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Abstract
BackgroundProximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature.ResultsThrough analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development.ConclusionsOur observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.
Highlights
Proximity ligation based techniques, like High-throughput Chromosome Conformation Capture (3C) (Hi-C), involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin
It is recognized that the eukaryotic genome is hierarchically organized into self-interacting topologically associated domains (TADs), which can have distinct chromatin states that are insulated from neighbourhood through boundaries marked with CCCTC-binding factor (CTCF), Cohesins, ZNF143 and TOP2b factors [11,12,13,14]
We show that the differential visibility of genomic loci to the restriction endonucleases used in HiC protocols induct potential bias in Hi-C data
Summary
Like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and the visibility in the contact maps, of engaged loci. The three-dimensional genome organization is tightly linked with the regulation of essential genomic functions like transcription, replication and genome integrity [1,2,3,4,5]. While the significance of genome organization has been realized for decades, the comprehensive evidence emerged somewhat recently through the advent of proximity ligation based techniques like Chromosome Conformation. It is recognized that the eukaryotic genome is hierarchically organized into self-interacting topologically associated domains (TADs), which can have distinct chromatin states that are insulated from neighbourhood through boundaries marked with CCCTC-binding factor (CTCF), Cohesins, ZNF143 and TOP2b factors [11,12,13,14]. CTCF binding is transiently lost during pro-metaphase, which coincides with the loss of TAD structures during M-phase [21,22,23]
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