Mechanisms That Control Antigen Receptor Variable Region Exon Assembly

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 or interstitial deletions frequently found in B and T lymphocyte cancers of developing
lymphocytes in humans.

Grant Number: 4R01AI020047-43
NIH Institute/Center: NIH
Principal Investigator: Frederick Alt