of LipofectAMINE 2000 reagents (Life Technologies, Inc.). Total plasmid amount was adjusted to 4.65 µg with pcDNA3 vector plasmid. Plasmids were also cotransfected into cardiac myocytes using the calcium phosphate method in a total amount of 3.1 µg: 1.2 µg of ANF(-638)-Luc reporter, 0.4 µg of MSV-beta -galactosidase, 0.5 µg of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) with or without 0.5 µg of pcDNA3-Csx/Nkx2.5(Wild). The adenovirus encoding Ile183 right-arrow Pro mutant, wild type, or control empty virus were infected on the next day of primary culture (day 2). On day 3, 2.4 µg of ANF(-638)-Luc reporter and 0.6 µg of MSV-beta -galactosidase were cotransfected using the calcium phosphate method for 2 h, and luciferase activity (Promega) and beta -galactosidase activity were measured after 48 h.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Csx/Nkx2.5 Forms a Homodimer on a Palindromic DNA Sequence in the ANF Promoter-- The ANF promoter contains three specific binding sites for Csx/Nkx2.5 that are located upstream of the transcription start site at positions -408, -242, and -87 bp. At position -242 bp, the promoter contains two binding sites spaced by 5 nucleotides (Fig. 1A, panel a). Because Csx/Nkx2.5 contacts DNA through its HD, we analyzed and compared the HD binding affinity for two sites in the ANF promoter at positions -242 bp (ANF -242) and -87 bp (ANF -87). Applying EMSA, we determined that the HD protein binds to the ANF -242 site with a Kd = 1 × 10-9 M (Fig. 1A, panel b), which is slightly lower than the previously reported Kd for the related Drosophila HD protein NK2 (52), but in the affinity range for the HD protein (Kd in the range of 10-9 to 10-10 M) (53). However, for the protein-DNA interaction with the ANF -87 site, we estimated a Kd = 8.2 × 10-8 M (Fig. 1A, panel c). Therefore, the HD bound to the ANF -242 site with more than 80 times higher affinity than to the ANF -87 site. Interestingly, we also observed that Csx/Nkx2.5 forms an additional specifically shifted band with a migration most likely corresponding to the occupation of the two specific DNA binding sites of the ANF -242 at higher protein concentrations (Fig. 1A, panel b, lanes 3-6). We asked whether the protein concentration at which the second band appears is physiologically relevant. Csx/Nkx2.5 forms a dimer at a protein concentration of 3.1 × 10-9 M (lane 3), which is comparable with the concentrations reported for other transcription factors (54-56).



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Fig. 1.   HD of Csx/Nkx2.5 homodimerizes on palindromic DNA sequences in the ANF promoter. A, schematics of two Csx/Nkx2.5 consensus binding sites in the ANF promoter (panel a). A palindromic DNA binding -242 bp site (panel b) and monomeric -87 bp sites (panel c) were used for gel shift analysis. The first lane shows free probe (F) without protein, followed by 3-fold serial increases in HD protein concentrations shown in lanes 1-6 (panels b and c). The HD forms monomer (M) as well as dimer (D) at -242 bp site with a binding affinity Kd = 1.0 × 10-9 M (panel a) and forms only monomers on -87 bp site with a Kd = 8.2 × 10-8 M (panel b). B, a mutated monomeric binding site was used for EMSA. HD protein binds as a monomer (M) on the mutated -242 bp site with a Kd = 1-3.0 × 10-9 M. C, HD protein was mixed with full-length Csx/Nkx2.5 to identify heterodimers. The HD protein (3.0 × 10-9 M) (lane 1) was mixed with 3-fold serially increased full-length Csx/Nkx2.5 protein (0.71, 2.1, or 6.4 × 10-9 M) (lanes 2-4). The HD and full-length Csx/Nkx2.5 form stable complexes (HD+Full) and migrated to an intermediate position between HD dimer (HD D) and full-length dimer (Full D). M, monomer; D, dimer; F, free probe.

To confirm that the appearance of the second shifted band is a result of the simultaneous occupation of two specific binding sites of the ANF -242, we performed EMSA using an oligonucleotide in which one of the DNA binding sites was deleted from the ANF -242 site. We found that HD bound to the mutated site (converted to a single DNA binding site) as a monomer (M in Fig. 1B) without showing the slow migrating bands. The binding affinity to the monomeric DNA binding site was slightly reduced (Kd = 1-3.1 × 10-9 M). Furthermore, we found that when the HD was mixed with the full-length Csx/Nkx2.5, the HD and the full-length protein produced newly shifted bands migrating to an intermediate position between the HD and full-length homodimers (Fig. 1C, HD+Full). These data indicate that the Csx/Nkx2.5 protein binds with a higher affinity to the palindromic ANF -242 site than to the mutated monomeric ANF -242 site or the monomeric ANF -87 site. Most likely Csx/Nkx2.5 forms a dimer on ANF -242 site, and dimerization stabilizes the protein-DNA interaction (see below).

