RESUMEN
Complex diseases, such as diabetes, are influenced by comprehensive transcriptional networks. Genome-wide association studies have revealed that variants located in regulatory elements for pancreatic transcription factors are linked to diabetes, including those functionally linked to the paired box transcription factor Pax6. Pax6 deletions in adult mice cause rapid onset of classic diabetes, but the full spectrum of pancreatic Pax6 regulators is unknown. Using a regulatory element discovery approach, we identified two novel Pax6 pancreatic cis-regulatory elements in a poorly characterized regulatory desert. Both new elements, Pax6 pancreas cis-regulatory element 3 (PE3) and PE4, are located 50 and 100 kb upstream and interact with different parts of the Pax6 promoter and nearby non-coding RNAs. They drive expression in the developing pancreas and brain and code for multiple pancreas-related transcription factor-binding sites. PE3 binds CCCTC-binding factor (CTCF) and is marked by stem cell identity markers in embryonic stem cells, whilst a common variant located in the PE4 element affects binding of Pax4, a known pancreatic regulator, altering Pax6 gene expression. To determine the ability of these elements to regulate gene expression, synthetic transcriptional activators and repressors were targeted to PE3 and PE4, modulating Pax6 gene expression, as well as influencing neighbouring genes and long non-coding RNAs, implicating the Pax6 locus in pancreas function and diabetes.
Asunto(s)
Diabetes Mellitus/genética , Factor de Transcripción PAX6/genética , Elementos Reguladores de la Transcripción/genética , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Diabetes Mellitus/patología , Regulación del Desarrollo de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Ratones TransgénicosRESUMEN
Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function.
Asunto(s)
Síndrome de Pierre Robin/genética , Elementos Reguladores de la Transcripción , Transgenes , Animales , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Sprouty proteins are regulators of cell growth and branching morphogenesis. Unlike mouse Spry3, which is X-linked, human SPRY3 maps to the pseudoautosomal region 2; however, the human Y-linked allele is not expressed due to epigenetic silencing by an unknown mechanism. SPRY3 maps adjacent to X-linked Trimethyllysine hydroxylase epsilon (TMLHE), recently identified as an autism susceptibility gene. We report that Spry3 is highly expressed in central and peripheral nervous system ganglion cells in mouse and human, including cerebellar Purkinje cells and retinal ganglion cells. Transient over-expression or knockdown of Spry3 in cultured mouse superior cervical ganglion cells inhibits and promotes, respectively, neurite growth and branching. A 0.7 kb gene fragment spanning the human SPRY3 transcriptional start site recapitulates the endogenous Spry3-expression pattern in LacZ reporter mice. In the human and mouse the SPRY3 promoter contains an AG-rich repeat and we found co-expression, and promoter binding and/or regulation of SPRY3 expression by transcription factors MAZ, EGR1, ZNF263 and PAX6. We identified eight alleles of the human SPRY3 promoter repeat in Caucasians, and similar allele frequencies in autism families. We characterized multiple SPRY3 transcripts originating at two CpG islands in the X-linked F8A3-TMLHE region, suggesting X chromosome regulation of SPRY3. These findings provide an explanation for differential regulation of X and Y-linked SPRY3 alleles. In addition, the presence of a SPRY3 transcript exon in a previously described X chromosome deletion associated with autism, and the cerebellar interlobular variation in Spry3 expression coincident with the reported pattern of Purkinje cell loss in autism, suggest SPRY3 as a candidate susceptibility locus for autism.
