RESUMO
The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.
Assuntos
Montagem e Desmontagem da Cromatina , Genoma , Imagem Molecular/métodos , Nucleossomos/química , Análise de Célula Única/métodos , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Elementos Facilitadores Genéticos , Fase G1 , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genoma/genética , Haploidia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Imagem Molecular/normas , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única/normas , CoesinasRESUMO
Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.
Assuntos
Cromatina/ultraestrutura , Cromossomos/ultraestrutura , Biologia Molecular/métodos , Conformação Molecular , Células-Tronco Embrionárias Murinas , Imagem Óptica/métodos , Animais , Células Cultivadas , Imageamento Tridimensional/métodos , Camundongos , Análise de Célula Única/métodosRESUMO
Recent developments have for the first time allowed the determination of three-dimensional structures of individual chromosomes and genomes in nuclei of single haploid mouse embryonic stem (ES) cells based on Hiâ»C chromosome conformation contact data. Although these first structures have a relatively low resolution, they provide the first experimental data that can be used to study chromosome and intact genome folding. Here we further analyze these structures and provide the first evidence that G1 phase chromosomes are knotted, consistent with the fact that plots of contact probability vs sequence separation show a power law dependence that is intermediate between that of a fractal globule and an equilibrium structure.