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Single-cell multi-omic profiling of chromatin conformation and DNA methylome

ABSTRACT: Recent advances in the development of single cell epigenomic assays have facilitated the analysis of the gene regulatory landscapes in complex biological systems. Single-cell variations of methods such as DNA methylation-sequencing and ATAC-seq hold tremendous promise for delineating distinct cell types and identifying their critical cis-regulatory sequences. Emerging evidence in recent years has shown that in addition to cis-regulatory sequences, dynamic regulation of 3D chromatin conformation is a critical mechanism for the modulation of gene expressions during development and disease. While assays for the investigation of single-cell 3D chromatin structure have been developed, cell-type specific chromatin conformation in primary human tissues has not been extensively explored. It remains unclear whether single-cell Chromatin Conformation Capture (3C) or Hi-C profiles are suitable for cell type identification and allow the reconstruction of cell-type specific chromatin conformation maps. To address these challenges, we have developed a multi-omic method single-nucleus methyl-3C sequencing (sn-m3C) to profile chromatin conformation and DNA methylation from the same cell. We have shown that bulk m3C and sn-m3C accurately capture chromatin organization information and robustly separate mouse cell types. We have developed a fluorescent-activated nuclei sorting strategy based on DNA content that eliminates nuclei multiplets caused by crosslinking. The sn-m3C-seq method allows high-resolution cell-type classification using two orthogonal types of epigenomic information and the reconstruction of cell-type specific chromatin conformation maps.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE124391 | GEO | 2018/12/26

REPOSITORIES: GEO

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Project description:Background: DNA in the nucleus of a living cell carries out its functions in the context of a complex, three-dimensional chromatin architecture. Several recently developed methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of loci in yeast, fruit fly, human and mouse. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication. Here we describe a novel assay to map genome-wide chromatin contacts, tethered multiple 3C (TM3C), that involves a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by deep DNA paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. Results: We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, as well as a low noise-to-signal ratio between inter- and intrachromosomal contacts. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Ph+ formation in KBM7. Furthermore, we develop a two-phase mapping strategy that separately maps chimeric subsequences within a single read, allowing us to identify contacts involving three or four loci simultaneously, potentially corresponding to combinatorial regulation events. This mapping strategy also greatly increases the number of distinct binary contacts identified and, therefore, the coverage obtained for a fixed number of mapped reads. We confirm a subset of the triplet contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Assaying the genome architecture of a near-haploid cell line allows us to create 3D models of a human cell line without averaging signal from two homologous copies of a chromosome. Our 3D models of KBM7 show clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines. Conclusion: TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and complex chromatin loops that may play a role in regulating reciprocal expression of the IGF2 and H19 genes. Analysis of the spatial organization of two human cell lines (KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line) using tethered multiple 3C (TM3C), a novel and simple protocol for ascertaining genome architecture which can be used to identify simultaneous contacts among three or four loci in addition to binary contacts that can be identified using traditional chromosome conformation capture coupled with next generation sequencing (Hi-C).

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Single-cell multi-omic profiling of chromatin conformation and DNA methylome

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