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Cardiovascular Research 2004 64(1):40-51; doi:10.1016/j.cardiores.2004.06.004
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Copyright © 2004, European Society of Cardiology

Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies

Hideko Kasahara*,a and D. Woodrow Benson*,b

aDepartment of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32610, USA
bCardiology Division, Cincinnati Children's Hospital Medical Center, Division of Cardiology ML7042, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

* Corresponding authors. D. Woodrow Benson is to be contacted at Tel.: +1-513-636-7716; fax: +1-513-636-5958. Hideko Kasahara is to be contacted at Tel.: +1-352-846-1503; fax: +1-352-846-0270. E-mail address: hkasahar{at}phys.med.ufl.edu, woody.benson{at}cchmc.org

Received 24 March 2004; revised 29 May 2004; accepted 2 June 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: There has been considerable interest in understanding determinants of the diverse cardiac phenotypes associated with heterozygous NKX2.5 mutations. We hypothesized that analysis of functional properties of NKX2.5 mutant proteins would result in the ability to classify mutations according to function in a scheme that would help to clarify genotype–phenotype correlations. We analyzed missense mutations in the conserved homeodomain. Methods: We studied in vitro biochemical characteristics, including nuclear localization, DNA binding, transcriptional activation and protein–protein interaction with transcriptional partners (GATA4, TBX5 and NKX2.5 itself), of eight homeodomain missense mutations. Associated phenotypes include atrioventricular (AV) block (98% penetrance), atrial septal defect (83% penetrance), and additional varied heart malformations. Results: Mutations were present at varied homeodomain locations in the putative nuclear localizing signal (1), helix 2 (1), a turn between helix 2 and 3 (1) and helix 3 (5); a spectrum of biochemical phenotypes was observed. All mutants localized to the nuclei but some exhibited anomalous nuclear distribution. While all mutants exhibited markedly decreased DNA binding and reduced transcriptional activation, interaction with transcriptional partners was varied. Conclusion: Each mutant protein had a unique spectrum of observed properties, but our data show that while dominant negative properties could be demonstrated in vitro, the best correlation with clinical phenotypes resulted from the markedly reduced DNA binding shared by all eight homeodomain mutations. This suggests that the principle determinant of the two most common phenotypes associated with homeodomain missense mutations is the total dose of NKX2.5 capable of binding to DNA.

KEYWORDS Conduction (block); Congenital defects

This article is referred to in the Editorial by C. Mittmann (pages 1–2) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Heterozygous NKX2.5 mutations have been associated with atrioventricular (AV) conduction block and varied defects in atrial, ventricular and conotruncal septation and AV valve formation. To date, 26 mutations have been reported [1–8]. In addition to DNA binding, the NKX2.5 homeodomain plays critical roles in transcriptional regulation by nuclear translocation of NKX2.5 protein [9] and protein–protein interaction with other transcription factors [10–17]. Mutant proteins that fail to bind DNA and also fail to interact with transcriptional cofactors result in haploinsufficiency, while mutants that fail to bind DNA yet continue to interact with transcriptional partners may have broader functional consequences including dominant negative effects. In mice, heterozygous loss-of-function Nkx2.5 mutations (haploinsufficiency) lead to a mild AV block and atrial septal dysmorphogenesis including ASD [18]. The higher penetrance of conduction abnormalities and greater diversity of cardiac malformations in humans with NKX2.5 mutations, as compared with heterozygous mutant mice, may reflect genetic background or the dominant negative nature of some human mutations [19].

To better define the spectrum of biochemical properties modified by NKX2.5 mutations, we studied structure–function relations in eight homeodomain missense mutations, selected in part because of a large body of prior work characterizing the homeodomain, a conserved DNA binding motif consisting of 60 amino acids and forming three {alpha}-helices [20,21]. Residues in the N-terminal arm preceding helix 1 establish specific contacts in the minor groove. Helix 1 is separated from helix 2 by a loop, which with helix 3, forms a helix–turn–helix motif. Helix 3, also known as the recognition helix, lies in the major groove establishing specific contact to bases and backbone phosphates. We hypothesized that the mutations modulate NKX2.5 biochemical properties in ways distinguishable from haploinsufficiency, and may be important determinants of variability of the resulting cardiac phenotypes.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Human subjects
The investigation conforms with the principles outlined in the Declaration of Helsinki. Informed consent was obtained from all participants in accordance with the Cincinnati Children's Hospital Medical Center Institutional Review Board. Participants were evaluated by medical history, physical examination and electrocardiogram (ECG). Reports of echocardiogram, cardiac catheterization, electrophysiology study, cardiac surgery and/or autopsy were reviewed when available. Clinical assessment was performed without knowledge of genotype.

