RESUMEN
The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.
Asunto(s)
Encéfalo/citología , Células/clasificación , Ensamble y Desensamble de Cromatina , Cromatina/química , Cromatina/genética , Genes , Conformación Molecular , Animales , Sitios de Unión , Células/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Familia de Multigenes/genética , Neuronas/clasificación , Neuronas/metabolismo , Desnaturalización de Ácido Nucleico , Factores de Transcripción/metabolismoRESUMEN
Changes in gene expression represent an important source of phenotypic innovation. Yet how such changes emerge and impact the evolution of traits remains elusive. Here, we explore the molecular mechanisms associated with the development of masculinizing ovotestes in female moles. By performing integrative analyses of epigenetic and transcriptional data in mole and mouse, we identified the co-option of SALL1 expression in mole ovotestes formation. Chromosome conformation capture analyses highlight a striking conservation of the 3D organization at the SALL1 locus, but an evolutionary divergence of enhancer activity. Interspecies reporter assays support the capability of mole-specific enhancers to activate transcription in urogenital tissues. Through overexpression experiments in transgenic mice, we further demonstrate the capability of SALL1 to induce kidney-related gene programs, which are a signature of mole ovotestes. Our results highlight the co-option of gene expression, through changes in enhancer activity, as a plausible mechanism for the evolution of traits.
Asunto(s)
Riñón , Topos , Animales , Femenino , Ratones , Riñón/metabolismo , Ratones Transgénicos , Topos/genéticaRESUMEN
The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.
Asunto(s)
Deformidades Congénitas de las Extremidades/genética , Quinasas Quinasa Quinasa PAM/genética , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Cocultivo , Endonucleasas , Exoma , Femenino , Humanos , Escala de Lod , Quinasas Quinasa Quinasa PAM/química , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación Missense , Linaje , Polimorfismo de Nucleótido Simple , Proteínas Quinasas/química , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Congenital central hypoventilation syndrome (CCHS) is a rare life-threatening disorder of respiratory and autonomic regulation. It is classically caused by dominant mutations in the transcription factor PHOX2B. The objective of the present study was to identify the molecular cause of a recessive form of central hypoventilation with autonomic dysfunction. METHODS: Here, we used homozygosity mapping and whole-genome sequencing in a consanguineous family with CCHS in combination with functional analyses in CRISPR/Cas9 engineered mice. RESULTS: We report on a consanguineous family with three affected children, all tested PHOX2B mutation negative, presenting with alveolar hypoventilation and symptoms of autonomic dysregulation. Whole-genome sequencing revealed a homozygous frameshift mutation in exon 25 of the MYO1H gene (c.2524_2524delA) segregating with the phenotype in the family. MYO1H encodes for the unconventional myosin IH, which is thought to function as a motor protein in intracellular transport and vesicle trafficking. We show that Myo1h is broadly expressed in the mouse lower medulla, including the CO2-sensitive Phox2b+ retrotrapezoid neurons. To test the pathogenicity of the variant, we engineered two Myo1h mutant mouse strains: the first strain (Myo1h*) resembling the human mutation and the second being a full knock-out (Myo1hFS ). Whole-body plethysmography studies in Myo1h* newborns with the re-engineered human mutation revealed hypoventilation and a blunted response to CO2, recapitulating the breathing phenotype observed in the kindred. CONCLUSIONS: Our results identify MYO1H as an important gene in CO2 sensitivity and respiratory control and as the cause of a rare recessive form of congenital central hypoventilation.