Homodimerization of Csx/Nkx2.5 in Vitro and in Cells-- We next examined whether Csx/Nkx2.5 proteins physically and specifically interact with each other in the absence of DNA. MBP-fused Csx/Nkx2.5 (Fig. 2A, lane 1), HD-MBP (lane 2), and MBP alone (lane 3) were mixed with in vitro translated 35S-labeled Csx/Nkx2.5 protein. After extensive washing, the protein complexes were resolved on SDS-PAGE and visualized by autoradiography. Approximately 25% of input 35S-labeled Csx/Nkx2.5 protein associated with MBP-Csx/Nkx2.5 (Fig. 2A, lane 1) as well as MBP-HD (lane 2), but not with MBP alone (lane 3). To examine whether Csx/Nkx2.5 homodimerizes in cells, we cotransfected FLAG epitope-tagged Csx/Nkx2.5 expression plasmid with HA epitope-tagged Csx/Nkx2.5 expression plasmid into the human embryonic kidney carcinoma cell line 293 and confirmed that both Csx/Nkx2.5 proteins coimmunoprecipitated with anti-FLAG Ab (Fig. 2B, lane 1). Thus, Csx/Nkx2.5 can homodimerize in solution as well as in cells, and binding to DNA is not required for this interaction.



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Fig. 2.   Homodimerization of Csx/Nkx2.5 without DNA. A, 35S-labeled Csx/Nkx2.5 protein was mixed with full-length (lane 1), HD (lane 2) fused to MBP, or MBP alone (lane 3). Bound protein was resolved on SDS-PAGE and autoradiographed. 50 and 25% input of 35S-labeled Csx/Nkx2.5 protein were loaded on lanes 4 and 5, respectively. MBP-full-length Csx/Nkx2.5 protein (lane 1), MBP-HD (lane 2), and MBP alone (lane 3) used for the assay are shown on the bottom panels. B, human embryonic kidney carcinoma 293 cells were transfected with two expression plasmids encoding FLAG-tagged Csx/Nkx2.5 and HA-tagged Csx/Nkx2.5. Cell lysates were immunoprecipitated with anti-FLAG Ab, and immunoprecipitants were blotted with anti-HA Ab to detect coimmunoprecipitated HA-Csx/Nkx2.5. Two transfectants expressing either FLAG-Csx/Nkx2.5 (lane 2) or HA-Csx/Nkx2.5 (lane 3) were also analyzed as controls.



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Fig. 3.   Csx/Nkx2.5 in the nuclear extract homodimerizes on DNA. A, endogenous Csx/Nkx2.5 protein from rat neonatal cardiac myocytes forms monomers (M) as well as dimers (D) at -242 bp site (lane 1). These bands correspond to the bands shifted by the nuclear extract from adenoviral infected rat cardiac myocytes expressing FLAG-tagged Csx/Nkx2.5 (lanes 2-4). The amounts of nuclear extracts used are shown. B, Western blot of the protein-DNA complex with anti-Csx/Nkx2.5 mAb. Endogenous Csx/Nkx2.5 (lane 1) was detected as monomer and dimer forms on DNA. Higher expression of Csx/Nkx2.5 was detected in adenoviral infected cardiac myocytes (lane 2). The amounts of nuclear extracts used are shown. C, EMSA of nuclear extracts from adenoviral infected cardiac myocytes. Cardiac myocytes were infected with adenoviral constructs encoding either FLAG-tagged Csx/Nkx2.5 (lane 1) or HA-tagged Csx/Nkx2.5 (lane 3) and mixture of two constructs (lane 2). D, protein-DNA complexes were transferred to a membrane followed by Western blot analysis. In the presence of DNA (lanes 1-3), FLAG-tagged Csx/Nkx2.5 or HA-tagged Csx/Nkx2.5 protein migrated faster than that without DNA (* in lanes 4-6) and was detected in the monomeric (M) or dimeric (D) protein-DNA complex. Both FLAG-tagged and HA-tagged Csx/Nkx2.5 were detected in the dimeric protein-DNA complex (lane 2) when both proteins were coexpressed in cardiac myocytes.

Homodimerization of Endogenous Csx/Nkx2.5 on the ANF -242 Site-- To examine whether endogenous Csx/Nkx2.5 homodimerizes on the ANF -242 site, nuclear extracts prepared from neonatal rat cardiac myocytes were used for EMSA. As shown in Fig. 3A, lane 1, endogenous Csx/Nkx2.5 forms monomers (M) as well as dimers (D). These two bands corresponded to the bands shifted by the nuclear extract from adenovirus infected rat cardiac myocytes that expressed FLAG-tagged Csx/Nkx2.5 (Fig. 3A, lanes 2-4). Because of the high expression levels of Csx/Nkx2.5 in the adenoviral vector-infected cardiac myocytes, 20-fold dilution of the nuclear extracts was required to shift the DNA probe to a similar level compared with that of the endogenous Csx/Nkx2.5 (Fig. 3A, lane 1 versus lane 3). When the protein-DNA complex was transferred to the membrane and blotted with anti-Csx/Nkx2.5 Ab, we detected the signal at the monomeric (M) and dimeric (D) protein-DNA complex both in the uninfected and the virus infected nuclear extracts (Fig. 3B).

To ascertain whether Csx/Nkx2.5 protein forms dimers with DNA in cardiac myocytes, adenovirus-encoding FLAG-tagged Csx/Nkx2.5 and/or HA-tagged Csx/Nkx2.5 were coinfected into cardiac myocytes, and the nuclear extracts were mixed with the DNA probe for EMSA analysis (Fig. 3C). The protein-DNA complex was transferred to a membrane followed by Western blot analysis (Fig. 3D). We detected the signal at the monomeric (M) and dimeric (D) protein-DNA complex, similar to Fig. 3B. Additional slow migrating bands observed in these experiments (* in Fig. 3D) corresponded to Csx/Nkx2.5 protein unbound DNA (lanes 4-6 in Fig. 3D). Both FLAG-tagged and HA-tagged Csx/Nkx2.5 were detected at the dimeric protein-DNA complex when both proteins were coexpressed in cardiac myocytes (Fig. 3D, lane 2), suggesting that two Csx/Nkx2.5 molecules homodimerize on DNA.