2.2. Human molecular genetic studies
As previously described [2], NKX2.5 was used as a candidate gene in two kindreds where subjects in multiple generations had AV block and congenital heart disease. Using the mutation as an allele, we performed linkage analysis for each family; two-point logarithm of odds (LOD) scores were calculated using LIPED software assuming an allele frequency of 0.1% and disease penetrance of 95%.

2.3. Plasmid construct
The mutations we studied are designated by their position in both the homeodomain and NKX2.5 protein (in parenthesis). FLAG-epitope tagged NKX2.5 was inserted to pcDNA3 (Invitrogen) or pMALC2 (New England Biolabs) [19]. Mutations were introduced into both plasmids using Quick Change Site-Directed Mutagenesis Kit (Strategene) with appropriate primers: Arg5(142)Cys (F, 5'-AGGAAGCCGTGCGTGCTCTTC-3'; R, 5'-GAAGAGCACGCACGGCTTCCT-3'): Leu34(171)Pro (F, 5'-CGAACGCGACCAGCCGGCCAGCGTGCTGA-3';R, 5' -TCAGCACGCTGGCCGGCTGGTCGCGTTCG-3'): Gln50(187)His (F, 5'-TCTGGTTCCACAACCGGCGCT-3'; R, 5'-AGCGCCGGTTGTGGAACCAGA-3'): Arg53(190)His (F, 5'-GTTCCAGAACCGGCACTACAAGTGCAAGC-3' R, 5'-GCTTGCACTTGTAGTGCCGGTTCTGGAAC-3'). BamHI fragments of pcDNA3 plasmids encoding either wild-type or mutants of NKX2.5 were subcloned into BamHI site of GAL4-fusion plasmid pFA-CMV (Strategene) to generate pFA-CMV-NKX2.5 expression plasmid.

Partial mouse TBX5 cDNA including T-box (331–1334) was generated by RT-PCR (F: 5'-GCTAAGAGGAAGAGGGGCGG-3', R: 5'-GCAGAAGGTCCTGGGAGGG-3') using neonatal mouse heart total RNA and cloned into pCRTMII (Invitrogen). NcoI–EcoRI fragment was subcloned into pGEX-CD (provided by G.J.Nabel) to generate glutathione-S-transferase (GST)-TBX5. GST-GATA4 (provided by D.Wilson) and maltose-binding protein (MBP)-NKX2.5 were described previously [19]. HA-epitope tagged full-length human TBX5 was subcloned into pcDNA3 plasmids as follows: a BamHI–EcoRV fragment of full-length human TBX5 (provided by J. Seidman) was subcloned into pcDNA3. pcDNA3-TBX5 was PCR amplified with two primers (F: 5'-GGGATCCCCCACCATGTACCCATACGATGTTCCAGATTACGCTGCCGACGCAGACGAG-3', R: 5'-TAGGCTGGGCACAGGCTCGC-3') and subcloned into pCR2.1TMTOPOTM (Invitrogen). A BamHI–BmgBI fragment was removed from pcDNA3-TBX5 and a BamHI–BmgBI fragment from pCR2.1TMTOPOTM-TBX5 was subcloned into pcDNA3-TBX5 resulting in pcDNA3-HA-TBX5.

2.4. Electrophoretic mobility shift assay (EMSA)
We examined DNA binding of NKX2.5 mutants by EMSA using 32 bp of the atrial natriuretic factor (ANF) promoter [19]. This oligonucleotide with paired NKX2.5 binding sites was [32P]-labeled and mixed with wild-type or mutant protein purified from Escherichia coli as an MBP fusion protein (0.006–1.5 µg/ml of MBP-NKX2.5) followed by separation in 5% native polyacrylamide gel. Protein-DNA binding affinity (Kd) was estimated by the protein concentration at which 50% of the DNA probe became bound [22]. Molecular mass of MBP fusion protein (77 kDa) was estimated by addition of MBP (42 kDa) and wild-type NKX2.5 (35 kDa).

