Functional analysis of TCF4 missense mutations that cause Pitt-Hopkins syndrome.
Pitt-Hopkins syndrome (PTHS) is a rare developmental disorder associated with severe mental retardation, facial abnormalities, and intermittent hyperventilation. Autosomal dominant PTHS is caused by mutations in the transcription factor 4 (TCF4) gene, whereas NRXN1 and CNTNAP2 mutations are associated with autosomal recessive PTHS. To determine the impact of missense mutations on TCF4 function, we tested a panel of PTHS-associated mutations using a range of quantitative techniques. Mutations in the basic helix-loop-helix (bHLH) domain of TCF4 alter the subnuclear localization of the mutant protein and can attenuate homo- and heterodimer formation in homogenous time-resolved fluorescence (HTRF) assays. By contrast, mutations proximal to the bHLH domain do not alter the location of TCF4 or impair heterodimer formation. In addition, we show that TCF4 can transactivate the NRXN1β and CNTNAP2 promoters in luciferase assays. Here we find variable, context-specific deficits in the ability of the different PTHS-associated TCF4 mutants to transactivate these promoters when coexpressed with different bHLH transcription factors. These data demonstrate that PTHS-associated missense mutations can have multiple effects on the function of the protein, and suggest that TCF4 may modulate the expression of NRXN1 and CNTNAP2 thereby defining a regulatory network in PTHS.
Institute of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Cardiff, UK.
Marc
M
Forrest
Ria M
RM
Chapman
A Michelle
AM
Doyle
Caroline L
CL
Tinsley
Adrian
A
Waite
Derek J
DJ
Blake
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D002135:Calcium-Binding Proteins; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D019066:Facies; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D006985:Hyperventilation / Q000235:genetics*; D008607:Intellectual Disability / Q000235:genetics*; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics; D019006:Neural Cell Adhesion Molecules; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C403049:CNTNAP2 protein, human; D002135:Calcium-Binding Proteins; D015816:Cell Adhesion Molecules, Neuronal; D008565:Membrane Proteins; C539628:NRXN1 protein, human; D009419:Nerve Tissue Proteins; D019006:Neural Cell Adhesion Molecules; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D049410:Luciferases, Renilla