Asunto(s)
Trastorno Autístico/genética , Cromosomas Humanos X , Predisposición Genética a la Enfermedad , Péptidos y Proteínas de Señalización Intracelular/genética , Regiones Promotoras Genéticas , Receptor PAR-2/genética , Alelos , Animales , Composición de Base , Secuencia de Bases , Línea Celular , Cerebelo/metabolismo , Islas de CpG , Metilación de ADN , Modelos Animales de Enfermedad , Exones , Ganglios/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Genes Ligados a X , Sitios Genéticos , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Neuritas/metabolismo , Polimorfismo Genético , Alineación de Secuencia , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
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
The strictly regulated expression of most pleiotropic developmental control genes is critically dependent on the activity of long-range cis-regulatory elements. This was revealed by the identification of individuals with a genetic condition lacking coding-region mutations in the gene commonly associated with the disease but having a variety of nearby chromosomal abnormalities, collectively described as cis-ruption disease cases. The congenital eye malformation aniridia is caused by haploinsufficiency of the developmental regulator PAX6. We discovered a de novo point mutation in an ultraconserved cis-element located 150 kb downstream from PAX6 in an affected individual with intact coding region and chromosomal locus. The element SIMO acts as a strong enhancer in developing ocular structures. The mutation disrupts an autoregulatory PAX6 binding site, causing loss of enhancer activity, resulting in defective maintenance of PAX6 expression. These findings reveal a distinct regulatory mechanism for genetic disease by disruption of an autoregulatory feedback loop critical for maintenance of gene expression through development.
Asunto(s)
Aniridia/genética , Aniridia/metabolismo , Elementos de Facilitación Genéticos , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Homeostasis/genética , Mutación , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Animales , Aniridia/diagnóstico , Secuencia de Bases , Ojo/patología , Regulación del Desarrollo de la Expresión Génica , Orden Génico , Humanos , Ratones , Datos de Secuencia Molecular , Factor de Transcripción PAX6 , Fenotipo , Alineación de Secuencia , Pez CebraRESUMEN
BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation. In this study, we postulated that mutations responsible for exerting a modifier effect on FSHD might reside within remotely acting regulatory elements that have the potential to interact at a distance with their cognate gene promoter via chromatin looping. To explore this postulate, genome-wide Hi-C data were used to identify genomic fragments displaying the strongest interaction with the SMCHD1 gene. These fragments were then narrowed down to shorter regions using ENCODE and FANTOM data on transcription factor binding sites and epigenetic marks characteristic of promoters, enhancers and silencers. RESULTS: We identified two regions, located respectively ~14 and ~85 kb upstream of the SMCHD1 gene, which were then sequenced in 229 FSHD/FSHD-like patients (200 with D4Z4 repeat units <11). Three heterozygous sequence variants were found ~14 kb upstream of the SMCHD1 gene. One of these variants was found to be of potential functional significance based on DNA methylation analysis. Further functional ascertainment will be required in order to establish the clinical/functional significance of the variants found. CONCLUSIONS: In this study, we propose an improved approach to predict the possible locations of remotely acting regulatory elements that might influence the transcriptional regulation of their associated gene(s). It represents a new way to screen for disease-relevant mutations beyond the immediate vicinity of the specific disease gene. It promises to be useful for investigating disorders in which mutations could occur in remotely acting regulatory elements.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Metilación de ADN/genética , Distrofia Muscular Facioescapulohumeral/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencia de Bases , Simulación por Computador , Epigénesis Genética , Femenino , Humanos , Masculino , Distrofia Muscular Facioescapulohumeral/patología , Mutación/genética , LinajeRESUMEN
Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a "small eye" phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.
Asunto(s)
Proteínas del Ojo/genética , Duplicación de Gen , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Tiburones/genética , Pez Cebra , Animales , Evolución Molecular , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Variación Genética , Genoma , Proteínas de Homeodominio/metabolismo , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Retina/metabolismo , Análisis de Secuencia de ADN , Vertebrados/genética , Vertebrados/crecimiento & desarrollo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/genética , Ojo/embriología , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , ARN no Traducido/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Línea Celular , Pollos/genética , Secuencia Conservada/genética , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes del Desarrollo/genética , Humanos , Ratones , Zarigüeyas/genética , Factor de Transcripción PAX6 , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Tiburones/genética , Vertebrados/genética , Xenopus/genética , Pez Cebra/genéticaRESUMEN
Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a â¼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at â¼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.