2.5. Reporter gene assays
We used the ANF(–638)-luciferase reporter construct (ANF-Luc, provided by K.R. Chien), containing multiple NKE sites, in transient transfection assays to examine the transcriptional activation function of NKX2.5 mutants. 10T1/2 cells cultured on six-well plates were co-transfected with 1.5 µg of ANF-Luc, 1 µg of empty pcDNA3 or pcDNA3 plasmids encoding wild-type or mutant NKX2.5, 0.5 µg of Rous sarcoma virus β-galactosidase plasmid (RSV-β-GAL, provided by B. Markham). The fold activation of luciferase activity is relative to activity in cells co-transfected with empty vector and ANF-Luc.

For co-transfection assays [19], 10T1/2 cells cultured on six-well plates were co-transfected with 2 µg of ANF-Luc, 0.4 µg of pcDNA3 expression vectors encoding mutant NKX2.5, 0.5 µg of RSV-β-GAL in the presence of 0.4 µg of expression plasmid encoding wild-type NKX2.5, GATA4 or TBX5.

To examine transcriptional activation of NKX2.5 independent from DNA binding, wild-type and mutants were subcloned into pFA-CMV expression vector (Stratagene) that contains the coding region of the GAL4 DNA binding domain (aa1–147) driven by CMV promoter and enhancer. Compared with control expression plasmid encoding only GAL4 DNA binding domain, NKX2.5 is expected to act as a repressor (similar to a previous study demonstrating repressor activity of NK4/tinman, a Drosophila homologue of NKX2.5 [23]). pFR-LUC reporter plasmid (Stratagene) contains 5 x GAL4 DNA binding sites upstream to the TATA box and luciferase reporter gene. COS 7 cells cultured in six-well plates were co-transfected with 3 µg of pFR-LUC, 0.5 µg of pFA-CMV or pFA-CMV-NKX2.5 (wild-type or mutant) chimeras and 0.5 µg of RSV-β-GAL. Transfection efficiency was normalized with β-galactosidase activity. Results are presented as mean±S.E. Results between groups were compared using ANOVA and Fisher PLSD post-hoc test. Statistical tests were performed using StatView version 5.01; p<0.05 was considered significant.

2.6. Immunostaining
COS7 cells transfected with pcDNA3-FLAG-NKX2.5 (wild-type or mutant) using Lipofectamine 2000 (Invitrogen) were stained with anti-FLAG mAb (SIGMA) followed by rhodamine-conjugated anti-mouse IgG (Jackson ImmunoResearch) and Hoechst dye (SIGMA) as described [24].

2.7. Protein–protein interaction
Interaction of NKX2.5, GATA4 and TBX5 has been described previously [19]. Briefly, bacterially produced MBP-NKX2.5, GST-GATA4 and GST-TBX5 fusion proteins bound on the beads were mixed with [35S]-labeled in vitro-transcribed and translated proteins of wild-type or its mutants of NKX2.5. After extensive washing, bound protein complexes were subjected to SDS-PAGE followed by autoradiography. Autoradiographs were scanned and their intensity measured by NIH Image. Bound relative to input protein was determined and compared to wild-type NKX2.5 (Table 1).


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  		Table 1 Genotype/phenotype summary

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Eight NKX2.5 homeodomain missense mutations
Six mutations were previously reported [1,2,4], and two novel mutations, Leu34(171)Pro and Arg53(190)His, are described below. Where appropriate, previous results of the in vitro biochemical characterization of four of the mutants are compared to current studies [19].

3.2. Candidate gene approach identifies two novel mutations in the NKX2.5 homeodomain
Sequence analysis identified distinct nucleotide changes, T512C and G569A, that altered the coding sense from leucine to proline, Leu34(171)Pro [CAA, Fig. 1A] and arginine to histidine, Arg53(190)His [BHC, Fig. 1B]. The sequence changes create a BsrFI restriction enzyme site (T512C) or abolish an HhaI restriction enzyme site (G569A) that allowed independent confirmation (data not shown). These sequence variants were considered mutations based on their cosegregation with disease status in each family, their absence in more than 100 chromosomes from unrelated normal subjects, and the alteration of highly conserved amino acid residues encoded in the homeodomain. Lod scores were 7.6 [CAA] and 1.1 [BHC] at recombination fraction ({theta}) 0.0.


