Research Interests:
The Regulation of Antigen Receptor
Gene Rearrangement and Lymphocyte Development. Immunoglobulin
(Ig) and T cell receptor (TCR) genes are assembled during lymphocyte
development from their component gene segments by a novel series
of highly regulated DNA rearrangement reactions known as V(D)J
recombination. (Figure
1) Understanding the regulation of gene rearrangement is important
because its products (Ig and TCR molecules) control the development
and function of B and T cells and because errors in the process
can result in leukemia, lymphoma, or immunodeficiency disease.
(Figure 2)
A single enzyme complex known as the V(D)J recombinase is required
for the rearrangement of each of the seven complex genetic loci
encoding antigen receptor chains. The recombinase recognizes a
highly conserved DNA sequence, the RSS, and introduces a dsDNA
break. The DNA break repair machinery expressed in all cells then
generates various molecular joints. (Figure
3) Our data suggests that transcription or transcription factor
binding targets the recombination reaction to a particular genetic
locus at the appropriate stage of development. My lab is interested
in determining the molecular basis of this link between transcription
and gene rearrangement during B cell development and in understanding
how a common recombinase recognizing a conserved DNA element can
result in highly regulated patterns of gene rearrangement. (Figure
4) In addition, we are interested in the reaction mechanism
of the V(D)J recombinase.
Using bone marrow, thymus and fetal liver from wild-type
or recombination-deficient Ig or TCR transgenic mice, we can purify
primary lymphoid cells "frozen" at different stages
of development. In addition, we can use gene targeting technologies
in embryonic stem cells to introduce specific mutations into key
genes. We are analyzing signal transduction pathways, patterns
of transcription factor activity and chromosomal DNA-protein interactions
to determine the critical receptors, protein factors and DNA sequences
involved in the regulation of B and T cell development and Ig
and TCR gene rearrangement. We have also devised a series of novel
assays which detect recombination reaction intermediates and are
using them to study both the developmental regulation and reaction
mechanism of the V(D)J recombinase. (Figure
5) Recently, we devised an in vitro assay system which recapitulates
the developmental regulation of V(D)J recombination and are engaged
in the dissection of its molecular components. (Figure
6)
Current Projects
Active
projects in the lab include 1) an analysis of the influence of
chromatin structure on the targeting of V(D)J recombination; 2)
reconstitution of regulated V(D)J recombination in vitro using
purified recombinase components and long DNA templates; 3) promoter
and enhancer activities involved in the transcriptional regulation
of genes encoding the V(D)J recombinase (RAG1 and RAG2); 4) the
use of gene targeting to determine the function of various domains
of RAG1 and RAG2; 5) identifying transcription factors and DNA
sequences involved in the regulated activation of rearrangement
at the Ig kappa locus and TCR beta locus; 6) determining the role
of transcriptional enhancer sequences in the targeting of recombination
and in coding joint formation; 7) elucidating the rules governing
receptor editing; 8) studying the mechanism and determining the
frequency of transposition events catalyzed by the recombinase
during lymphocyte development; and 9) determining the role of
the c-Abl protein tyrosine kinase in lymphocyte development and
cellular transformation
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