Lys193-Arg194 within the HD Is Required for Dimerization-- To confirm the specificity and to identify the regions that are required for dimerization, we mapped the dimerization domain of Csx/Nkx2.5 using in vitro binding assays. Initially, four [35S]methionine-labeled COOH-terminal deletion mutants were mixed with MBP-HD (Fig. 4A). Two COOH-terminal deletion mutants of Csx/Nkx2.5, 1-230 and 1-199, associated with MBP-HD, whereas the further deletions to 1-159 or 1-149 abolished the association (Fig. 4B, top panel). These results indicate that amino acids between 159 and 199 are necessary for dimerization. Next, 5 amino acid serial deletion mutants from the carboxyl terminus of HD, 1-196, 1-191, 1-186, and 1-181, were examined. The 1-196 protein interacted with the HD, but the 1-191, 1-186, and 1-181 proteins did not (Fig. 4B, middle panel). Further single amino acid deletions revealed that 1-193 dramatically reduced the interaction, and 1-192 completely abolished the interaction (Fig. 4B, bottom panel). Therefore, two basic amino acids, Lys193 and Arg194 are necessary for the interaction with the HD.



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Fig. 4.   Lys193 and Arg194 are required for homodimerization with HD. A, schematics of deletion mutants used for the in vitro binding assays and summarized results. B, various 35S-labeled Csx/Nkx2.5 deletion mutant proteins were mixed with MBP-HD protein, and bound proteins were resolved on SDS-PAGE and autoradiographed. Bound 35S-labeled Csx/Nkx2.5 mutants to HD or 50% input of 35S-labeled protein are shown. Top panel shows that the deletion between aa 199 and aa 159 abolished the association. Middle panel shows the 5 amino acid deletion series from the carboxyl terminus of HD. Amino acids between aa 196 and aa 191 are necessary for the association. Bottom panel shows single amino acid deletions between aa 196 and aa 191. 1-195 and 1-194 associated with HD; however, 1-193 markedly reduced the association, and 1-192 further decreased the association. C, two basic amino acids Lys193 and Arg194 were mutated into Ile or Asp and examined for protein-protein interactions with MBP-HD and MBP-full-length Csx/Nkx2.5. Bound 35S-labeled Csx/Nkx2.5 mutants to HD or full-length Csx/Nkx2.5 or 50% input of 35S-labeled protein are shown on the upper panels, and MBP-HD and MBP-full-length Csx/Nkx2.5 protein used for the assay are shown on the bottom panels.

We further mutated Lys193 and Arg194 into neutral or acidic amino acids (Lys193 to Ile, Arg193 to Ile, and Lys193-Arg194 to Ile193-Asp194) and examined them for dimerization with the HD as well as with full-length Csx/Nkx2.5 (Fig. 4C). The Lys193 right-arrow Ile mutant markedly reduced the interaction with the HD, and an ~50% reduction was observed in Arg194 right-arrow Ile mutant. The interaction with the HD was undetectable when both amino acids were mutated (Fig. 4C, HD). However, we still detected a weak interaction between Lys193-Arg194 mutant and full-length Csx/Nkx2.5 (Fig. 4C, Full). These findings confirm that two amino acids Lys193 and Arg194, are required for the dimerization of the HD, and additional protein domain(s) outside of HD are also likely to be involved in dimerization.

Involvement of the Region(s) Outside of HD for Dimerization-- To identify the domain(s) outside of the HD of Csx/Nkx2.5 that are involved in homodimerization on DNA, we examined DNA binding affinity of HD and full-length protein on the palindromic ANF -242 site or a mutated monomeric ANF -242 site shown in Fig. 1. The HD protein bound DNA predominantly as a monomer at a low protein concentration (Fig. 5A, panel a, lanes 1-3) and dimerized more at a higher protein concentration (Fig. 5A, panel a, lanes 5 and 6). The monomer to dimer transition was observed between lanes 4 and 5 at a protein concentration of 0.91-2.7 × 10-8 M in the HD protein (arrow in Fig. 5A, panel a). However, with the full-length Csx/Nkx2.5 (Fig. 5A, panel b), the monomer-dimer transition occurred more abruptly between lane 2 and lane 3 (protein concentration 0.71-2.1 × 10-9 M). We tested and confirmed that HD and full-length protein bound to the mutated monomeric site with similar affinity (Kd = 1.0-3.0 × 10-9 M for HD; 0.71-2.1 × 10-9 M for full-length Csx/Nkx2.5) (Fig. 5A, panels c and d).