Figure 1
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  		Fig. 1 Phenotypes associated with Leu34(171)Pro and Arg53(190)His. In kindreds CAA (A) and BHC (B) males are denoted by squares, females by circles. Darkened quadrants indicate AV block, ASD, VSD, or "other" phenotypes (determined from records of deceased individuals who could not be genotyped). Open symbols denote normal genotype and phenotype. ECG examples of first (C, D and E) and second (F) degree AV block are shown for some family members.

 
3.3. Phenotypes associated with eight homeodomain missense mutations
Nine members of family CAA had a Leu34(171)Pro mutation (Fig. 1A). AV block (first, second or third degree) was present in all genotype positive individuals (Fig. 1C–F). Seven individuals had undergone ASD closure, and one individual had ventricular septal defect (VSD) surgery. Because of electrocardiograpic abnormalities similar to a family member who died suddenly, four individuals were treated with an implantable cardioverter defibrillator (ICD, Fig. 1A). Three members of family BHC had an Arg53(190)His mutation (Fig. 1B), and all exhibited ECG evidence of AV block. Individual II-2 suffered pulmonary hypertension from an undiagnosed ASD. She was treated with a single lung transplantation and ASD closure. Individuals III-2 and III-3 had ASD and VSD. In both kindreds, some individuals were known to have been affected, but because they were deceased could not be genotyped; individuals III-7 (CAA) and III-1 (BHC) had tricuspid atresia (type IB), a defect not previously associated with NKX2.5 mutation.

Clinical features of genotype positive individuals for six mutations have been reported [1,2,4] and are summarized in Table 1 along with individuals from kindreds CAA and BHC. Although associated cardiac anomalies were quite varied, among 54 genotype positive individuals, AV block (53 of 54, 98%) and ASD (45 of 54, 83%) were noted most often.

3.4. All eight mutant proteins are localized in the nucleus, but Arg53(190)His accumulates in distinct nuclear compartments
To address functional significance, we examined intracellular localization of NKX2.5 mutant proteins in transfected cells. Homeodomain proteins typically have a nuclear localization signal (NLS) near the homeodomain N-terminus [25–27]; the NKX2.5 NLS is a stretch of 7 amino acids (R64-1-40PR, Fig. 2A) [9]. Because of its position in the NLS, Arg5(142)Cys might be expected to prevent nuclear localization, but all eight mutant proteins localized to the nucleus (Fig. 2B).


Figure 2
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  		Fig. 2 Nuclear localized wild-type and mutant proteins. (A) Human NKX2.5 protein structure including 60 amino acids of homeodomain (shaded box, residues 138 to 197). Two arginine residues preceeding the homeodomain are also shown. Mutation positions are underlined and codon changes listed below. Four mutation "hot spots" are indicated (*) [39]. (B) COS cells, transfected with pcDNA3-FLAG-mutant NKX2.5, were stained with anti-FLAG antibody (FLAG, top panels) and Hoechst dye (Nuc, bottom panels). (C) Enlarged image of FLAG (red) and Hoechst dye (green) staining and merged images of COS cells transfected with pcDNA3-FLAG-wild-type NKX2.5 (left), Arg52(189)Gly (middle) and Arg53(190)His mutants (right). Wild-type NKX2.5 protein is diffusely localized in the nucleoplasm, whereas some Arg52(189)Gly mutant protein and all Arg53(190)His in the observed field showed concentrated FLAG staining in the isolated compartment in the nucleus (arrowheads). Anomalous nuclear localization may limit NKX2.5 protein accessibility to its normal transcriptional target. Bars=20 µm.

 
Wild-type NKX2.5 and some mutants (Arg5(142)Cys, Gln50(187)His, Asn51(188)Lys and Tyr54(191)Cys) appear diffusely in the nucleoplasm (Fig. 2B and C, left panel, wild). In contrast, the majority of Arg53(190)His protein accumulates in nuclear compartments (Fig. 2C, right panel, Arg53(190)His). Other mutants (Leu34(171)Pro, Thr41(178)Met and Arg52(189)Gly) appear to localize in nucleoplasm but with some accumulation in nuclear compartments (Fig. 2C, middle panel, Arg52(189)Gly).

3.5. DNA binding affinity of NKX2.5 mutants
Among various NK class proteins, the three-dimensional structure has only been analyzed in Drosophila vnd/NK2 (ventral nervous system defective) protein (Fig. 3A, NCBI MMDB No: mmdbsrv9716) [28–30]. Since the homeodomain is conserved [21,30], the homeodomains of vnd/NK2 and NKX2.5 are expected to form a similar structure. Fig. 3B shows the positions of the mutations on three-dimensional structure of homeodomain–DNA complex.


