5C Chromatin interactions in the Igh locus in B cell progenitors

These tracks include chromatin interaction data produced using the 5C (chromatin conformation capture carbon copy) method by the Kenter Lab located at the University of Illinois College of Medicine, Chicago, IL. These tracks show looping interactions across the Igh locus using alternating forward and reverse primer design scheme. The 3C method uses formaldehyde crosslinking to covalently associate interacting chromatin segments in intact cells. The cells are then lysed and chromatin is digested with the Hind III restriction enzyme. The digested fragments are ligated under dilute conditions to promote intra-molecular ligation. A genome-wide interaction library of ligation products corresponding to all possible chromatin interactions is produced by this method. In 5C assays, over 12,000 potential ligation products specific to the Igh locus are detected by PCR using multiplexed primer pairs. These studies have revealed three conserved topological sub-domains. Each topological folds encompasses a major VH gene family. The Pax5 transcription factor organizes the topological sub-domain that spans the VHJ558 gene family. In the absence of Pax5, the distal J558 VH genes fail to associate with the proximal VH genes, thus providing an explanation for reduced VHJ558 gene rearrangements in Pax5-deficient pro-B cells. We propose that Igh locus contraction is the cumulative effect of several independently controlled chromatin sub-domains that provide the structural infrastructure to coordinate optimal antigen receptor assembly (Montefiori et al. 2015). We also note that the configuration of the 3’ end of the Igh locus spanning the CH region genes assumes a developmental stage specific configuration in pro-B cells that appreas to skew class switch recombination to specific isotypes. This 3D spatial configuration is lost in resting splenic B cells that are capable of class switch recombination to all isotypes (Kumar et al. 2013). The aim of this study was to create a comprehensive map of chromatin interactions spanning the entire 2.9 Mb Igh locus in murine embryonic fibroblasts (MEF), pro-B cells and resting splenic B cells. 5C primers follow an alternating pattern and were designed at HindIII restriction sites using the 5C primer design tools that are available online (http://my5C.umassmed.edu) (Lajoie et al., 2009) and were previously reported (Kumar et al., 2013). In this experimental scheme, 112 forward primers and 113 reverse primers were designed for all possible HindIII restriction fragments. This design allowed for the interrogation of many (but not all) restriction fragments, thus an unbiased interaction map across the locus and for potentially identifying new regulatory elements. An alternating scheme of forward and reverse primers was also included for a gene desert on chromosome 5 using 24 forward and 25 reverse 5C primers for a total of 525 potential cis interactions. Primers settings were: U-BLAST: 3; S-BLAST: 80: 15-MER: 800; MIN_FSIZE: 1000; MAX_FSIZE: 15,000; OPT_TM: 65; OPT_PSIZE: 30. Several primers with somewhat relaxed parameters were included to provide a more complete coverage of the Igh locus. Currently, data for two biological replicates have been generated for murine embryonic fibroblasts (MEF), progentior B cells and resting splenic B cells spanning the murine Igh locus and a gene desert on chromosome 5 using high-throughput paired-end sequencing in the Illumina GAII platform. The 5C looping interactions that are detected in both the biological replicates are considered significant. ------------------------ Extremely long range chromatin loops link topological domains to facilitate a diverse antibody repertoire Lindsey Montefiori, Robert Wuerffel, Damian Roqueiro, Bryan Lajoie, Changying Guo, Tatiana Gerasimova, Supriyo De, William Wood, Kevin G. Becker, Job Dekker, Jie Liang, Ranjan Sen, and Amy L. Kenter, Cell Reports in press, 2015. Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny. Kumar, S., Wuerffel, R., Achour, I., Lajoie, B., Sen, R., Dekker, J., Feeney, A.J., Kenter, A.L., Genes Dev. 2013 Nov 15;27(22):2439-44. doi: 10.1101