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Fig. 5.   Regions outside the HD facilitate homodimerization on DNA. A, HD and full-length Csx/Nkx2.5 were analyzed for DNA binding using palindromic ANF -242 site (panels a and b) and mutated monomeric site (panels c and d). Panel a, HD protein bound ANF -242 site predominantly as a monomer in low protein concentrations and gradually transitioned into a dimer. An equivalent monomer-dimer ratio was observed between lanes 4 and 5 in the HD (approximate protein concentration, 0.91-2.7 × 10-8 M). Panel b, with full-length protein, the monomer to dimer transition occurred rather abruptly between lanes 2 and 3 (protein concentration, 0.71-2.1 × 10-9 M). HD (panel c) and full-length Csx/Nkx2.5 (panel d) bound a mutated monomeric binding sites with similar affinity (Kd = 1.0-3.0 × 10-9 M for HD; Kd = 0.71-2.1 × 10-9 M for full-length Csx/Nkx2.5). B, in the COOH-terminal deletion mutant (panel a), the transition from monomer to dimer occurred between lanes 4 and 5, similar to HD (protein concentration, 0.66-2.1 × 10-8 M). In the amino-terminal deletion (panel b), the transition occurred between lanes 3 and 4 (protein concentration, 2.4-7.4 × 10-9 M). 3-Fold serial increases in protein concentration (0.018-4.4 µg/ml) were used. M, monomer; D, dimer; F, free probe.

To further examine the regions responsible for dimeric DNA binding, we constructed two deletion mutants, a carboxyl terminus deletion mutant () and an amino-terminal deletion mutant () and examined their DNA binding on the ANF -242 site (Fig. 5B). In the COOH-terminal deletion mutant (Fig. 5B, panel a), the monomer-dimer transition was observed between lanes 4 and 5 (0.66-2.0 × 10-8 M), which was similar to that of HD protein. The amino-terminal deletion showed the monomer-dimer transition between lanes 3 and 4 (2.4-7.3 × 10-9 M) (Fig. 4B, panel b); therefore it required 3-fold lower protein concentration than that of the HD or the carboxyl terminus deletion, but still required 3-fold higher protein concentration than that of the full-length protein. Taken together, although the HD and full-length Csx/Nkx2.5 binds the monomeric DNA binding site with a similar affinity, full-length Csx/Nkx2.5 preferentially forms dimers at ~13-fold lower protein concentration than the HD alone. Thus, regions outside of the HD, particularly the COOH-terminal region of Csx/Nkx2.5, seem to facilitate protein-protein interactions involved in the dimerization on DNA.

Lys193 Is Necessary for Association with GATA4-- We and others have reported that Csx/Nkx2.5 interacts with the transcription factor GATA4 (42-45). It was demonstrated that the second zinc finger of GATA4 is involved in the specific interaction with the HD of Csx/Nkx2.5, and amino acids between 182 and 199 are responsible for the direct interaction with GATA4 (43). Our data presented in Fig. 4 revealed that this domain is also responsible for homodimerization. Therefore, we examined whether GATA4 associates with Lys193-Arg194 mutants (Fig. 6A). As shown in Fig. 6A, lane 1, the wild type Csx/Nkx2.5 (1-318) associated with GATA4-GST protein, whereas Lys193 right-arrow Ile (lane 2) and Lys193 right-arrow Ile/Arg194 right-arrow Asp (lane 4) mutants abolished the interaction. Interestingly, the Arg194 right-arrow Ile mutant (lane 3) associated with GATA4 with an apparent higher affinity than wild type Csx/Nkx2.5, in contrast to its lower homodimerization ability (Fig. 4C). These data demonstrate that Lys193 in the HD of Csx/Nkx2.5, which is critical for homodimerization, is also essential for the interaction with GATA4.



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Fig. 6.   Protein interactions of Csx/Nkx2.5 between GATA4, Nkx2.3, and Nkx2.6/Tix: Lys193 is required for interaction with GATA4. A, 35S-labeled wild type, Lys193, Arg194, and Lys193-Arg194 mutants were mixed with GST-GATA4 (lanes 1-4) or GST protein (lanes 5-8) to examine whether Csx/Nkx2.5 Lys193-Arg194 mutations affect the protein interaction with GATA4. Bound proteins were resolved on SDS-PAGE. Wild type (lane 1), Arg194 mutant (lane 3) interacted with GATA4, whereas Lys193 (lane 2) and Lys193-Arg194 mutant (lane 4) did not interact. B, 35S-labeled Csx/Nkx2.5 (lanes 1 and 4), Nkx2.3 (lanes 2 and 5), and Nkx2.6 (lanes 3 and 6) were mixed with MBP-full-length Csx/Nkx2.5 (lanes 1-3) and MBP fusion proteins (lanes 4-6) to detect heterodimers. 50% input of proteins is also shown (lanes 7-9).

Heterodimerization of Csx/Nkx2.5 with Other NK2 Class HD Proteins-- Since Xenopus XNkx2.3 and XNkx2.5 are coexpressed in the heart, and Csx/Nkx2.5 and Nkx2.6/Tix are coexpressed in restricted areas in the mouse heart (31, 32, 41), we examined the potential interaction of Csx/Nkx2.5 with Nkx2.3 and Nkx2.6/Tix by using in vitro binding assay. As shown in Fig. 6B, full-length Csx/Nkx2.5 associated with Csx/Nkx2.5 as well as Nkx2.6/Tix, and weakly with Nkx2.3. Therefore, Csx/Nkx2.5 demonstrates the potential to interact with other NK2 class proteins with varying binding affinity depending on the partner.

