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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Figure 5

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Effect of ten mutations on transcriptional activation. 10T1/2 cells were...
Effect of ten mutations on transcriptional activation. 10T1/2 cells were transfected with pcDNA3 expression vectors encoding wild-type or each of ten mutations with the reporter gene ANF-luciferase. When the wild-type CSX/NKX2.5 was transfected with the ANF reporter gene, luciferase activity was increased 23-fold compared with cells transfected with the empty expression vector pcDNA3. M112, group 1, and group 2 expression vectors did not activate the ANF promoter. M25 and M198 transactivated the reporter gene similarly to the wild-type CSX/NKX2.5; however, M259 transactivated only 5.2-fold. Another COOH-terminus deletion mutant, CSX/NKX2.5(1–200), transactivated the reporter construct approximately 136-fold. Bars represent means ± SEM of at least three separate transfection assays done in duplicate.