Elucidating Mechanisms of RAG Endonuclease Mediated Feedback Inhibition of V(D)J Recombination

PROJECT SUMMARY/ABSTRACT
A hallmark of adaptive immunity is mono-allelic expression (allelic exclusion) of B and T cell antigen receptor
proteins, which is thought to allow highly-specific immune responses and inhibit autoimmunity. Allelic exclusion
is achieved via mono-allelic initiation of RAG1/RAG2 (RAG) nuclease-mediated V gene segment recombination,
followed by protein from a V(D)J rearrangement signaling permanent feedback inhibition of V recombination on
the other allele. A shortcoming of this control is that it needs time for completion of recombination, expression
and signaling of protein, and changes on the second allele. The applicant demonstrated that RAG DNA double
strand breaks (DSBs) induced during Vk-to-Jk recombination in pre-B cells signal via the ATM kinase to inhibit
Rag1/Rag2 expression, Jk accessibility, and RAG cleavage of the other allele until after the first allele is repaired.
He showed that ATM deficiency in mice increases bi-allelic Igk IgH, or TCRb expression. While these data imply
that ATM enforces allelic exclusion by signaling DSB feedback inhibition of V recombination, they neither prove
this model nor rule out a role for ATM-stimulated DSB repair. The applicant shows new data that ATM enforces
Igk allelic exclusion by signaling via the NFkB essential modulator (NEMO) protein, with inhibition of Rag1/Rag2
transcription likely key for inter-allelic control of Vk recombination. The applicant shows that RAG DSBs during
IgH D-to-J recombination in pro-B cells or TCRb D-to-J, TCRg, and/or TCRd recombination in DN thymocytes do
not require ATM to repress Rag1/Rag2 expression. However, these RAG TCR DSBs signal via ATM to repress
expression of a Vb region anti-sense long non-coding RNA, which the applicant shows is expressed only in DN
cells where Vb and Db-Jb segments interact and rearrange over vast genomic distances. Based on these data,
the applicant hypothesizes that RAG DSBs feedback inhibit V(D)J recombination through complementary
mechanisms, including cell type-specific signaling pathways that repress Rag1/Rag2 transcription and antigen
receptor locus-specific alterations that suppress V rearrangements. He proposes two independent aims to test
fundamental aspects of his model. Aim 1 proposes to elucidate how RAG DSBs induced during different types
of rearrangements in different cell types signal repression of Rag1/Rag2 transcription to coordinate initiation of
V-to-(D)J recombination between alleles and thereby enforce allelic exclusion. Aim 2 proposes to determine the
role of V region anti-sense long non-coding RNAs in promoting long-range V-to-(D)J rearrangements and serving
as a DSB-responsive switch to transiently inhibit these rearrangements and thereby orchestrate allelic exclusion.
The proposed work will employ powerful mouse models to rigorously elucidate mechanisms by which RAG DSBs
trigger transient feedback inhibition of V recombination to help enforce allelic exclusion. The project will provide
novel mechanistic insights into one understudied and one completely novel line of research in the field, the latter
relevant to the biology all cells. Beyond advancing understanding of a hallmark of adaptive immunity, the findings
could identify mechanisms important for suppressing autoimmunity and/or lymphoid malignancies.

Grant Number: 5R01AI172163-04
NIH Institute/Center: NIH
Principal Investigator: CRAIG BASSING