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. 2013 Jun;6(3):238-47.
doi: 10.1161/CIRCGENETICS.113.000057. Epub 2013 May 9.

Functional Characterization of a Novel Mutation in NKX2-5 Associated With Congenital Heart Disease and Adult-Onset Cardiomyopathy

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Free PMC article

Functional Characterization of a Novel Mutation in NKX2-5 Associated With Congenital Heart Disease and Adult-Onset Cardiomyopathy

Mauro W Costa et al. Circ Cardiovasc Genet. .
Free PMC article

Abstract

Background: The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown.

Methods and results: Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein.

Conclusions: Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.

Keywords: NKX2-5; UBIQUITIN-proteasome system; dilated cardiomyopathy; gene mutations; transcription factors.

Figures

Figure 1
Figure 1
Novel I184M NKX2-5 mutation with high association with DCM. (A) Pedigree showing DO family members. The family proband is indicated by an arrow. Phenotypes are indicated as: conduction-system abnormalities (black, right half, upper quadrant), dilated cardiomyopathy (red, left half, upper quadrant), congenital heart disease (ASD or PFO, black, right half lower quadrant; Tricuspid atresia, black, left half lower quadrant), unknown (gray symbols). (B) Sequence electropherograms showing wild type sequence (upper trace) and a C to G substitution that alters the amino acid 184 from Isoleucine to Methionine (lower trace) identified in the Family DO proband, (DO-II-4). (C) The presence of the variant G allele in affected family members was confirmed by loss of a BglII restriction enzyme site. CD – conduction defect; DCM – dilated cardiomyopathy; CHD – congenital heart disease; ASD – atrial septal defects; PFO – patent foramen ovale.
Figure 2
Figure 2
Structural consequences of aminoacid variation in position I184 in the NKX2-5 HD. (A) Alignment of NKX2-5 HD sequence in various species showed complete evolutionary conservation of isoleucine (I) at amino acid 184 (arrow). Homologous amino acids are highlighted in blue. Positions of previously reported human HD mutations (*) are shown. (B) Conformational model of human NKX2-5, shows helix 3 (arrowhead) in close contact with the major groove of DNA. (C) I184 is located into a pocket formed by Q187 and N188. The underlying interacting DNA binding strand (pink ribbon) and deoxyribonucleic acid are shown. Atoms indicated are: carbon (green, except at position 184, where WT is shown as orange and mutant as cyan), nitrogen (blue), oxygen (red), sulphur (yellow), phosphorus (dark pink) and hydrogen (white). (D) One possible conformation of 184M has the sulphur atom in close proximity to the oxygen atom of the Q187 side chain, resulting in disruption of the underlying helical structure. (E) An alternative 184M conformation allows a hydrogen bond to be formed between the sulphur atom and one of the amide hydrogens of N188, altering position of the N188 side chain and disrupting the structure of the helix. HD – homeodomain
Figure 3
Figure 3
I184M Nkx2-5 mutation causes decreased DNA affinity. (A) EMSA assay using [35S]-labelled Nkx2-5 showed specific interaction of WT Nkx2-5 with the NKE site present in the Nppa promoter (lane 2). Binding was inhibited by competition with self-oligonucleotide (lane 3) or anti-Nkx2-5 antibody (lane 4). No interaction was observed when the I184M variant was used (lanes 5-7). (B) EMSA performed with increasing amounts of GST-purified Nkx2-5 HD showed that WT Nkx2-5HD (arrowheads, lanes 2-6) displayed at least 10-fold greater DNA binding affinity than Nkx2-5 I184M (lanes 7-11).
Figure 4
Figure 4
I184M mutation leads to partial transcriptional impairment. (A-C) I184M Nkx2-5 has decreased activation of the cardiac promoters Nppa and Gja5 in transfected COS-7 cells, while I184P and Y191C variants showed no detectable activation. (D) In the presence of WT Nkx2-5, increasing doses of I184M Nkx2-5 allowed transcriptional activation in transfected COS-7 cells but did not restore normal levels of Nppa activation, while I184P and Y191C caused strong dominant effects by further depressing promoter function. (E) Quantification of Gata4, Nppa and Gja1 transcripts in HL-1 cells following transient overexpression of WT or I184M Nkx2-5 mutant protein. qPCR showed a 3-fold increase in Nppa transcript level when WT protein was transfected, while addition of I184M decreased basal activation. (F) GST-pull down showed that I184M Nkx2-5 had normal self-dimerization and interaction with Gata4, Tbx5 and Tbx20. Input represents 20% of the in vitro translated protein used in each pull-down assay. Transfections were performed in triplicates in a least two independent experiments. Data represented as average mean and standard deviation.
Figure 5
Figure 5
I184M Nkx2-5 is localized to the nucleus but displays increased levels when compared to WT Nkx2-5. (A) Normal nuclear localization of both WT and I184M Nkx2-5 in COS-7 cells was evident by immunofluorescence microscopy, but mutant protein displayed more intense signal. (B-C) Levels of WT and I184M Nkx2-5 in transfected cells by western blot in both COS-7 and cardiac HL-1 cells showed much higher expression of I184M and I184P when compared to WT Nkx2-5. (D) Increased stability of I184 mutants was confirmed by CHX treatment. WT protein was dramatically reduced after 24 hours, while mutant protein expression was sustained. α-tubulin was used as a loading control in all experiments. (E) Increased protein expression was also observed for a cluster of mutations located in helices 2 and 3 of the Nkx2-5 HD, as per marked asterisks in yellow strand.
Figure 6
Figure 6
Nkx2-5 protein levels are regulated by the UPS. (A) Protein levels in transfected COS-7 cells were evaluated by western blot before and after treatment with the specific proteasome inhibitor Lactacystin. WT Nkx2-5 levels were increased by 3.1 fold (**, P<0.01, Students t-test, Prism Software) upon UPS inhibition. I184M Nkx2-5 levels were unchanged (1.1 fold), indicating that the mutant had decreased proteasome degradation before the drug is added. Effective inhibition of the UPS was confirmed increased cyclin D1 levels; α-tubulin was used as a loading control. (B,C) Immunoprecipitation to detect poly-ubiquitylated Nkx2-5 conjugates. FLAG antibody was used to pull-down tagged Nkx2-5 protein, while western blotting has performed using anti-HA antibodies that detected tagged ubiquitin. Similar amounts of input Nkx2-5 were confirmed using an anti-Nkx2-5 antibody (lower panel). (D) In vitro ubiquitylation kinetics assay for WT, I184M, I184P and Y191C Nkx2-5 variants confirmed that these mutations did not impair ubiquitylation.
Figure 7
Figure 7
Comparison of expression levels among Nkx2-5 protein variants and other HD proteins. (A) Western blots showing protein levels of I184 mutant were still elevated upon deletion of the C-terminal domain (ΔC) as compared to the full length (FL) WT or I184P protein. (B) Alignment of HD proteins showing molecular divergence among family members and presence of isoleucine or valine at position 184. (C) High protein levels associated with variation at position 184 were observed only for Nkx2-5 and closely related Bapx1, but not for the more distantly related Pax7 and Islet1, even though all proteins were ubiquitylated in vitro (D).

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