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High order chromatin contact in regulatory regions with the Pore C approach
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International audience. In humans, gene transcription is controlled by the promoter, located immediately upstream of the genes, and enhancers, which are localized at a distance from their target genes. Recent studies suggest the existence of complex interactions involving multiple enhancers and promoters [1]. The precise nature of these hubs remains largely unknown: the Hi-C (High-throughput Chromosome Conformation Capture), currently the preferred approach for analyzing physical interactions between chromatin regions at the genome scale, is highly ineffective in detecting interactions involving more than two regions. The Pore-C approach [2] has recently been developed to overcome the aforementioned limitation. Similar to Hi-C, Pore-C begins with cross-linking cells to stabilize physical interactions that naturally occur within the nucleus. Chromatin is digested into smaller fragments by a selected restriction enzyme and digested fragments located close to each other are then associated by ligation. The resulting long chimeric DNA fragments (>10kb), also called concatemers, are finally sequenced using the Oxford Nanopore Technology (ONT).We carried out Pore-C experiments on the human B lymphocyte line (NA12878). We tested two different restriction enzymes for chromatin fragmentation. We selected DpnII, which produces fragments of 0.5-5kb and NlaIII, which digests the genome into smaller fragments. We sequenced the Pore-C libraries on the ONT promethION platform. We mapped fragments (monomers) in chimericPore-C long reads to the reference genome hg38 using the Pore-C Workflow[2] to reconstitute the original concatemers.To assess the quality of our Pore-C data, we re-analyzed public Pore-C datasets[2], generated from the same NA12878 cell line with DpnII and NlaIII, with an equivalent library depth. We showed that stratum-adjusted correlation coefficients were very high between our in-house and their corresponding public Pore-C datasets, indicating a high level of reproducibility.We also used a standard Hi-C dataset generated in our lab on NA12878 (with DpnII enzyme). To facilitate the comparison with Hi-C, we generated virtual pairwise contacts by decomposing each Pore-C concatemers into all possible monomer pairs. Pore-C datasets (public and in-house) have a significantly lower number of virtual pairwise contacts than in Hi-C. Despite this, we visualized in the “virtual” contact maps the same high-order structures as in Hi-C.We analyzed the cardinality of concatemers, which corresponds to the number of monomers per concatemer, between public and in-house datasets and we found that the cardinality distributions were very similar. As previously described in Deshpande et al., Pore-C datasets have higher cardinalities with NlaIII compared to DpnII with an equivalent distribution of read length. This indicates that NlaIII is more suitable for identifying multi-way contacts in chromatin.The Chromunity package [2] identifies high-order contacts from Pore-C data by detecting regions that were frequently associated in concatemers. We utilized Chromunity on both our in house and public NlaIII Pore-C datasets, focusing on intra-chromosomal interactions. We optimized various parameters (such as bin size, sequencing depth, and the number of cardinality tested) to ensure significant results while taking into account our computational system's limitations. By analyzing concatemers overlapping enhancer and promoter regions, we detected significant multi-way contacts (cardinality>4) that are reproducible between our in house and public datasets. Additionally, we conducted hierarchical clustering of multi-way contacts in a region of interest to detect alternative chromatin conformations that could correspond to various cell states [3]. Our analysis revealed consistent and complementary outcomes by overlapping HiPore-C clusters with frequently interacting regions identified by Chromunity.
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