RESUMEN
Zinc finger of the cerebellum (Zic) proteins act as classic transcription factors to promote transcription of the Foxd3 gene during neural crest cell specification. Additionally, they can act as co-factors that bind proteins from the T-cell factor/lymphoid enhancing factor (TCF/LEF) family (TCFs) to repress WNT-ß-catenin-dependent transcription without contacting DNA. Here, we show that ZIC activity at the neural plate border is influenced by WNT-dependent SUMOylation. In the presence of high canonical WNT activity, a lysine residue within the highly conserved zinc finger N-terminally conserved (ZF-NC) domain of ZIC5 is SUMOylated, which reduces formation of the ZIC-TCF co-repressor complex and shifts the balance towards transcription factor function. The modification is crucial in vivo, as a ZIC5 SUMO-incompetent mouse strain exhibits neural crest specification defects. This work reveals the function of the ZF-NC domain within ZIC, provides in vivo validation of target protein SUMOylation and demonstrates that WNT-ß-catenin signalling directs transcription at non-TCF DNA-binding sites. Furthermore, it can explain how WNT signals convert a broad region of Zic ectodermal expression into a restricted region of neural crest cell specification.
Asunto(s)
Cresta Neural , Sumoilación , Animales , Diferenciación Celular , Ratones , Cresta Neural/metabolismo , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The ZIC proteins are a family of transcription regulators with a well-defined zinc finger DNA-binding domain and there is evidence that they elicit functional DNA binding at a ZIC DNA binding site. Little is known, however, regarding domains within ZIC proteins that confer trans-activation or -repression. To address this question, a new cell-based trans-activation assay system suitable for ZIC proteins in HEK293T cells was constructed. This identified two previously unannotated evolutionarily conserved regions of ZIC3 that are necessary for trans-activation. These domains are found in all Subclass A ZIC proteins, but not in the Subclass B proteins. Additionally, the Subclass B proteins fail to elicit functional binding at a multimerised ZIC DNA binding site. All ZIC proteins, however, exhibit functional binding when the ZIC DNA binding site is embedded in a multiple transcription factor locus derived from ZIC target genes in the mouse genome. This ability is due to several domains, some of which are found in all ZIC proteins, that exhibit context dependent trans-activation or -repression activity. This knowledge is valuable for assessing the likely pathogenicity of variant ZIC proteins associated with human disorders and for determining factors that influence functional transcription factor binding.
Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , Elementos de Respuesta , Factores de Transcripción , Transcripción Genética , Células HEK293 , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.
Asunto(s)
Codón sin Sentido/genética , Síndrome de Heterotaxia/embriología , Síndrome de Heterotaxia/genética , Proteínas de Homeodominio/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/genética , Factores de Transcripción/metabolismo , Alelos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Difusión , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Endodermo/anomalías , Endodermo/embriología , Gastrulación/genética , Proteínas de Homeodominio/genética , Humanos , Mesodermo/anomalías , Mesodermo/embriología , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación/genética , Organogénesis/genética , Estabilidad Proteica , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Transcripción Genética , beta Catenina/metabolismoRESUMEN
Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.