ABSTRACT
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.
Subject(s)
Chromatin Assembly and Disassembly , Genome , Molecular Imaging/methods , Nucleosomes/chemistry , Single-Cell Analysis/methods , Animals , CCCTC-Binding Factor , Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Mammalian/chemistry , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , Enhancer Elements, Genetic , G1 Phase , Gene Expression Regulation , Gene Regulatory Networks , Genome/genetics , Haploidy , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Mice , Models, Molecular , Molecular Conformation , Molecular Imaging/standards , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Promoter Regions, Genetic , Repressor Proteins/metabolism , Reproducibility of Results , Single-Cell Analysis/standards , CohesinsABSTRACT
Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment.
