RESUMEN
Heterozygous loss-of-function (LOF) mutations in the gene encoding the DNA-binding protein, SATB2, result in micrognathia and cleft palate in both humans and mice. In three unrelated individuals, we show that translocation breakpoints (BPs) up to 896 kb 3' of SATB2 polyadenylation site cause a phenotype which is indistinguishable from that caused by SATB2 LOF mutations. This syndrome comprises long nose, small mouth, micrognathia, cleft palate, arachnodactyly and intellectual disability. These BPs map to a gene desert between PLCL1 and SATB2. We identified three putative cis-regulatory elements (CRE1-3) using a comparative genomic approach each of which would be placed in trans relative to SATB2 by all three BPs. CRE1-3 each bind p300 and mono-methylated H3K4 consistent with enhancer function. In silico analysis suggested that CRE1-3 contain one or more conserved SOX9-binding sites, and this binding was confirmed using chromatin immunoprecipitation on cells derived from mouse embryonic pharyngeal arch. Interphase bacterial artificial chromosome fluorescence in situ hybridization measurements in embryonic craniofacial tissues showed that the orthologous region in mice exhibits Satb2 expression-dependent chromatin decondensation consistent with Satb2 being a target gene of CRE1-3. To assess their in vivo function, we made multiple stable reporter transgenic lines for each enhancer in zebrafish. CRE2 was shown to drive SATB2-like expression in the embryonic craniofacial region. This expression could be eliminated by mutating the SOX9-binding site of CRE2. These observations suggest that SATB2 and SOX9 may be acting together via complex cis-regulation to coordinate the growth of the developing jaw.
Asunto(s)
Proteínas de Unión a la Región de Fijación a la Matriz/genética , Síndrome de Pierre Robin/diagnóstico , Factor de Transcripción SOX9/genética , Factores de Transcripción/genética , Adulto , Animales , Sitios de Unión , Células Cultivadas , Niño , Preescolar , Epistasis Genética , Femenino , Humanos , Lactante , Masculino , Ratones , Mutación , Síndrome de Pierre Robin/genética , Elementos Reguladores de la Transcripción , Adulto Joven , Pez CebraRESUMEN
During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.
Asunto(s)
Redes Reguladoras de Genes , Sistema Nervioso , Animales , Sistema Nervioso/metabolismo , Pollos , Desarrollo Embrionario , Organizadores Embrionarios , VertebradosRESUMEN
Mutation of SATB2 causes cleft palate in humans. To understand the role of SATB2 function in palatogenesis, SATB2 analyses in vertebrate model systems will be essential. To facilitate these analyses, we have performed a cross-species comparison of SATB2 structure and function across three vertebrate model systems: mouse, chick, and zebrafish. We find that the SATB2 transcript is highly conserved across human, mouse, chick, and zebrafish, especially within the Satb2 functional domains. Furthermore, our expression analyses demonstrate that SATB2 is likely to have similar functions in vertebrate model organisms and humans during development of the facial processes and secondary palate. Together, these data suggest an evolutionary conserved role for SATB2 during development of the face and palate across vertebrates. Moreover, expression of zebrafish satb2 in the anterior neurocranium supports the utility of the anterior neurocranium as a simplified model of amniote palatogenesis.