Asunto(s)
Displasia Campomélica/genética , Elementos de Facilitación Genéticos , Síndrome de Pierre Robin/genética , Factor de Transcripción SOX9/genética , Adulto , Animales , Secuencia de Bases , Displasia Campomélica/patología , Niño , Cromosomas Humanos Par 17 , Femenino , Sitios Genéticos , Humanos , Masculino , Mandíbula/anomalías , Mandíbula/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Linaje , Síndrome de Pierre Robin/patología , Pez Cebra , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The precise control of gene expression programs is crucial for the establishment of the diverse gene activity patterns required for the correct development, patterning and differentiation of the myriad of cell types within an organism. The crucial importance of non-coding regions of the genome in the control of gene regulation is well established and depends on a diverse group of sequence fragments called cis-regulatory elements that reside in these regions. Advances in novel genome-wide techniques have greatly increased the ability to identify potential regulatory elements. In contrast, their functional characterisation and the determination of their diverse modes of action remain a major bottleneck. Greater knowledge of gene expression control is of major importance for human health as disruption of gene regulation has become recognised as a significant cause of human disease. Appreciation of the role of cis-regulatory polymorphism in natural variation and susceptibility to common disease is also growing. While novel techniques such as GWAS and NGS provide the ability to collect large genomic datasets, the challenge for the twenty-first century will be to extract the relevant sequences and how to investigate the functional consequences of disease-associated changes. Here, we review how studies of transcriptional control at selected paradigm disease gene loci have revealed general principles of cis-regulatory logic and regulatory genome organisation, yet also demonstrate how the variety of mechanisms can combine to result in unique phenotypic outcomes. Integration of these principles with the emerging wealth of genome-wide data will provide enhanced insight into the workings of our regulatory genome.
Asunto(s)
Regulación de la Expresión Génica , Enfermedades Genéticas Congénitas/genética , Animales , Eliminación de Gen , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , Ratones , Modelos Genéticos , Familia de Multigenes , Neoplasias/genética , Fenotipo , Polimorfismo Genético , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The characterization of transcriptional networks (TNs) is essential for understanding complex biological phenomena such as development, disease, and evolution. In this study, we have designed and implemented a procedure that combines in silico target screens with zebrafish and mouse validation, in order to identify cis-elements and genes directly regulated by Pax6. We chose Pax6 as the paradigm because of its crucial roles in organogenesis and human disease. We identified over 600 putative Pax6 binding sites and more than 200 predicted direct target genes, conserved in evolution from zebrafish to human and to mouse. This was accomplished using hidden Markov models (HMMs) generated from experimentally validated Pax6 binding sites. A small sample of genes, expressed in the neural lineage, was chosen from the predictions for RNA in situ validation using zebrafish and mouse models. Validation of DNA binding to some predicted cis-elements was also carried out using chromatin immunoprecipitation (ChIP) and zebrafish reporter transgenic studies. The results show that this combined procedure is a highly efficient tool to investigate the architecture of TNs and constitutes a useful complementary resource to ChIP and expression data sets because of its inherent spatiotemporal independence. We have identified several novel direct targets, including some putative disease genes, among them Foxp2; these will allow further dissection of Pax6 function in development and disease.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Animales , Sitios de Unión , Linaje de la Célula , Inmunoprecipitación de Cromatina , Secuencia Conservada , Desarrollo Embrionario , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Cadenas de Markov , Ratones , Ratones Noqueados , Neuronas/metabolismo , Factor de Transcripción PAX6 , Transcripción Genética , Transgenes , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
BACKGROUND: Hyperglycemia induces chromatin remodeling with consequences on differential gene expression in mouse hepatocytes, similar to what occurs during aging. The liver is the central organ for the regulation of glucose homeostasis and xenobiotic and lipid metabolism and is affected by insulin signaling. The precise transcriptional profiling of the type-1 diabetic liver and its comparison to aging have not been elucidated yet. METHODS: Here, we studied the differential genomic expression of mouse liver cells under adult hyperglycemic and aged normoglycemic conditions using expression arrays. RESULTS: Differential gene expression involved in an increase in glucose and impaired lipid metabolism were detected in the type-1 diabetic liver. In this regard, Ppargc1a presents an increased expression and is a key gene that might be regulating both processes. The differential gene expression observed may also be associated with hepatic steatosis in diabetic mouse liver, as a secondary disease. Similarly, middle-aged mice presented differential expression of genes involved in glucose, lipid and xenobiotic metabolism. These genes could be associated with an increase in polyploidy, but the consequences of differential expression were not as drastic as those observed in diabetic animals. CONCLUSIONS: Taken together, these findings provide new insights into gene expression profile changes in type-1 diabetic liver. Ppargc1a was found to be the key-gene that increases glucose metabolism and impairs lipid metabolism impairment. The novel results reported here open new areas of investigation in diabetic research and facilitate the development of new strategies for gene therapy.
