# Chromatin looping directed RAG targeting during V(D)J recombination

> **NIH NIH R01** · WESTERN MICHIGAN UNIV SCHOOL OF MEDICINE · 2021 · $377,500

## Abstract

Project Abstract
As a critical part of adaptive immunity, generation of a highly diverse antibody repertoire begins with efficient
assembly of the immunoglobulin heavy chain locus (IgH) through V(D)J recombination during early B cell
development. At IgH locus, hundreds of VHs are widely-spread within a 2.4 Mb upstream region and multiple
DHs and JHs are located within a downstream 0.3 Mb domain. The lymphoid-specific RAG1/RAG2 (RAG)
endonuclease initiates V(D)J recombination by cutting paired recombination signal sequences (RSSs) flanking
V, D, and J segments. A major knowledge gap is what mechanisms bring the RSS pairs, which are widely
separated in chromosomal distance, into spatial proximity for RAG cleavage. Although 3D genome topology
and long-range chromatin interaction at IgH locus has been implicated to play important roles in directing RAG
targeting, understanding the underlying mechanisms is greatly hindered due to lack of high-resolution
chromatin interaction maps and efficient systems to comprehensively characterize the putative regulatory
elements. The overall objective of this proposal is to determine the molecular mechanisms driving functional
chromatin interactions mediating IgH RAG targeting. We have recently revealed a novel mechanism driving D
to JH recombination, whereby cohesin-mediated chromatin loop extrusion propels RAG scanning within D-JH
domain to promote physiologically deletional D-to-JH joining. With new compelling evidence, we propose that
this cohesin-mediated dynamic chromatin looping also operates during V to DJH recombination to ensure
generation of a diverse VH repertoire. With ultra-sensitive chromatin interaction and V(D)J recombination
assays combined with comprehensive genetic studies, in Aim1, we will address the functional importance of
chromatin loop extrusion in V to DJH recombination. In Aim2, we will determine the molecular mechanisms
underlying cohesin-mediated IgH long-range looping. In Aim3, we will identify and characterize novel IgH cis-
regulatory elements in directing RAG long-range targeting. These studies will be greatly facilitated by a novel
v-Abl pro-B cell line we generated that shows efficient IgH long-range chromatin looping and diverse VH
utilization across IgH locus. This will provide a flexible cell-based system to systematically characterize
complex interplay between IgH cis-regulatory elements and trans-acting factors, which is difficult to achieve in
animal models. Completion of this project will provide new mechanistic insights on how the dynamic 3D
genome topology harnesses a major immune process for generation of diverse antibody repertoires.

## Key facts

- **NIH application ID:** 10099796
- **Project number:** 1R01AI155775-01
- **Recipient organization:** WESTERN MICHIGAN UNIV SCHOOL OF MEDICINE
- **Principal Investigator:** Yu Zhang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $377,500
- **Award type:** 1
- **Project period:** 2020-12-01 → 2025-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10099796

## Citation

> US National Institutes of Health, RePORTER application 10099796, Chromatin looping directed RAG targeting during V(D)J recombination (1R01AI155775-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10099796. Licensed CC0.

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