Unravelling global genome organization by 3C-seq

Abstract

Eukaryotic genomes exist in the cell nucleus as an elaborate three-dimensional structure which reflects various nuclear processes such as transcription, DNA replication and repair. Next-generation sequencing (NGS) combined with chromosome conformation capture (3C), referred to as 3C-seq in this article, has recently been applied to the yeast and human genomes, revealing genome-wide views of functional associations among genes and their regulatory elements. Here, we compare the latest genomic approaches such as 3C-seq and ChIA-PET, and provide a condensed overview of how eukaryotic genomes are functionally organized in the nucleus.

Fig. 1

Application of NGS for capturing genome-wide associations. The experimental procedures for GCC, Hi-C, biotin-based yeast method, ELP and ChIA-PET are schematically illustrated. All the methodologies start with fixation of in vivo chromatin structures. The 3C-seq method employs 3C, followed by the procedures designed for concentration of small hybrid DNA molecules reflecting genomic associations. In the ChIP-PET method, the fixed samples are immunoprecipitated (ChIP) and the hybrid DNA molecules are enriched by biotin–streptavidin bead purification. The resultant samples are processed by paired-end sequencing.

Fig. 2

Processing of 3C-seq raw data to detect genome-wide associations. NGS with PE module provides more than 10 million paired sequence information for hybrid DNA molecules, which reflect genomic associations. Those paired-end reads are mapped to genomic positions, and reads are eliminated based on four different filtering processes (labeled a–d). The paired-end reads reflect short-range associations (a), and associations involving repetitive DNA sequences (b) are eliminated. The paired reads assigned to regions where restriction enzyme sites are not present nearby are also eliminated, because it is not clear how these hybrid DNA molecules are produced during the 3C-seq procedures (c). The redundant paired-end reads with the exact same sequences are counted as a single hybrid molecule to avoid PCR bias (d). The remaining paired-end reads are then examined to identify long-range genomic associations. Numbers of paired-end reads assigned to genomic combinations are used for score calculation.

Fig. 3

Genome organization from three points of view. (A) Chromosome territories are depicted in different colors. An enlarged view indicates intermingling chromatin fibers derived from two chromosome territories. (B) Nuclear domains: Multiple architectural components and various nuclear bodies are involved in global genome organization. (C) Cohesin mediates enhancer–promoter interaction through its interaction with mediator (left), and also associations among genes and their regulatory elements via CTCF (right).

Fig. 4

Gene associations mediated by transcription factors. Recent studies from fission yeast and mammals imply that transcription factors mediate colocalization of their target genes at RNA Pol II-enriched subnuclear domains. This gene arrangement potentially facilitates coordinated regulation and efficient expression of a group of genes dispersed throughout the genome.