RESUMEN
Gene duplication is a major driver of evolutionary divergence. In most vertebrates a single PAX6 gene encodes a transcription factor required for eye, brain, olfactory system, and pancreas development. In zebrafish, following a postulated whole-genome duplication event in an ancestral teleost, duplicates pax6a and pax6b jointly fulfill these roles. Mapping of the homozygously viable eye mutant sunrise identified a homeodomain missense change in pax6b, leading to loss of target binding. The mild phenotype emphasizes role-sharing between the co-orthologues. Meticulous mapping of isolated BACs identified perturbed synteny relationships around the duplicates. This highlights the functional conservation of pax6 downstream (3') control sequences, which in most vertebrates reside within the introns of a ubiquitously expressed neighbour gene, ELP4, whose pax6a-linked exons have been lost in zebrafish. Reporter transgenic studies in both mouse and zebrafish, combined with analysis of vertebrate sequence conservation, reveal loss and retention of specific cis-regulatory elements, correlating strongly with the diverged expression of co-orthologues, and providing clear evidence for evolution by subfunctionalization.
Asunto(s)
Proteínas del Ojo/genética , Duplicación de Gen , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , Biología Computacional , Cartilla de ADN/genética , Elementos de Facilitación Genéticos , Evolución Molecular , Anomalías del Ojo/embriología , Anomalías del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Genes Reporteros , Prueba de Complementación Genética , Ligamiento Genético , Ratones , Ratones Transgénicos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación Missense , Factor de Transcripción PAX6 , Fenotipo , Homología de Secuencia de Ácido Nucleico , Pez Cebra/anomalías , Pez Cebra/embriologíaRESUMEN
BACKGROUND: The mouse corneal epithelium is a continuously renewing 5-6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age. RESULTS: The initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks. CONCLUSION: Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.
Asunto(s)
Epitelio Corneal/citología , Células Madre/citología , Cicatrización de Heridas/fisiología , Animales , Diferenciación Celular , Movimiento Celular , Epitelio Corneal/fisiología , Femenino , Limbo de la Córnea/citología , Ratones , Ratones Transgénicos , Células Madre/fisiología , Inactivación del Cromosoma XRESUMEN
The transcription factor Pax6 is crucial for the development of the central nervous system, eye, olfactory system and pancreas, and is implicated in human disease. While a single Pax6 gene exists in human and chicken, Pax6 occurs as a gene family in other vertebrates, with two members in elephant shark, Xenopus tropicalis and Anolis lizard and three members in teleost fish such as stickleback and medaka. However, the complement of Pax6 genes in jawless vertebrates (cyclostomes), the sister group of jawed vertebrates (gnathostomes), is unknown. Using a combination of BAC sequencing and genome analysis, we discovered three Pax6 genes in lampreys. Unlike the paired-less Pax6 present in some gnathostomes, all three lamprey Pax6 have a highly conserved full-length paired domain. All three Pax6 genes are expressed in the eye and brain, with variable expression in other tissues. Notably, lamprey Pax6α transcripts are found in the pancreas, a vertebrate-specific organ, indicating the involvement of Pax6 in development of the pancreas in the vertebrate ancestor. Multi-species sequence comparisons revealed only a single conserved non-coding element, in the lamprey Pax6ß locus, with similarity to the PAX6 neuroretina enhancer. Using a transgenic zebrafish enhancer assay we demonstrate functional conservation of this element over 500 million years of vertebrate evolution.
