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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
Specific Aims NIH Grant I
Specific Aims NIH Grant II
Specific Aims NIH Grant III
Schlissel Lab Members
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