Figure 3
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  		Fig. 3 Positions of eight homeodomain mutations and DNA binding affinity of four mutants. (A) Structure of Drosophila vnd/NK2 homeodomain protein binding to double stranded DNA (NCBI MMDB No: mmdbsrv9716) (28–30). (B) Positions of eight mutated amino acids are depicted. Arg5 (#1) is N-terminus of the homeodomain, and binds minor groove of DNA. Leu34 (#2) is located in helix 2, and Thr41 (#3) is located between helix 2 and 3. Five mutations are located in helix 3 (#4–8). (C) Sequence of two consensus NKX2.5 binding sites in rat ANF promoter: a paired binding site (–242 to –224) used for the EMSA and a single binding site (–87 to –81). (D) DNA binding affinity of (a) wild-type NKX2.5, (b) Arg5(142)Cys, (c) Leu34(171)Pro, (d) Gln50(187)His and (e) Arg53(190)His. Wild-type bound as a monomer (M) as well as a dimer (D) depending on the protein concentration. The EMSA of these mutants shows at least 81 fold (34-fold) reduced DNA binding affinity compared to wild-type NKX2.5. Similar results were reported for Thr41(178)Met, Asn51(188)Lys, Arg52(189)Gly and Tyr54(191)Cys (19). D, dimer; M, monomer; F, free probe.

 
We examined DNA binding of NKX2.5 mutants by EMSA using the ANF promoter (Fig. 3C) [19]. Depending on protein concentration (estimated Kd{approx}7 x 10–10), NKX2.5 binds as a monomer or a dimer (M, D, Fig. 3D-a) to palindromic binding sites (–242 to –224) in the ANF promoter but only as a monomer on ANF (–87 to –81) site (data not shown) [14,19]. Arg5(142)Cys (Fig. 3D-b, estimated Kd{approx}6.5 x 10–9) and Gln50(187)His (Fig. 3D-d, Kd{approx}2.1–6.5 x 10–9) exhibited reduced DNA binding. For Leu34(171)Pro and Arg53(190)His DNA-protein binding was barely detectable (Fig. 3D-c and -e). Results for Thr41(178)Met, Asn51(188)Lys, Arg52(189)Gly and Tyr54(191)Cys were previously reported and compared to wild-type showed markedly reduced (Thr41(178)Met) to barely detectable (Arg52(189)Gly) DNA binding [19]. Taken together, these results indicate that all eight mutations exhibit decreased DNA binding affinity.

3.6. Transcriptional function of NKX2.5 mutants
We previously reported that transcriptional activation function of four mutants (Thr41(178)Met, Asn51(188)Lys, Arg52(189)Gly and Tyr54(191)Cys) showed reduced activation properties compared to wild-type NKX2.5 [19]. As shown in Fig. 4, Arg5(142)Cys, Leu34(171)Pro, Gln50(187)His and Arg53(190)His exhibited similar findings which is not unexpected given the reduced DNA binding of these mutants (Fig. 3).


Figure 4
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  		Fig. 4 Transcriptional activity of mutants on ANF promoter (–638) containing multiple NKE sites. Wild-type NKX2.5 transactivates the ANF-Luc reporter (22±1.4 fold, mean±S.E.) compared with the empty expression vector pcDNA3. Four mutants transactivate the reporter gene weaker than wild-type NKX2.5; Arg5(142)Cys=3.9±0.18, Leu34(171)Pro=0.67±0.17, Gln50(187)His=6.0±0.72, Arg53(190)His=0.65±0.11. Transfection assays were done in duplicate. ANOVA demonstrated significant difference among groups (F=164, p<0.0001, *p<0.05 versus wild-type NKX2.5). In previous studies (19), four mutants, Thr41(178)Met, Asn51(188)Lys, Arg52(189)Gly and Tyr54(191)Cys, showed reduced activation function compared to that of wild-type NKX2.5: wild=24±2.2 (mean±S.E.), Thr41(178)Met=1.8±0.66, Asn51(188)Lys=2.8±0.54, Arg52(189)Gly=2.1±0.56, Tyr54(191)Cys=1.9±0.26.

