# Mechanisms Underlying the Control of Recombination and Gene Regulation

> **NIH NIH R35** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2021 · $713,367

## Abstract

Summary
My lab's work has been at the forefront of studies showing that nuclear organization and long-range chromatin
interactions play an essential role in recombination and gene regulation. In this application we have
incorporated two distinct projects that extend this work. The first project focuses on understanding the
mechanisms underlying feedback control of RAG activity in individual lymphocytes and the
consequence of de-regulated cleavage. V(D)J recombination has to be tightly regulated to ensure that
cleavage does not continue in cis, or in trans on accessible target loci that undergo recombination at
overlapping stages of development as well as on actively transcribed off-target loci with cryptic recombination
signal sequence (RSS) sites that bind the RAG proteins. Our recent studies reveal that ATM and the C
terminus of RAG2 have an important role in feedback control of cleavage in individual cells through modulation
of nuclear organization. This limits the number of potential substrates for translocation and provides an
important mechanism for protecting genome stability. Given that an absence of the C terminus of RAG2 and
inhibition of ATM kinase activity lead to similar phenotypes we hypothesized they could act in the same
pathway. In our most recent unpublished work we identified a conserved SQ target phosphorylation site on
RAG2 (residues 365-366) that recapitulates the function of the RAG2 C-terminus and ATM in regulating
cleavage. However, in contrast to these two mutants, RAG2-S356A has a stable RAG post cleavage complex.
Thus, for the first time we have a tool to study feedback regulation in the absence of any confounding repair
defect. Here we aim to determine!(i) the mechanism underlying feedback control of RAG activity in individual
cells, (ii) the consequences of cleavage deregulation on allelic exclusion, genome instability and gene
regulation and (ii) the mechanism by which deregulated RAG activity contributes to oncogenesis. The second
project focuses on understanding the mode of action of enhancers in controlling gene regulation in the
context of 3D chromatin structure. Enhancers play a fundamental role in ensuring precise control of
transcriptional patterns in development and differentiation. Physical contacts between genes and these
regulatory elements are essential for proper transcriptional control and maintenance of these interactions is
critical for preventing aberrations in physiological processes that could manifest as disease states. Using new
tools developed under the support of GM086852 and GM112192 we are now able to investigate these
interactions using live imaging systems and high-resolution chromosome conformation capture (4C).
Specifically, our aim is to investigate the mode of action of enhancers in the context of: (i) enhancers that
control the regulation of more than one target gene, (ii) the functional relevance of clusters of enhancers that
constitute super-enhancers in regulating target loci and t...

## Key facts

- **NIH application ID:** 10119293
- **Project number:** 5R35GM122515-05
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** Jane Amanda Skok
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $713,367
- **Award type:** 5
- **Project period:** 2017-04-03 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10119293

## Citation

> US National Institutes of Health, RePORTER application 10119293, Mechanisms Underlying the Control of Recombination and Gene Regulation (5R35GM122515-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10119293. Licensed CC0.

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