# Mechanisms That Control Antigen Receptor Variable Region Exon Assembly

> **NIH NIH R01** · BOSTON CHILDREN'S HOSPITAL · 2020 · $597,511

## Abstract

Immunoglobulin (Ig) heavy chain locus (IgH) VH, D, and JH segments are clustered over megabase
(Mb) regions of the mouse IgH locus. Their assembly via V(D)J recombination is regulated in multiple contexts,
including ordered D to JH joining prior to VH to DJH joining, with the latter step regulated to promote utilization of
proximal and distal VHs to promote diverse repertoires. The IGCR1 control region in VH to D inter-genic region
mediates V(D)J recombination control via two CTCF-binding elements ("CBEs") proposed to mediate
regulatory chromatin loops. To initiate V(D)J recombination, the RAG1/2 endonuclease (RAG) cleaves V(D)J
gene segments flanked by complementary recombination signal sequence (RSS) pairs and mediates their
subsequent joining. RAG can directionally track across Mb chromosomal loops, which could allow it to locate
proper RSS pairs for cleavage activities. RAG tracking was discovered with our new LAM-HTGTS technique,
which is so sensitive that it reveals multitudes of low frequency RAG-initiated cleavage/joining events invisible
to prior assays. We have adapted the LAM-HTGTS for V(D)J repertoire sequencing (“HTGTS-Rep-seq”). We
propose four specific aims designed to build on these findings and new technologies to elucidate mechanisms
that promote joining of IgH Vs, Ds, and Js across Mb distances to generate diverse antibody repertoires. We
also propose to extend these studies to other mouse Ig loci and to human antigen receptor loci. Many
proposed studies will employ G1-arrested, RAG-inducible v-Abl transformed pro-B cell lines as a test system,
but key results will be confirmed or extended by studies of normal B lineage cells. Aim 1 proposes to elucidate
the mechanism and functional consequences of RAG tracking by using a large c-Myc locus loop domain as a
test system to obviate potential confounding effects of numerous RSSs and other regulatory elements in
antigen receptor loci. Proposed studies will evaluate contribution of RAG tracking versus other mechanisms for
capture of distant RSS pairs and elucidate factors that propel RAG tracking. Aim 2 proposes to elucidate
mechanisms that regulate IgH D to JH recombination. In IgH, Ds are located upstream of JHs within an IGCR1-
dependent domain that isolates them from upstream Vs. In this domain, D to JH recombination initiates from a
RAG-bound recombination center ("RC") comprising JHs and the most proximal D (DQ52). We will test the
hypothesis that RAG is activated in the RC by pairing of DQ52 and JH RSSs, with convergent 12RSSs of other
Ds then captured by other mechanisms, potentially including tracking. We also propose to define cis elements
required for RC formation. Aim 3 proposes to elucidate mechanisms that bring distant VHs into V(D)J
recombination domains, including unique aspects of IgH CBE organization across the IgH locus and potential
roles of VH-associated CBEs. This aim also proposes to extend studies to the Igκ light chain locus. Aim 4
proposes to extend Aim 2 an...

## Key facts

- **NIH application ID:** 9817675
- **Project number:** 5R01AI020047-38
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** Frederick W. Alt
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $597,511
- **Award type:** 5
- **Project period:** 1983-04-01 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9817675

## Citation

> US National Institutes of Health, RePORTER application 9817675, Mechanisms That Control Antigen Receptor Variable Region Exon Assembly (5R01AI020047-38). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9817675. Licensed CC0.

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