 
Because NKX2.5 can be brought to transcriptional complexes by protein–protein interaction independent of DNA binding, we generated an expression vector that contains GAL4 DNA binding domain fused to the N-terminus of NKX2.5; wild-type and mutant forms of NKX2.5 act as repressors in this promoter context. As shown in Fig. 5, none of the mutations was strong enough to modify NKX2.5 function into a transcriptional activator, and most mutants were less efficient as a repressor than wild-type Nkx2.5; Thr41(178)Met and Asn51(188)Lys mutants exhibited the least repressor activity (2.9 and 2.6 fold increased reporter activation compared with wild-type NKX2.5, respectively, p<0.05). We interpret these findings to indicate that disturbances in homeodomain structure affect other aspects of the tertiary structure of the NKX2.5 protein.


Figure 5
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  		Fig. 5 GAL4-NKX2.5 chimeras act as repressors. Compared to control plasmid (pFA-CMV), wild-type and mutant NKX2.5 act as repressors. Most mutants were less efficient repressors than wild-type Nkx2.5; Thr41(178)Met and Asn51(188)Lys exhibited the least repressor activity (2.9 and 2.6 fold increased reporter activation compared with wild-type NKX2.5, respectively). ANOVA demonstrated significant difference among groups (F=34.7, p<0.0001, *p<0.05 versus wild-type NKX2.5). Results are presented as percentage of pFR-Luc activity when cotransfected with pFA-CMV empty vector. Six separate transfection assays were done in duplicate. Values are means±S.E. Since pFR-Luc activation is very low in 10T1/2 cells, COS7 cells were utilized in these experiments.

 
3.7. Protein–protein interaction of mutants with wild-type NKX2.5, GATA4, and TBX5
The homeodomain of NKX2.5 also plays critical roles in protein–protein interactions with other transcription factors [11–16]. NKX2.5 is expected to transactivate or repress its target in different combinations with these factors in a stage-specific manner [17]. In our previous study, four mutants, Thr41(178)Met, Asn51(188)Lys, Arg52(189)Gly and Tyr54(191)Cys, were found to associate with NKX2.5 itself and GATA4 similar to wild-type NKX2.5 [19]. Since an interaction between NKX2.5 and TBX5 [15,16] has been reported recently, we examined protein–protein interactions for all eight mutants with NKX2.5, GATA4 and TBX5.

As expected from the diverse homeodomain locations, protein–protein interactions among the eight mutants were varied. Both Arg5(142)Cys (Fig. 6, lane 2) and Arg53(190)His (lane 8) showed reduced interaction with NKX2.5, GATA4 and TBX5. Leu34(171)Pro (lane 3) interacted with GATA4 and TBX5 less efficiently, and Gln50(187)His (lane 5) had reduced interaction with GATA4. In contrast, under the same experimental conditions, four other mutants, Thr41(178)Met (lane 4), Asn51(188)Lys (lane 6), Arg52(189)Gly (lane 7) and Tyr54(191)Cys (lane 9), associated with these transcriptional partners similar to wild-type NKX2.5.


Figure 6
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  		Fig. 6 Interaction of wild-type and NKX2.5 mutants with transcriptional partners. [35S]-labeled wild-type (lane 1) and mutants (lanes 2–9) were mixed with MBP-NKX2.5, GST-GATA4 and GST-TBX5 proteins. Fifty percent input of [35S]-labeled proteins is shown at the top panel showing similar amount of wild-type and mutant input proteins. Wild-type NKX2.5 (lane 1) associated with NKX2.5, GATA4 and TBX5 proteins. Pull-down assays of the eight mutants are shown in (lanes 2–9) demonstrate a spectrum of defects: some mutants, e.g. Arg5(142)Cys, demonstrate altered interaction with NKX2.5, GATA4 and/or TBX5, while for others, e.g. Thr41(178)Met, the interactions appear similar to wild-type NKX2.5.

 
To examine whether DNA binding deficient mutants affect transcriptional activity through protein–protein interactions, we co-transfected four mutants exhibiting very low transcriptional activity and varied interactions with the transcriptional partners. The transcriptional target was the ANF-Luc reporter plasmid. NKX2.5 dependent transcriptional activity was little changed in the presence of Leu34(171)Pro and moderately reduced (~50%) in the presence of mutant NKX2.5 (Fig. 7A). Therefore, these mutants act as repressors for wild-type NKX2.5, but the degree of repression appears to be independent of the interaction with wild-type NKX2.5 (Fig. 6).