Generation of an Inhibitory Mutant-- To examine the effects of protein dimerization on transcriptional activity, we attempted to create a mutant protein that does not dimerize, but does bind, DNA. As shown in Fig. 4, we constructed Csx/Nkx2.5 mutants that do not dimerize to the HD. We next examined the DNA binding of these mutants using ANF -242 site and found that the Lys193 right-arrow Ile mutant completely abolished DNA binding (Fig. 6A). Lys193-Arg194 mutant bound DNA, but the binding affinity was significantly lower than that of wild type Csx/Nkx2.5 (Fig. 7A).



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Fig. 7.   A non-DNA binding mutant with preserved homodimerization acts in an inhibitory manner in vitro. A, two mutants with protein dimerization defects were examined for DNA binding. EMSA shows Lys193 right-arrow Ile completely abolished DNA binding (middle panel), and Lys193 right-arrow Ile/Arg194 right-arrow Asp significantly decreased DNA binding (right panel) compared with wild type (left). B, an Ile183 right-arrow Pro mutant was created by mutating Ile183 into Pro in the third helix of HD (see Fig. 3A). EMSA shows Ile183 right-arrow Pro completely abolished DNA binding (right panel). C, protein dimerization of wild type (W) and Ile183 right-arrow Pro mutant (IP). 35S-Labeled wild type (W) (lanes 1, 3, 5, 7) or Ile183 right-arrow Pro mutant (IP) protein (lanes 2, 4, 6, 8) were mixed with MBP-wild type Csx/Nkx2.5 (lanes 1 and 2), MBP (lanes 3 and 4), GATA4-GST (lanes 5 and 6), or GST fusion protein (lanes 7 and 8). Complexes were resolved by SDS-PAGE and autoradiographed. The Ile183 right-arrow Pro mutant associated with MBP-Csx/Nkx2.5 protein (lane 2) similar to wild type Csx/Nkx2.5 (lane 1). The association between Ile183 right-arrow Pro mutant and GATA4 (lane 6) was markedly reduced compared with wild type (lane 5). D, inhibition of Csx/Nkx2.5-dependent transactivation in the presence of increasing amount of Ile183 right-arrow Pro expression plasmid. 10T1/2 cells were transiently transfected with 0.4 µg of pcDNA3-wild type Csx/Nkx2.5 expression plasmid, ANF-Luc, and RSV-beta -GAL plasmid and the indicated amount of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) expression plasmid. Approximately 44% reduction in transactivation was seen upon transfection with 0.4 µg, and ~53% reduction with 0.8 µg of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) (left panel). The right panel shows the inhibitory effect of Ile183 right-arrow Pro in the presence of GATA4 expression vector. Cotransfection with GATA4 plasmid increased ANF-Luc activity as reported previously (43). The Ile183 right-arrow Pro mutant reduced ANF-Luc by ~20% at a 1:1 ratio (0.4 µg) and by 44% at a 2:1 ratio (0.8 µg). Values are means ± S.E.

As an alternative to examine the effect of protein dimerization, we generated a converse mutant in which protein dimerization is preserved, but DNA binding is abolished. By mutating Ile183 in the third helix of HD into Pro, DNA binding of Csx/Nkx2.5(Ile183 right-arrow Pro) mutant was completely abolished (Fig. 7B), but this mutant associated with MBP-Csx/Nkx2.5 protein with a similar affinity as that of wild type protein (Fig. 7C, lane 1 versus lane 2). In contrast, this Ile183 right-arrow Pro mutant markedly reduced the interaction with GATA4 (Fig. 7C, lane 6).

We tested the function of the Ile183 right-arrow Pro mutant by transient transfection assays in 10T1/2 fibroblasts using ANF(-638)-luciferase reporter construct (ANF-Luc), which includes the -87 and -242 bp sites shown in Fig. 1A. The Ile183 right-arrow Pro mutant did not bind DNA (Fig. 7B), and the mutant itself did not activate or repress the ANF-Luc (data not shown). When we cotransfected the expression plasmid encoding Ile183 right-arrow Pro mutant protein with wild type Csx/Nkx2.5 at 1:1 ratio (0.4 µg), the luciferase activity of wild type Csx/Nkx2.5 decreased by ~44%. A slight further reduction (~53%) of ANF-Luc activity was observed when the Ile183 right-arrow Pro expression plasmid was increased to 2:1 (0.8 µg) (Fig. 7D). In the presence of GATA4 expression plasmid, we observed a further increase of ANF-Luc activity from the ANF promoter as reported previously (43). We found that the Ile183 right-arrow Pro mutant reduced luciferase activity by ~20% at 1:1 ratio of plasmid amount and by ~44% at 2:1 ratio. These data demonstrate that the non-DNA binding mutant, Ile183 right-arrow Pro, acts in an inhibitory manner on wild type Csx/Nkx2.5 in transient transfection assays in 10T1/2 cells.

Ile183 right-arrow Pro Mutant Does Not Inhibit the Csx/Nkx2.5-dependent ANF Promoter Activation in Neonatal Cardiac Myocytes-- We further examined the inhibitory effect of the Ile183 right-arrow Pro mutant on endogenous Csx/Nkx2.5 as well as wild type Csx/Nkx2.5 in cultured neonatal cardiac myocytes. In rat neonatal cardiac myocytes, the base-line ANF-Luc activity was high. When we used the LipofectAMINE transfection method, the base-line ANF-Luc activity was approximately the same as that detected in 10T1/2 cells transfected with the wild type Csx/Nkx2.5 expression plasmid. ANF-Luc activation was suppressed by the cotransfection of Ile183 right-arrow Pro expression plasmid by 29%. (Fig. 8A, 183I-P). When we cotransfected the wild type Csx/Nkx2.5 expression plasmid, ANF-Luc activity was increased by 50% (Fig. 8A, Wild). However, cotransfection of Ile183 right-arrow Pro expression plasmid did not inhibit the wild type Csx/Nkx2.5 function (Fig. 8A, 183I-P+Wild). Similar results were obtained using the calcium phosphate methods (data not shown).



