The genome of higher eukaryotes has a complex three-dimensional spatial structure, such as chromatin loops (Chromatin loops), topologically associated domains (TADs), active / inactive chromatin compartments (A / B compartments), and chromosomal domains (Chromosome territories) at different scales. These structures are important for the maintenance of genome stability, the precise regulation of gene expression, thereby influencing cell fate determination and phenotype establishment. The classical 3 D structure of the genome is mainly revealed by various high-throughput technologies represented by chromosome conformation capture (3C) and its derived methods such as 4Cs, 5C, Hi-C, and ChIA-PET. These technologies can capture pairwise DNA sequences in the nucleus, but fail to capture synergistic multisite interactions (multi-way contact) and single-molecule topology (single-allele topology) in cell populations. Furthermore, genomic 3D structures change dynamically during cell cycle, development and differentiation and are associated with chromatin interactions in multiple genes and regulatory intervals. Obtaining the chromosome single-molecule topology in a cell population is important to probe the dynamic folding mechanism of the genome and the association with gene regulatory functions.
Hou Chunhui, a researcher of Kunming Institute of Zoology of Chinese Academy of Sciences, and Xiao Chuanle, an associate researcher of Zhongshan Ophthalmology Center of Sun Yat-sen University, published a research paper entitled High-throughput Pore-C reveals the single-allele topology and cell type-specificity of 3D genome folding in Nature Communications (Nature Communications). This work optimizes a high-throughput Pore-C method, significantly increases the detection flux of higher-order chromatin interactions, and reveals the single-molecule topological diversity and cell specificity of the three-dimensional genome.
The reason for the relatively low sequencing throughput of the Pore-C technology may be the blockage of the sequencing nanopore core because the protein crosslinked to the DNA is not completely removed. To address this issue, studies optimized enzymatic conditions, tested the strategy of multiple proteolysis and using mixed proteases, increased the sequencing yield by about 80%, and nearly multiplied the use cost of this technology. In addition, we developed the MapPore-C alignment process by integrating NGMLR and Minimap2 alignment algorithms, which significantly improved the alignment accuracy and low data utilization. Meanwhile, we verified that HiPore-C can highly reproduce Hi-C capture-based chromatin loops, topology-associated domains and chromatin region compartments by comparison with Hi-C data. Further, the study explored the high order interaction between chromosomes, most interactions is not occur between telomeres and centrioles, but in genomic regions, and form two transcriptional activity different interaction hub, one hub gene density, enhancer density and active state of chromatin related epigenetic modification levels are higher. The study also found that high-frequency interactions across chromosomes occur between tRNA gene-enriched regions of multiple chromosomes. HiPore-C high-order interactions not only occur within TAD and compartment, but also span multiple compartments, topologically-related domains and chromatin loops. Chromatin interaction maps based on direct and indirect DNA fragment interactions are generally similar to conventional Hi-C maps, but indirect DNA fragment interactions are more likely to span multiple structural units. The above studies reveal the universality of the existence of interactions across chromatin domains and highlight the advantages and importance of HiPore-C technology in resolving three-dimensional high-order interactions at the single-molecule level.
We discuss the single molecule topology clusters in the topology of different types of cells. These structural clusters are the basis for the formation of sub-TAD-like (subTAD-like) domains, with obvious cell specificity, indicating that single molecule topological diversity is the basis of TAD domain division in cell populations, and is of great significance to explore the relationship between spatial organization of genome and cell-specific gene expression. Furthermore, studies used HiPore-C data to compare higher-order interactions in β -globin locus in erythroid GM12878 cells. We found that multi-site simultaneous, cell-specific enhancer-promoter centers form between the human ε -and γ -globin gene promoters and multiple enhancers, and that this interaction may be dynamic. The ability of HiPore-C to capture both high-order interaction and DNA methylation status, and the positive correlation between DNA methylation signal and the interaction strength between chromatin loop anchors, in addition, the type of chromatin compartment can be accurately distinguished according to DNA methylation level (A vs B). This study established the HiPore-C technique, which can comprehensively describe the diversity of single molecule topology, and revealed the dynamic folding of single molecule topology is more complex than previously thought, which further enhanced the cognition of the law of three-dimensional genome folding.
