7. Project Summary/Abstract. During the past funding period, we discovered that the cohesin-mediated loop extrusion process involved in genome-wide modulation of chromosome architecture plays fundamental roles in V(D)J recombination and the generation of antibody diversity. In developing progenitor B cells, we found that cohesin-mediated loop extrusion linearly presents Ig heavy chain locus (Igh) VH, D, and JH gene segments to the RAG endonuclease for V(D)J recombination. Our preliminary data indicate that long-range V(D)J recombination in the Igk light chain locus may occur, at least in part, by a mechanistically distinct process from that of Igh. We propose 2 specific aims to elucidate the potentially differential mechanisms of Igh versus Igk long-range V(D)J recombination. A major hypothesis guiding Aim 1 and Aim2 studies is that Igh achieves long range V(D)J recombination via linear RAG chromatin scanning that leads to predominantly deletional recombination events. A major hypothesis to be tested for Aim 2 is that Igk is structurally-optimized to employ a related loop extrusion-based mechanism that accommodates both robust deletional and inverted Vk-to- Jk joining. These hypotheses are supported by a wealth of published and preliminary data derived in large part from powerful new technologies that we developed during the current funding period. In particular we developed LAM-HTGTS-V(D)J-Seq to assay V(D)J recombination with unprecedented sensitivity and LAM-3C- HTGTS to map sequence interactions across chromatin domains at far higher resolution than prior assays. We further developed G1-arrested, RAG inducible v-Abl transformed pro-B cell cell ("v-Abl cell") approaches to test roles specific cis elements or trans-acting factors in long-range RAG chromatin-scanning through introduced Igh or Igk locus modifications and/or targeted protein depletion. Aims 1 and 2 experiments together will compare and contrast, in depth, the long-range mechanisms used by Igh and Igk to incorporate Vs into the V(D)J recombination reaction. While most initial Aim 1 and 2 studies will employ v-Abl cells; all key results will be confirmed/extended by studies of normal progenitor and precursor B cell populations These studies may reveal new paradigms for understanding V(D)J recombination in vivo and illuminate the range of mechanisms employed for long-range V(D)J recombination in antigen receptor loci. Addressing Aim 1 and 2 goals should greatly impact the immunology field by providing major new insights into fundamental mechanisms that establish highly diverse primary antibody repertoires. The studies proposed in Aim 1 and 2 will also further elucidate how impediments in the scanning path focus cryptic RSS targets within impeded areas of chromatin for incorporation into the RAG complex and subsequent rearrangement. Thus, these studies should provide critical information for understanding how RAG targets cryptic RSSs in other genes and promotes common translocations ...