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Fig. 8.   Ile183 right-arrow Pro mutant does not inhibit coexpressed wild type Csx/Nkx2.5 function in cardiac myocytes. A, rat neonatal cardiac myocytes cultured in six-well plates were cotransfected with 1.8 µg of ANF(-638)-Luc, 0.6 µg of MSV-beta -GAL, 0.75 µg of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) and/or 0.75 µg of pcDNA3-Csx/Nkx2.5(Wild) using 10 µl of LipofectAMINE 2000 reagents. pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) reduced ANF-Luc activation by 29%; in contrast pcDNA3-Csx/Nkx2.5 increased the activation by 50% (Wild). Cotransfection of pcDNA3-Csx/Nkx2.5(Ile183 right-arrow Pro) with pcDNA3-Csx/Nkx2.5(Wild) did not reduce ANF-Luc activation (183I-P+Wild). Total plasmid amount was adjusted to 4.65 µg with pcDNA3 vector plasmid. Values are means ± S.E. B, adenovirus encoding either Csx/Nkx2.5(Ile183 right-arrow Pro) or wild type Csx/Nkx2.5 or empty vector were transfected and stained with anti-FLAG Ab. In most of the cardiac myocytes infected with either Csx/Nkx2.5(Ile183-Pro) mutant or wild type Csx/Nkx2.5 adenoviral vector, FLAG staining was colocalized with nuclear staining (arrows in b, c, e, and f). A few cells without FLAG staining were observed (* in b, c, e, and f). The virus titer was between 5 and 15 multiplicity of infection in each construct Bars = 20 µm. C, Western blot of cardiac myocyte cell lysates expressing Csx/Nkx2.5(Ile183 right-arrow Pro, lane 2), wild type Csx/Nkx2.5 (Wild, lane 3) and both (183I-P+Wild, lane 4). D, the next day of adenoviral infection, 2.4 µg of ANF(-638)-Luc reporter and 0.6 µg of MSV-beta -GAL were cotransfected using the calcium phosphate method for 2 h, and luciferase and beta -galactosidase activity were measured after 48 h. When the Ile183 right-arrow Pro mutant protein was expressed, ANF-Luc activity was suppressed by 31% (183I-P). In contrast, wild type Csx/Nkx2.5 activated the ANF-Luc reporter by 250% (Wild), which was not suppressed by Ile183 right-arrow Pro mutant (183I-P+Wild). Values are means ± S.E. E, DNA binding affinity of wild type Csx/Nkx2.5 expressed by adenoviral vector was not modified by coexpressed Ile183 right-arrow Pro mutant protein. EMSA of nuclear extracts from adenoviral infected cardiac myocytes expressing wild type Csx/Nkx2.5 alone (panel a) or coexpressing wild type and Ile183 right-arrow Pro mutant proteins (panel b). 3-Fold serial increases in protein concentration (0.015-0.13 µg/µl) were used. M, monomer; D, dimer; F, free probe.

Transfection efficiency of primary cardiac myocytes is known to be very low when plasmid vectors are used. Therefore, we infected cardiac myocytes with adenoviral vectors, which exhibit a high efficiency of gene transfer. More than 90% of cardiac myocytes expressed either Ile183 right-arrow Pro mutant or wild type Csx/Nkx2.5 (Fig. 8B), and each construct expressed a similar protein amount determined by Western blotting (Fig. 8C). Twenty-four hours after adenovirus infection, ANF-Luc reporter gene was transfected into cardiac myocytes, and the transcriptional activation was measured by luciferase activity. When Ile183 right-arrow Pro mutant protein was expressed by the adenoviral vector, ANF-Luc activity was suppressed by 31% (Fig. 8D, 183I-P). In contrast, wild type Csx/Nkx2.5 activated the ANF-Luc reporter by 3.5-fold (Fig. 8D, Wild), which was not suppressed by coexpression of Ile183 right-arrow Pro mutant (Fig. 8D, 183I-P+Wild). When we examined DNA binding of the nuclear extract from cardiac myocytes expressing wild type alone (Fig. 8E, panel a) or wild type with Ile183 right-arrow Pro mutant (Fig. 8E, panel b), there was no significant difference in DNA binding of wild type Csx/Nkx2.5 either as monomers or dimers. These data indicate that the expression of Ile183 right-arrow Pro mutant weakly suppresses ANF-Luc activity in cardiac myocytes; however, Ile183 right-arrow Pro mutant does not suppresses ANF-Luc activity or reduce DNA binding of wild type Csx/Nkx2.5 when coexpressed with wild type Csx/Nkx2.5 (see "Discussion").


