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Genome Organization Variation Uncovered With Molecular Mapping Methods

NEW YORK (GenomeWeb) – Genome organization can vary significantly within cells and by allele, according to a team from the National Cancer Institute, Harvard University, the University of Massachusetts, and the Massachusetts Institute of Technology.

"Our findings reveal a remarkably high degree of cell-to-cell and allele-to-allele heterogeneity in spatial genome organization," senior author Tom Misteli, director of NCI's Center for Cancer Research, and his co-authors wrote.

The researchers used high-throughput chromosome conformation capture (Hi-C) mapping and high-throughput fluorescence in situ hybridization (hiFISH) optical mapping to analyze genome organization and self-associating topologically associating domain (TAD) patterns in human foreskin fibroblast cells. Together with single-cell, bacterial artificial chromosome probe-based TAD profiling, allele-specific single nuclei probing, and published Hi-C data for another cell type, the data offered a look at genome organization patterns within and between alleles. 

The team's results, reported online today in Cell, revealed genome organization, TAD structure flexibility, and allele interaction independence, though physical interactions involving distinct genome regions appeared to occur only rarely.

Using existing Hi-C chromosome cross-linking protocols and Illumina sequencing, the researchers generated four technical replicates of human foreskin fibroblasts, each composed of 25 million cells. The resulting data were binned to distinguish between nearby, mid-range, or distant interactions in those cells, they explained, and subsequently segmented and diluted in situ to assess TAD boundary domains in the human foreskin fibroblasts.

Along with comparisons to available dilution and in situ datasets for the human lung fibroblast cell line IMR90, the team used hiFISH imaging, automated imaging analyses, and statistical approaches to explore the two-dimensional and three-dimensional interactions within the cells profiled.

"Our method overcomes the limitations of traditional biochemical mapping methods that generate population averaged datasets," they explained, "and at the same time elevates traditional imaging methods beyond their anecdotal nature due to small sample sizes."

To that, the team added interaction frequencies in relation to physical distance between alleles for 125 pairs of interacting loci with Hi-C data as well as allele-focused TAD interaction profiles in single cell nuclei, demonstrating that the heterogeneity observed reflected variation by allele within individual cells rather than variation between cells alone.

From these and other findings, including between-TAD interaction profiles, the authors proposed a model involving "dynamic and variable chromatin structures, interacting with dynamic and variable protein complexes" that "act in concert to provide a stable transcriptional response across a population, or in single cells over time."

While additional data is needed to explore the mechanisms used to maintain functional stability despite such organizational heterogeneity and variation, they explained, the results "support a view of genome organization as plastic, heterogeneous, and marked by variability between individual cells and alleles."