Figure 7
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  		Fig. 7 Effect of protein–protein interaction on transcriptional activity. Co-transfectation assays were done with mutant NKX2.5 and wild-type NKX2.5 (A), GATA4 (B) and TBX5 (C). Results are presented as percent of ANF-Luc reporter activity normalized with RSV-β-GAL activity compared with that of pcDNA3 empty vector. Mutant protein and binding results from Fig. 6 are shown as +'s. Transfection assays were done in duplicate. Values are means±S.E. (A) Wild-type NKX2.5 transactivates ANF-Luc by 30±4.8 fold compared with empty pcDNA3 (defined as 100%). NKX2.5 dependent transcriptional activity was little changed in the presence of Leu34(171)Pro and moderately reduced in the presence of Thr41(178)Met (47% reduction), Asn51(188)Lys (45% reduction) or Thy54(191)Cys (46% reduction). ANOVA demonstrated significant difference among groups (F=8.72, p<0.0001, *p<0.05 versus wild-type NKX2.5). (B) GATA4 transactivates ANF-Luc by 16±1.7 fold compared with empty pcDNA3 (defined as 100%); the addition of wild-type NKX2.5 further transactivates by ~390% (63±15 fold). In contrast, ANF-Luc activation was slightly modified by mutants: Leu34(171)Pro (6% reduction), Thr41(178)Met (38% induction), Asn(188)Lys (24% reduction) and Tyr54(191)Cys (32% reduction). ANOVA demonstrated significant difference among groups (F=10.0, p<0.0001, *p<0.05 versus wild-type NKX2.5). (C) TBX5 weakly transactivates ANF-Luc by 2.8±0.22 fold but is markedly activated by addition of wild-type NKX2.5 by ~2000% (58±17). Co-transfection of plasmids encoding mutants increased ANF-Luc activity but much less than wild-type NKX2.5 [Leu34(171)Pro (270%), Thr41(178)Met (210%), Asn(188)Lys (180%) and Tyr54(191)Cys (160%)] ANOVA demonstrated significant difference among groups (F=12.7, p<0.0001, *p<0.05 versus wild-type NKX2.5).

 
GATA4 alone transactivates the ANF-Luc reporter gene and further transactivates the reporter in the presence of wild-type NKX2.5 (~3.9 fold induction) (Fig. 7B). In contrast, co-transfection with the expression plasmid encoding Leu34(171)Pro, which showed no interaction with GATA4 (Fig. 6), did not modify ANF-Luc activity. In three other mutants with preserved binding to GATA4, ANF-Luc activity was only slightly modified.

TBX5 alone transactivates the ANF-Luc reporter (~2.8 fold induction), but in the presence of wild-type NKX2.5, luciferase activity was markedly increased (~20 fold induction) (Fig. 7C). Slight increases of ANF-Luc activity (1.6–2.6 fold induction) were observed with each NKX2.5 mutant, all of which exhibited interaction with TBX5. Our findings suggest that NKX2.5 transcriptional synergy with TBX5 and GATA4 requires an intact homeodomain, possibly for its DNA binding properties.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Results of this study bring to 28 the total of NKX2.5 mutations that have been identified in humans with varied congenital heart malformations [1–8]; eight homeodomain missense mutations are characterized by high disease penetrance: 98% for AV block and 83% for ASD. Our study represents the most comprehensive analysis of NKX2.5 homeodomain mutations to date and provides further definition of molecular mechanisms of NKX2.5 mutant protein dysfunction. The mutants we studied alter homeodomain function and, as predicted from the wide range of locations, exhibit a spectrum of in vitro biochemical phenotypes.

Most of the mutations we studied are in conserved residues. For example, among 346 homeoproteins, 98% of amino acids at position 5 are Arg [20]. In addition to comprising a portion of the NLS, Arg5 is predicted to contact bases in both the minor groove and DNA backbone and contribute to the affinity of the homeodomain and DNA interaction [21]. Leu34 contributes to the hydrophobic core responsible for the tertiary structure of the homeodomain [21]; Leu34(171)Pro is expected to lose its DNA binding ability despite the distance of helix 2 from DNA. At position 34, among 346 homeoproteins examined, 44% of amino acids are Leu and 38% of amino acids are Ile; Pro has not been reported at this position [20]. Thr41, located in the loop between helix 2 and 3, is not highly conserved; among 346 homeoproteins, residues frequently positioned at this location are Thr (47%) and Ser (20%) [20]. Thr41(178)Met may result in a change in direction of helix 3, which directly contacts the major groove. Residues 50 to 53 are highly conserved: among 346 homeoproteins examined Gln50 (84%), Asn51 (100%), Arg52 (87%), and Arg53 (99%) were found frequently [20]. Tyr54 is a signature amino acid in the NK2 class homeodomain and is known to specify the DNA binding sequence [31].

