Abstract
There is an established relationship between primary DNA sequence, secondary and tertiary chromatin structure, and transcriptional activity, suggesting that observed differences in one of these properties may reflect changes in the others. Here, we exploit these relationships to show that variations in DNA structure can be used to identify a wide range of genomic alterations in mammalian samples. In this proof-of-concept study we characterized and compared genome-wide histone occupancy by ChIP-Seq, DNA accessibility by ATAC-Seq, and chromosomal conformation by Hi-C for five CRISPR/Cas9-modified mammalian cell lines and their unmodified parent strains, as well as in one modified tissue sample and its parent strain. The results showed that the impact of genomic alterations on each of the levels of DNA organization varied depending on mutation type (insertion or deletion), size, and genomic location. The largest genomic alterations we identified included chromosomal rearrangements and deletions (greater than 200 Kb) in four of the modified cell lines, which can be difficult to identify by standard whole genome sequencing analysis. This multi-level DNA organizational analysis provides a sensitive approach for identifying a wide range of genomic and epigenomic perturbations that can be utilized for biomedical and biosecurity applications.
Highlights
DNA does not exist in the cell as a naked, linear molecule, but rather is compacted into a highly organized three-dimensional structure that exerts genome-wide influence over gene expression levels [1, 2]
The mutant samples consisted of three edited human cell lines (BRD4, TOP2B, SMARCA4) derived from a single HAP1 parent strain, two edited mouse embryonic stem cell lines (Sox2, SE15) derived from a single F123 parent strain [33,34], and one edited mouse liver tissue sample (Kcnc3) derived from a C57BL/6NJ parent strain
We describe how multi-level DNA structural analysis can be leveraged to identify mutations in the genome that range in scale from small nucleotide variations (SNVs) to small and large (Kb) indels and chromosomal rearrangements (Mb) (Fig 5)
Summary
DNA does not exist in the cell as a naked, linear molecule, but rather is compacted into a highly organized three-dimensional structure that exerts genome-wide influence over gene expression levels [1, 2]. The DNA accessibility, and subsequently transcriptional activity, of a given locus can vary between cell types or physiological conditions depending on the presence of specific DNAbinding proteins that can covalently modify histone proteins and disrupt nucleosome stability [5, 6]. These strings of nucleosomes are further organized into a higher level tertiary structure consisting of loops that are formed by protein-mediated promoter-enhancer interactions, which in turn are contained within larger topological associating domains (TADs) [7, 8]. Gene Description Histone binding protein that helps maintain chromatin structure during mitosis
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