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The NK2 class homeobox-containing transcription factor Csx/Nkx2.5 is one of the earliest cardiogenic markers from insects to vertebrates (28, 31, 34, 38, 39, 57, 58). Recently, human CSX/NKX2.5 mutations were identified in patients with congenital heart disease (22, 23). These patients show progressive conduction delays and in some cases left ventricular dysfunction after birth, suggesting that Csx/Nkx2.5 also functions in the later stages of heart development and maturation. While evidence is accumulating that Csx/Nkx2.5 mutations inhibit normal cardiac development and maturation in both humans and Xenopus (22, 23, 25), the molecular mechanisms for these phenotypes remain to be explained. In this study, we report the new finding of protein dimerization of Csx/Nkx2.5, which may yield insights into the dominant effects of CSX/NKX2.5 mutations found in humans.

Csx/Nkx2.5 binds to the palindromic Csx/Nkx2.5 consensus binding sites in the ANF promoter as a monomer as well as a dimer, and dimer formation increases the protein-DNA binding affinity. Csx/Nkx2.5 physically interacts with each other in vitro as well as in a cell. Two basic amino acids, Lys193-Arg194, located in the third helix of HD are necessary for dimerization, and Lys193 is also indispensable for the association with GATA4, which is a cofactor for Csx/Nkx2.5 function. To examine the functional significance of dimerization, we generated a converse mutant (Ile183 right-arrow Pro) that does not bind DNA, but has preserved homodimerization ability. In transient transfection assays in 10T1/2 fibroblast cells, this mutant acts in an inhibitory manner on wild type Csx/Nkx2.5. Also, this mutant suppresses the ANF-Luc activity in cardiac myocytes, but does not inhibit the ANF-Luc activity that is further induced by wild type Csx/Nkx2.5.

Protein Dimerization of Csx/Nkx2.5 through the HD-- By using various deletion and point mutants, we found that two positively charged amino acids, Lys193-Arg194 at the COOH-terminal end of HD (Lys57-Arg58 in HD), are critical for protein-protein interaction. As expected, these two amino acids were highly conserved within NK2 class HD (29). It was shown that three amino acids (Arg58-Val59-Lys60) located at the carboxyl end of the HD of Pit1 are involved in the homodimerization on DNA by forming a protein-protein interface with the POU-specific domain (15). In contrast to Csx/Nkx2.5, Pit1 requires DNA to homodimerize (59).

The Lys193-Arg194 mutation, located at the carboxyl end of the HD in Csx/Nkx2.5, markedly reduced DNA binding, which is consistent with the NMR structure of another NK2 class HD protein Drosophila NK-2. The third alpha -helix (helix III) of NK-2 extends up to amino acid 62 in the presence of DNA (52, 60). We demonstrated that Lys193 is required for the Csx/Nkx2.5 and GATA4 interaction as well as for homodimerization of Csx/Nkx2.5.

Regions Outside the HD Facilitate Dimerization on DNA-- Cooperative dimerization of HD proteins has been characterized in paired and paired-like HD proteins (11). Paired HD proteins cooperatively bind DNA with a palindromic TAAT sequence separated by 3 bp. The presence of Arg28 or Arg43 prevents cooperative dimerization, and paired class HD proteins do not have Arg residues at the 28 and 43 positions (11). In contrast, 50% of HD proteins have conserved Arg28 or Arg43 residues among ~350 HD proteins (53). NK class HD proteins, as well as engrailed, bcd, POU, and msh class proteins do not have Arg28 or Arg43, suggesting the possibilities for cooperative dimerization in these classes of HD proteins.

In Csx/Nkx2.5, regions outside of the HD, particularly the region carboxyl terminus to the HD (aa 251-318), appear to facilitate cooperative dimerization on DNA. Compared with the DNA binding of HD of Csx/Nkx2.5 or the COOH-terminal deletion mutant, we found that the full-length protein facilitated dimerization, and the monomer-dimer transition occurred at ~13-fold lower protein concentrations than that of the HD or the carboxyl terminus deletion mutant. Our observation that Csx/Nkx2.5 homodimerizes through the HD as well as outside of the HD supports the hypothesis that protein-protein interactions play important roles in cooperative dimerization. Alternatively, a full-length Csx/Nkx2.5 molecule may "bend" DNA to facilitate the binding of a second Csx/Nkx2.5 molecule to DNA, or full-length Csx/Nkx2.5 proteins may be more stable than HD proteins in a dimerized form on DNA. It is also possible that these effects may function cooperatively to regulate the transcriptional activation of target genes.

Palindromic NK2-specific binding sites are also identified in the enhancer of the Drosophila ind gene, which is the target of another NK2 class HD protein Vnd (61). Also, Tinman, the Drosophila homologue of Csx/Nkx2.5, binds the sequence located at -5.4 kilobases of the Dmef2 gene, which contains two NK2 binding sites separated by 165 bp (62). Mutations that disrupt either one of two Tinman binding sites caused loss of activation of the Dmef2 gene, leading to the hypothesis that the physical interaction of Tinman molecules occurs by looping of the 165-bp intervening segment (62). Although it has yet to be shown that Tinman protein homodimerizes, our data are consistent with this hypothesis.

