Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease
Hideko Kasahara, … , Christine E. Seidman, Seigo Izumo
Hideko Kasahara, … , Christine E. Seidman, Seigo Izumo
Published January 15, 2000
Citation Information: J Clin Invest. 2000;106(2):299-308. https://doi.org/10.1172/JCI9860.
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Article

Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

Abstract

CSX/NKX2.5 is an evolutionarily conserved homeodomain-containing (HD-containing) transcription factor that is essential for early cardiac development. Recently, ten different heterozygous CSX/NKX2.5 mutations were found in patients with congenital heart defects that are transmitted in an autosomal dominant fashion. To determine the consequence of these mutations, we analyzed nuclear localization, DNA binding, transcriptional activation, and dimerization of mutant CSX/NKX2.5 proteins. All mutant proteins were translated and located to the nucleus, except one splice-donor site mutant whose protein did not accumulate in the cell. All mutants that had truncation or missense mutations in the HD had severely reduced DNA binding activity and little or no transcriptional activation function. In contrast, mutants with intact HDs exhibit normal DNA binding to the monomeric binding site but had three- to ninefold reduction in DNA binding to the dimeric binding sites. HD missense mutations that preserved homodimerization ability inhibited the activation of atrial natriuretic factor by wild-type CSX/NKX2.5. Although our studies do not characterize the genotype-phenotype relationship of the ten human mutations, they identify specific abnormalities of CSX/NKX2.5 function essential for transactivation of target genes.

Authors

Hideko Kasahara, Bora Lee, Jean-Jacques Schott, D. Woodrow Benson, J.G. Seidman, Christine E. Seidman, Seigo Izumo

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Inhibition of ANF-luciferase transcriptional activity of wild-type CSX/N...
Inhibition of ANF-luciferase transcriptional activity of wild-type CSX/NKX2.5 by group 1, 2, and 3 mutants but not by group 4 mutant. (a) Inhibition of transactivation of the ANF-luciferase reporter gene in the presence of both 1:1 and 2:1 ratio of M170, M189, M191, and M259 expression plasmid to wild-type expression plasmid. 10T1/2 cells were transiently transacted with 1.2 μg of ANF-luciferase(-638), 0.3 μg of RSV–β-galactosidase, 0.7 μg of wild-type CSX/NKX2.5 expression plasmid, and 0.7 μg (hatched bars) or 1.4 μg (filled bars) of mutant expression plasmid or empty pcDNA3 plasmid to adjust the total amount of plasmid. A moderate reduction of luciferase activity was observed in M170 mutants, and a further decrease of luciferase activity was detected in M189, M191, and M250 mutants. In contrast, M25 mutant increased luciferase activity. Results are presented as a percent of the ANF reporter activity when cotransfected with wild-type and mutant plasmid compared with that of the wild type alone (open bar). Bars represent means ± SEM of the means of at least three separate transfection assays done in duplicate. (b) Protein-protein interaction of wild-type CSX/NKX2.5 with mutant proteins. MBP-fused wild-type CSX/NKX2.5 protein was mixed with [35S]-labeled wild-type (lane 1) or ten mutants (lanes 2–11). After washing five times with binding buffer, the protein complexes were resolved on SDS-PAGE and autoradiographed (top panel). Group 1 (lanes 2 and 3) and group 5 (lane 11) mutants did not associate with MBP-CSX/NKX2.5, whereas group 2, 3, and 4 mutants associated with MBP-CSX/NKX2.5 (lanes 4–10). Fifty percent input of [35S]-labeled proteins are shown in the middle panel, and Coomassie blue–stained MBP-fused wild-type CSX/NKX2.5 proteins are shown in the bottom panel.