Although associated cardiac anomalies were quite varied among 54 genotype positive individuals, AV block (53 of 54, 98%) and ASD (45 of 54, 83%) were noted most often. The total number of subjects is too small to allow detailed comparison of genotype, biochemical phenotype and cardiac phenotype for all eight mutants, but nearly half of the individuals studied were in two kindreds. The cardiac phenotypes are very similar in kindreds with Arg5(142)Cys (13 heterozygous carriers) or Thr41(178)Met (12 heterozygous carriers); AV block and ASD are the most common problems, but some members have VSD or tetralogy of Fallot. However, these two mutants demonstrated different molecular properties, including nucleoplasm distribution and interaction with transcriptional partners. Arg5(142)Cys showed reduced interaction with all three partners while Thr41(178)Met demonstrated an interaction similar to wild-type. The greatest similarity between these two mutants was the marked reduction in DNA binding affinity noted for all mutant proteins studied.

Based on observed DNA binding deficiency, NKX2.5 haploinsufficiency has been suggested as the cause of congenital heart disease. In support of this, heterozygous deletion of 5q34, which includes the NKX2.5 locus, was found in a patient with ASD, AV block and ventricular non-compaction [32]. Extensive analyses of mice heterozygous for Nkx2.5-null alleles (Nkx2.5+/-) [33,34] in different genetic backgrounds demonstrated similar features including increased frequency of patent foramen ovale and mild prolongation of the P–R interval (1° AV block). However, heterozygous mice do not show advanced AV block, high penetrance of ASD, or other cardiac malformations seen in humans [18]. One interpretation of these observations has been that the effects of Nkx2.5 haploinsufficiency in mice are much weaker than in human patients. In contrast, when the DNA nonbinding mutant of mouse Nkx2.5-Ile47(183)Pro was expressed in transgenic mice under the beta-myosin heavy chain promoter, postnatal heart failure and advanced AV block developed [35]. In Xenopus embryos, overexpression of a DNA nonbinding Nkx2.5 mutant resulted in small or complete loss of heart, suggesting dominant inhibitory effects of DNA nonbinding Nkx2.5 mutant [36]. Taken together, these studies suggest that expression of a DNA nonbinding Nkx2.5 mutant is not silent and may be involved in Nkx2.5-dependent transcriptional regulation either in a dominant inhibitory manner or in a hypomorphic gain-of-function manner.

Mutant proteins that fail to interact with NKX2.5 transcriptional cofactors and fail to bind DNA may result in haploinsufficiency. On the other hand, mutants that preserve protein–protein interactions but fail to bind DNA may have broader functional consequences including dominant negative effects. It might be anticipated that for such diverse effects, i.e. haploinsufficiency versus dominant-negative, there would be associated variation in clinical phenotypes. Our data show that although dominant negative properties could be demonstrated in vitro, clinical phenotypes did not correlate with any property other than DNA binding ability. This, therefore, argues that the principle determinant of the AV block phenotype, and to a lesser extent the ASD phenotype, is the total dose of NKX2.5 capable of binding to DNA. The presence of additional cardiac defects, including VSD and tricuspid valve abnormalities were not correlated with any protein property we determined. However, the possibility that dominant negative effects, modifying alleles or environmental factors contribute to other phenotypes cannot be completely excluded because of the small sample size per mutation. Resolution of these questions will require careful analysis in experimental models permitting tight dosing control of NKX2.5 wild-type and mutant proteins [37,38].


   Acknowledgements
 
We thank N. Horikoshi, J. Ma, T. McQuinn, M.Tennant, E. O. Weinberg, C.Wood and K. Yutzey for critical reading of the manuscript and valuable suggestions and M. Dyment, L. Etter, K. Gauthier and J. Reyna for technical help. We are indebted to participating family members; C. Cottrill, D.A. Dodd and W.H. Franklin provided invaluable assistance in collecting clinical material. This work was supported by Biomedical Research Support Program for Medical Schools Award to the University of Florida College of Medicine by the Howard Hughes Medical Institute (HK), AHA/National SDG grant (HK) and NIH [HL69712 (DWB), HD39946 (DWB)].


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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