RESUMO
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 NRXN1ß 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.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Hiperventilação/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Fatores de Transcrição/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células COS , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Fácies , Genes Reporter , Células HEK293 , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa , Regiões Promotoras Genéticas , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Genome-wide association studies have linked common variation in ZNF804A with an increased risk of schizophrenia. However, little is known about the biology of ZNF804A and its role in schizophrenia. Here, we investigate the function of ZNF804A using a variety of complementary molecular techniques. We show that ZNF804A is a nuclear protein that interacts with neuronal RNA splicing factors and RNA-binding proteins including RBFOX1, which is also associated with schizophrenia, CELF3/4, components of the ubiquitin-proteasome system and the ZNF804A paralog, GPATCH8. GPATCH8 also interacts with splicing factors and is localized to nuclear speckles indicative of a role in pre-messenger RNA (mRNA) processing. Sequence analysis showed that GPATCH8 contains ultraconserved, alternatively spliced poison exons that are also regulated by RBFOX proteins. ZNF804A knockdown in SH-SY5Y cells resulted in robust changes in gene expression and pre-mRNA splicing converging on pathways associated with nervous system development, synaptic contact, and cell adhesion. We observed enrichment (P = 1.66 × 10-9) for differentially spliced genes in ZNF804A-depleted cells among genes that contain RBFOX-dependent alternatively spliced exons. Differentially spliced genes in ZNF804A-depleted cells were also enriched for genes harboring de novo loss of function mutations in autism spectrum disorder (P = 6.25 × 10-7, enrichment 2.16) and common variant alleles associated with schizophrenia (P = .014), bipolar disorder and schizophrenia (P = .003), and autism spectrum disorder (P = .005). These data suggest that ZNF804A and its paralogs may interact with neuronal-splicing factors and RNA-binding proteins to regulate the expression of a subset of synaptic and neurodevelopmental genes.
Assuntos
Regulação da Expressão Gênica/genética , Fatores de Transcrição Kruppel-Like/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Esquizofrenia/genética , Transtorno do Espectro Autista/genética , Transtorno Bipolar/genética , Proteínas CELF/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Musculares/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Genome-wide association studies allied with the identification of rare copy number variants have provided important insights into the genetic risk factors for schizophrenia. Recently, a meta-analysis of several genome-wide association studies found, in addition to several other markers, a single nucleotide polymorphism in intron 4 of the TCF4 gene that was associated with schizophrenia. TCF4 encodes a basic helix-loop-helix transcription factor that interacts with other transcription factors to activate or repress gene expression. TCF4 mutations also cause Pitt-Hopkins Syndrome, an autosomal-dominant neurodevelopmental disorder associated with severe mental retardation. Variants in the TCF4 gene may therefore be associated with a range of neuropsychiatric phenotypes, including schizophrenia. Recessive forms of Pitt-Hopkins syndrome are caused by mutations in NRXN1 and CNTNAP2. Interestingly, NRXN1 deletions have been reported in schizophrenia, whereas CNTNAP2 variants are associated with several neuropsychiatric phenotypes. These data suggest that TCF4, NRXN1, and CNTNAP2 may participate in a biological pathway that is altered in patients with schizophrenia and other neuropsychiatric disorders.