Heterodimerization with Other NK2 Class Proteins-- Several NK2 class HD proteins are coexpressed both temporally and spatially, suggesting that NK2 class HD proteins may heterodimerize. As shown in Fig. 6, Csx/Nkx2.5 and Nkx2.6/Tix associated with each other, but the association of Csx/Nkx2.5 and Nkx2.3 was significantly weaker. Although further quantitative analyses are necessary, these results suggest that NK2 class HD proteins potentially interact with each other, and the affinity of the interaction is different depending on heterodimer pairs. In this study, we did not examine the possible heterodimerization of Csx/Nkx2.5 with other classes of HD proteins. Of note, mouse Nkx2.3 is not expressed in the heart, and Nkx2.6/Tix expression is restricted to the sinus venosa and outflow tract of the mouse heart. Other NK2 class HD proteins coexpressed temporally and spatially similar to Csx/Nkx2.5 in the heart have not been described in mouse and human (29, 41, 63).

The Effect of Non-DNA Binding Mutant on Wild Type Csx/Nkx2.5-- Based on the studies of phenotypes caused by the non-DNA binding mutants of Csx/Nkx2.5 in patients and Xenopus (22-25), and on the evidence for the formation of homodimers of Csx/Nkx2.5 in our study, non-DNA binding mutants might act in a dominant inhibitory manner. We generated a single missense mutation in the third helix of the HD, Csx/Nkx2.5(Ile183 right-arrow Pro), which abolishes DNA binding, but preserves dimerization ability. The interaction between GATA4 and Csx/Nkx2.5(Ile183 right-arrow Pro) was significantly weaker than that of wild type Csx/Nkx2.5; therefore, it is likely that Csx/Nkx2.5(Ile183 right-arrow Pro) will not sequester GATA4 from wild type Csx/Nkx2.5 when it is overexpressed. The transcriptional activation of wild type Csx/Nkx2.5 on ANF(-638) promoter was indeed suppressed by the coexpressed Csx/Nkx2.5(Ile183 right-arrow Pro) in a dose-dependent manner in 10T1/2 cells.

ANF(-638) promoter activity was slightly suppressed by Csx/Nkx2.5(Ile183 right-arrow Pro) in neonatal cardiac myocytes where endogenous Csx/Nkx2.5 is expressed. However, when both wild type and Csx/Nkx2.5(Ile183 right-arrow Pro) mutants were overexpressed in cardiac myocytes, transcriptional activation by wild type was not suppressed by the Csx/Nkx2.5(Ile183 right-arrow Pro) mutant. Thus, unlike in 10T1/2 cells, Csx/Nkx2.5(Ile183 right-arrow Pro) does not seem to act as a typical dominant inhibitory mutant on the ANF(-638) promoter in cultured cardiac myocytes. The EMSA using cell lysates prepared from adenovirus-infected cardiac myocytes revealed that coexpression of Csx/Nkx2.5(Ile183 right-arrow Pro) mutant does not inhibit the specific binding of wild type Csx/Nkx2.5 to the ANF -242 site (Fig. 8E). Since the Csx/Nkx2.5(Ile183 right-arrow Pro) mutant expressed in cardiac myocytes did not bind to the ANF -242 site (data not shown), it is possible that Csx/Nkx2.5(Ile183 right-arrow Pro) mutant loses the ability to form dimers with wild type Csx/Nkx2.5 on DNA and, therefore, does not inhibit the function of wild type Csx/Nkx2.5 on the ANF promoter in cardiac myocytes. It is also possible that the inhibitory effect of Csx/Nkx2.5(Ile183 right-arrow Pro) observed in 10T1/2 cells may occur through a mechanism independent of wild type Csx/Nkx2.5. Mutant Csx/Nkx2.5 protein may squelch a transcription factor that is critical for the ANF(-638) promoter activity in 10T1/2 cells.

Protein homodimerization of Csx/Nkx2.5 yields the potential for it to precisely regulate a number of genes by utilizing monomeric and dimeric binding. It is possible that the genetically dominant effect of the human CSX/NKX2.5 missense mutations (22-24) may in part be due to an inhibitory effect of the mutant protein over the wild type protein on target genes that require dimeric binding.


    ACKNOWLEDGEMENTS

We thank E. O. Weinberg for critical reading of the manuscript; S. Hardy, T. Shioi, and A. Horwitz for valuable suggestions; H. Liao for Tix/Nkx2.6 plasmid; M. Schinke for Nkx2.3 plasmid; and B. Lee and J. Hampe for technical help.


    FOOTNOTES

* This work was supported by the Charles H. Foundation and American Heart Association Massachusetts Affiliate Fellowship and Beginning Grant-in-aid (to H. K.), an American Heart Association National Grant (to A. U.), and by National Institutes of Health (NIH) Grant R01-HL51253 and a Specialized Center for Research in Atherosclerosis in Congenital Heart Disease grant from NIH Grant P50-HL61036 (to S. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed: Beth Israel Deaconess Medical Center, 330 Brookline Ave. SL215, Boston, MA 02215. Tel.: 617-667-4862; Fax: 617-975-5268; E-mail: hkasahar@caregroup.harvard.edu.

Dagger Dagger To whom correspondence may be addressed: Beth Israel Deaconess Medical Center, 330 Brookline Ave. SL201, Boston, MA 02215. Tel.: 617-667-4858; Fax: 617-975-5268; E-mail: sizumo@caregroup.harvard.edu.

Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M004995200


    ABBREVIATIONS

The abbreviations used are: HD, homeodomain; bp, base pair(s); ANF, atrial natriuretic factor; HA, hemagglutinin; PCR, polymerase chain reaction; MBP, maltose-binding protein; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; Ab, antibody; mAb, monoclonal antibody; aa, amino acid(s); GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MSV, murine sarcoma virus.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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