Asunto(s)
Encéfalo/metabolismo , Ojo/metabolismo , Lampreas/metabolismo , Factor de Transcripción PAX6/metabolismo , Páncreas/inervación , Factores de Transcripción/metabolismo , Vertebrados/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Evolución Molecular , Factor de Transcripción PAX6/química , Factores de Transcripción/química , Pez CebraRESUMEN
The nuclease-deactivated variant of CRISPR-Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable orthologous dCas9 or Cpf1 effector proteins, thus opening options for multidimensional control of functional activities through combinations of orthogonal, drug-tunable artificial transcription factors.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Genes Sintéticos/genética , ARN Guía de Kinetoplastida/genética , Animales , Proteínas Bacterianas/metabolismo , Células CHO , Proteína 9 Asociada a CRISPR , Cricetinae , Cricetulus , Endonucleasas/metabolismo , Células HEK293 , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The Pax6 transcription factor is expressed during development in the eyes and in specific CNS regions, where it is essential for normal cell proliferation and differentiation. Mice lacking one or both copies of the Pax6 gene model closely humans with loss-of-function mutations in the PAX6 locus. The sequence of the Pax6/PAX6 protein is identical in mice and humans and previous studies have shown structural conservation of the gene's regulatory regions. RESULTS: We generated a transgenic mouse expressing green fluorescent protein (GFP) and neomycin resistance under the control of the entire complement of human PAX6 regulatory elements using a modified yeast artificial chromosome (YAC). Expression of GFP was studied in embryos from 9.5 days on and was confined to cells known to express Pax6. GFP expression was sufficiently strong that expressing cells could be distinguished from non-expressing cells using flow cytometry. CONCLUSION: This work demonstrates the functional conservation of the regulatory elements controlling Pax6/PAX6 expression in mice and humans. The transgene provides an excellent tool for studying the functions of different Pax6/PAX6 regulatory elements in controlling Pax6 expression in animals that are otherwise normal. It will allow the analysis and isolation of cells in which Pax6 is activated, irrespective of the status of the endogenous locus.
Asunto(s)
Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Ratones Transgénicos/genética , Factores de Transcripción Paired Box/genética , Elementos Reguladores de la Transcripción , Proteínas Represoras/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Cromosomas Artificiales de Levadura , Secuencia Conservada , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Sustancias Luminiscentes/análisis , Ratones , Ratones Transgénicos/metabolismo , Factor de Transcripción PAX6 , Proteínas tau/genéticaRESUMEN
Pax6 is a key transcriptional regulator in eye, olfactory system, forebrain, pituitary cerebellum, spinal cord and pancreas development. Alternative splicing, promoter usage and multiple enhancers regulate the complex Pax6 spatio-temporal expression pattern. Chromosomal rearrangements which abolish PAX6 gene expression have been characterised downstream of the coding region. Through evolutionary sequence comparison and transgenic reporter studies, we have identified a new Pax6 3' cis-regulatory region. This region, C1170 Box 123, contains three distinct modules of human-mouse sequence conservation, while only Box 1 is conserved to Fugu. Both the human and the orthologous Fugu sequence direct similar reporter gene expression in the developing pretectum, neural retina and olfactory region, indicating evolutionary conservation of Pax6 regulatory mechanisms despite the low level of overall sequence conservation.