Asunto(s)
Enfermedades del Sistema Nervioso Autónomo/genética , Genes Recesivos , Hipoventilación/genética , Miosina Tipo I/genética , Animales , Enfermedades del Sistema Nervioso Autónomo/complicaciones , Enfermedades del Sistema Nervioso Autónomo/congénito , Consanguinidad , Mutación del Sistema de Lectura , Humanos , Hipoventilación/complicaciones , Hipoventilación/congénito , Ratones , Mutagénesis Sitio-Dirigida , Linaje , Secuenciación Completa del GenomaRESUMEN
Catel-Manzke syndrome is characterized by Pierre Robin sequence and a unique form of bilateral hyperphalangy causing a clinodactyly of the index finger. We describe the identification of homozygous and compound heterozygous mutations in TGDS in seven unrelated individuals with typical Catel-Manzke syndrome by exome sequencing. Six different TGDS mutations were detected: c.892A>G (p.Asn298Asp), c.270_271del (p.Lys91Asnfs(∗)22), c.298G>T (p.Ala100Ser), c.294T>G (p.Phe98Leu), c.269A>G (p.Glu90Gly), and c.700T>C (p.Tyr234His), all predicted to be disease causing. By using haplotype reconstruction we showed that the mutation c.298G>T is probably a founder mutation. Due to the spectrum of the amino acid changes, we suggest that loss of function in TGDS is the underlying mechanism of Catel-Manzke syndrome. TGDS (dTDP-D-glucose 4,6-dehydrogenase) is a conserved protein belonging to the SDR family and probably plays a role in nucleotide sugar metabolism.
Asunto(s)
Deformidades Congénitas de la Mano/genética , Oxidorreductasas/genética , Síndrome de Pierre Robin/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Preescolar , Exoma/genética , Femenino , Deformidades Congénitas de la Mano/enzimología , Haplotipos , Heterocigoto , Homocigoto , Humanos , Lactante , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxidorreductasas/metabolismo , Linaje , Síndrome de Pierre Robin/enzimología , Alineación de Secuencia , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
The brain comprises many different cell types with specialized functions which respond and adapt to the continuously changing environment, through tight spatiotemporal regulation of gene expression. The three-dimentional (3D) organisation of the genome is increasingly recognized as a major feature of gene regulation in brain cells, for the activation, repression and poising of gene expression, and in coupling transcription with RNA processing and transport. Here, we discuss the importance of dynamic chromatin organisation in the developmental patterning of the brain, and its role in fine tuning brain activity and plasticity. A better understanding of how disease-associated mutations interfere with chromatin organisation and long-range gene regulation will help reveal the molecular mechanisms underlying complex neurodevelopmental and neuropsychiatric disorders.
Asunto(s)
Encéfalo/ultraestructura , Cromatina/ultraestructura , Trastornos Mentales/genética , Trastornos del Neurodesarrollo/genética , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Cromatina/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genoma Humano/genética , Humanos , Mutación/genética , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
The genome requires tight regulation in space and time to maintain viable cell functions. Advances in our understanding of the 3D genome show a complex hierarchical network of structures, involving compartments, membraneless bodies, topologically associating domains, lamina associated domains, protein- or RNA-mediated loops, enhancer-promoter contacts, and accessible chromatin regions, with chromatin state regulation through epigenetic and transcriptional mechanisms. Further technology developments are poised to increase genomic resolution, dissect single-cell behaviors, including in vivo dynamics of genome folding, and provide mechanistic perspectives that identify further 3D genome players by integrating multiomics information. We highlight recent key developments in 4D nucleome methodologies and give a perspective on their future directions.
Asunto(s)
Nucleosomas/metabolismo , Investigación , Animales , Cromatina/metabolismo , Genoma , Genómica , HumanosRESUMEN
Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.
Asunto(s)
Adaptación Fisiológica/genética , Factor 9 de Crecimiento de Fibroblastos/genética , Topos/genética , Elementos Reguladores de la Transcripción , Diferenciación Sexual/genética , Esteroide 17-alfa-Hidroxilasa/genética , Animales , Inversión Cromosómica , Conjuntos de Datos como Asunto , Femenino , Regulación de la Expresión Génica , Genoma , Ratones , Ratones Transgénicos , Secuencias Repetidas en Tándem , Testosterona/sangre , Testosterona/genéticaRESUMEN
The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity and specificity in vivo and its disturbance can result in gene misexpression and disease.
Asunto(s)
Cromatina/química , Elementos de Facilitación Genéticos/fisiología , Miembro Posterior/embriología , Conformación Molecular , Morfogénesis/genética , Factores de Transcripción Paired Box/fisiología , Animales , Sistemas CRISPR-Cas , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , ADN/química , ADN/metabolismo , Embrión de Mamíferos , Miembro Anterior/embriología , Miembro Anterior/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Miembro Posterior/metabolismo , Ratones , Ratones Transgénicos , Conformación de Ácido Nucleico , Factores de Transcripción Paired Box/genéticaRESUMEN
Structural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements.