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Recombineering Homologous Recombination Constructs in Drosophila
Published on: July 13, 2013
Enhancer control of local accessibility to V(D)J recombinase
M T McMurry1, C Hernandez-Munain, P Lauzurica
1Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Molecular and Cellular Biology
|August 1, 1997
Summary
Transcriptional enhancers control V(D)J recombination by ensuring accessibility of recombination signal sequences (RSSs) to the V(D)J recombinase. The TCR delta enhancer (Edelta) specifically facilitates J segment accessibility, not D segment accessibility.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- V(D)J recombination is a crucial process for adaptive immune system development.
- Transcriptional enhancers are known regulators of gene expression and recombination.
- The T-cell receptor (TCR) delta enhancer (Edelta) plays a role in TCR delta gene rearrangement.
Purpose of the Study:
- To investigate the role of transcriptional enhancers in providing accessibility of recombination signal sequences (RSSs) to the V(D)J recombinase.
- To determine if the TCR delta enhancer (Edelta) is necessary for J segment accessibility during V(D)J recombination.
- To elucidate the mechanism by which enhancers regulate V(D)J recombination.
Main Methods:
- Generation of transgenic mice carrying a human TCR delta gene minilocus with or without a functional Edelta.
- Analysis of V(D)J recombination by assessing the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs using ligation-mediated PCR.
- Comparison of DSB frequencies at RSSs flanking Ddelta3 and Jdelta1 segments in wild-type and enhancer-deficient miniloci.
Main Results:
- Deletion or inactivation of Edelta significantly reduced DSB formation at the RSS 5' of Jdelta1.
- DSB formation at the RSS 3' of Ddelta3 was unaffected by the absence of Edelta.
- Efficient DSB formation at the Ddelta3 RSS required an accessible partner RSS, suggesting synapsis is necessary in vivo.
Conclusions:
- Edelta is essential for the accessibility of the Jdelta1 RSS but not the Ddelta3 RSS to the V(D)J recombinase.
- Enhancers regulate V(D)J recombination by providing local accessibility to specific RSSs.
- Additional cis-acting elements likely maintain Ddelta3 accessibility in the absence of Edelta.
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