Michael S. Krangel

The process of V(D)J recombination assembles T cell receptor (TCR) genes (α,β,γ,δ) from variable (V), diversity (D) and joining (J) gene segments during T cell development, and is essential for the formation of diverse antigen receptor repertoires on αβ and γδ T lymphocytes. We are interested in the molecular basis for developmentally regulated rearrangement and expression of murine TCR genes. One focus of our studies is the TCRα/δ locus, because it represents an intriguing model with two sets of gene segments that are differentially activated for recombination during T cell development. We are also studying the TCRβ locus, because this locus presents a model in which there is a developmental inactivation of V(D)J recombination associated with the process of allelic exclusion. V(D)J recombination depends on the ability of recombinase proteins RAG1 and RAG2 to recognize and generate double-strand breaks at recombination signal sequences that flank TCR gene segments. Our main focus has been on the role of chromatin structure in defining the portions of these loci that are accessible to the RAG recombinase and therefore active for V(D)J recombination, and on the mechanisms by which cis-regulatory elements within these loci (enhancers, promoters) function as developmental regulators of chromatin structure. Our primary approach has been to manipulate cis-acting elements within these loci by gene targeting, and to study the effects of these manipulations on locus chromatin structure and recombination events in developing thymocytes in vivo. An important outcome of this work has been our demonstration that enhancer- and promoter-directed transcription through recombination signal sequences can displace and covalently modify nucleosomes to provide accessibility for RAG binding and V(D)J recombination.

Recent work in our laboratory and elsewhere has highlighted additional properties of antigen receptor loci that likely to play important roles in developmental regulation. One area of interest is subnuclear positioning. We have used three-dimensional fluorescence in situ hybridization (3D-FISH) to show that TCRβ alleles interact stochastically and at high frequency with the nuclear lamina and with foci of pericentromeric heterochromatin, and that these interactions are inhibitory to V(D)J recombination. We suspect that these inhibitory interactions help to promote allelic exclusion by diminishing the likelihood of simultaneous V to DJ recombination on both alleles. Current work is aimed at developing a better understanding of how the TCRβ locus interacts with the nuclear lamina and the mechanism by which this interaction impacts recombination events.

A second area of interest is locus conformation. It is now appreciated that recombination events at antigen receptor loci depend on locus conformational changes that bring into proximity gene segments that may be widely separated in the linear DNA sequence. Conformational states can be defined using 3D-FISH or a chemical crosslinking approach called chromosome conformation capture (3C). Recent studies indicate that developmental changes in locus conformation contribute to allelic exclusion at the TCRβ locus and mediate a transition from TCRδ to TCRα rearrangement at the TCRα/δ locus. Current work aims to address at a molecular level how locus conformational states are maintained and modified during T cell development and how these changes impact long-distance transactions including enhancer-promoter communication and V(D)J recombination.