CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning [dataset 3]

RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function. We performed 3C-HTGTS (high throughput, genome-wide translocation sequencing) on v-Abl kinase transformed pro-B cell lines using different baits to examine the interaction profiles of different loci and their dependence on VH-CBEs. All lines analyzed were RAG2-deficient derived from a parental WT line that harbors a non-productive VDJH rearrangement involving a distal VHJ558 (VH1-2P) which deletes the entire proximal VH domain on one allele while the other allele harbors a DHFL16.1 to JH4 rearrangement (DJH allele). The DJ allele was genetically manipulated to derive the various mutants.