# Impact of novel enhancers on Igh repertoire diversity

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2023 · $612,259

## Abstract

ABSTRACT
Antigen receptor genes are assembled from several gene segments via V(D)J rearrangement during
early lymphoid cell development to generate a diverse repertoire of antibodies. During early B cell
development in the bone marrow (BM), V(D)J and VJ joining occurs on the IgH and L chain genes,
respectively and is mediated by the RAG recombinase. VH genes are dispersed through 2.5 Mb of the
Igh locus. Locus compaction serves to facilitate spatial proximity between the rearranged DHJH join
and distal VH genes. Furthermore, V genes rearrange with very different intrinsic frequencies.
However, little is known about the precise looping structure of the Igh locus that leads to locus
contraction. We undertook an analysis of the entire Igh locus using chromosome conformation capture
(3C) based methodology to systematically characterize three-dimensional (3D) chromatin
organization on several genomic scales. We found that the Igh locus is compartmentalized into a
topologically associated domain (TAD) that is partitioned into three sub-domains. A set of pro-B cell-
specific very-long range looping interactions bridge the sub-domains and are Pax5-dependent. These
looping interactions are anchored at Sites I, II, II.5 and III and which are critical facilitators of Igh
locus contraction. We have now defined the DNA motifs in these loop anchors and discovered a series
of pro-B cell specific novel enhancers (NEs) that participate in a NE-NE-VH gene promoter
interactome. We have systematically characterized locus compaction using specific KO mice in
combination with chromatin-loop mapping methods and newly constructed NE1 KO mice and cell
lines. To begin to understand NE interactome function we asked whether those NEs engaged in E-E
and E-Pr looping are in an active state as defined by the H3K27Ac histone marks in single cells. We
discovered that the NEs are marked by remarkably large H3K27Ac foci. Here we will 1)
systematically characterize NEs individually and in the NE interactome as it relates to VH gene usage
during repertoire formation, and 2) examine the relationship between the NE interactome and
H3K27Ac foci. The presence of H3K27Ac foci on NEs may indicate the participation of the Igh locus
in transcriptional condensates which may define the molecular environment for V(D)J recombination.

## Key facts

- **NIH application ID:** 10716628
- **Project number:** 1R01AI177507-01
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO
- **Principal Investigator:** Amy L Kenter
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $612,259
- **Award type:** 1
- **Project period:** 2023-09-08 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10716628

## Citation

> US National Institutes of Health, RePORTER application 10716628, Impact of novel enhancers on Igh repertoire diversity (1R01AI177507-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10716628. Licensed CC0.

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