Department of Immunology
The process of VDJ recombination assembles T-cell receptor genes (TCR alpha, beta, gamma, delta) from variable (V), diversity (D), and joining (J) gene segments during thymic differentiation of T cells, leading to the production of alpha/beta and gamma/delta T lymphocytes. Our goal is to elucidate the molecular basis for the developmentally regulated rearrangement and expression of TCR genes. Our focus is on the complex genetic locus that carries TCR alpha and TCR delta gene segments (TCR alpha/delta locus), because these two sets of gene segments are differentially regulated during T-cell development.
We engineered a human TCR delta gene minilocus, composed of unrearranged V, D, J, and constant gene segments, to serve as a VDJ recombination reporter substrate in transgenic mice. We then analyzed the role of transcriptional enhancers as developmental regulators of TCR gene segment accessibility to the VDJ recombinase, by measuring VDJ recombination in versions of our minilocus that carry wild-type or mutant versions of the TCR delta enhancer, wild-type or mutant versions of the TCR alpha enhancer or no enhancer. Our studies identified an enhancer-dependent step of transgene rearrangement and showed that the TCR delta and TCR alpha enhancers confer developmental specificity to the process of transgene rearrangement that mimics the regulation of VDJ recombination at the endogenous TCR alpha/delta locus. We want to identify specific enhancer-binding factors that are responsible for the observed developmental regulation. To date we have implicated CBF/PEBP2 and Myb family transcription factors in TCR delta gene rearrangement, and TCF/LEF and Ets family transcription factors in TCR alpha gene rearrangement. Ongoing studies that test the effects of additional enhancer mutations and that use in vivo footprinting to measure the occupancy of transcription factor binding sites in wild-type and mutant enhancers and in different populations of developing thymocytes, should help us to define better the molecular basis for the distinct developmental properties of the two enhancers.
As our studies implicate the TCR delta and TCR alpha enhancers as region-specific developmental regulators of VDJ recombination within the TCR alpha/delta locus, they raise the question of how the regulatory effects of these enhancers are limited to discrete regions of the locus. This led us to consider the possibility that there may be boundary (or enhancer-blocking) elements within the TCR alpha/delta locus that delimit the regulatory domains influenced by the TCR alpha and TCR delta enhancers. We recently identified one such element (BEAD-1, for blocking element alpha/delta-1) situated between TCR delta and TCR alpha gene segments. BEAD-1 is defined by its ability to block the TCR delta enhancer from activating a promoter when located between the two in a chromatin integrated reporter construct in transfected cells. We will test a regulatory role for BEAD-1 in vivo by using gene targeting to eliminate it from the endogenous TCR alpha/delta locus and will investigate the molecular basis for BEAD-1 function using transfection, mutagenesis and biochemical approaches.
A second research focus of the laboratory is the expanding family of inflammatory cytokines known as chemokines (short for "chemotactic cytokines"). These molecules are important in host defense and play essential roles in the recruitment and activation of neutrophils, monocytes, subsets of lymphocytes, basophils, and fibroblasts to sites of inflammation. Secreted chemokines are thought to be immobilized both on cell surfaces and in the extracellular matrix on the basis of their ability to bind to glycosaminoglycans, and are then thought to be "presented" to target cells carrying specific chemokine receptors. Our current efforts are directed toward testing this model of chemokine action. Specifically, we are using in vitro mutagenesis to map the amino acids of chemokines that determine glycosaminoglycan and receptor binding. We will then ask whether chemokines that can bind to their specific receptors but not to glycosaminoglycans display altered biological properties in vivo.
McMurry MT, Hernandez-Munain C, Lauzurica P, Krangel MS. Enhancer control of local accessibility to VDJ recombinase. Mol Cell Biol 1997;17:4553-61.
Koopmann W, Krangel MS. Identification of a glycosaminoglycan binding site in chemokine MIP-1alpha. J Biol Chem 1997;272:10103-9.
Lauzurica P, Zhong X-P, Krangel MS, Roberts JL. Regulation of T-cell receptor delta gene rearrangement by CBF/PEBP2. J Exp Med 1997;185:1193-1202.
Selvan RS, Zhou L-J, Krangel MS. Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1. Eur J Immunol 1997;27:687-94.
Roberts JL, Lauzurica P, Krangel MS. Developmental regulation of VDJ recombination by the core fragment of the T-cell receptor alpha enhancer. J Exp Med 1997;185:131-40.
Zhong X-P, Krangel MS. An enhancer-blocking element between alpha and delta gene segments within the T-cell receptor alpha/delta locus. Proc Natl Acad Sci USA 1997;94:5219-24.
Hernandez-Munain C, Lauzurica P, Krangel MS. Regulation of T-cell receptor delta gene rearrangement by c-Myb. J Exp Med 1996;183:289-93.
Hernandez-Munain C, Krangel MS. c-Myb and core binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer. Mol Cell Biol 1995;15:3090-9.
Buchwalder A, Krangel MS, Wang G, Hao P, Diamond DJ. Direct binding of antigen to lipid anchored and soluble forms of an MHC independent human alpha/beta T-cell receptor. Mol Immunol 1994;31:857-72.
Hernandez-Munain C, Krangel MS. Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core binding factors. Mol Cell Biol 1994;14:473-83.
Lauzurica P, Krangel MS. Enhancer dependent and independent steps in the rearrangement of a human T-cell receptor delta transgene. J Exp Med 1994;179:43-55.
Lauzurica P, Krangel MS. Temporal and lineage-specific control of TCR alpha/delta rearrangement by TCR alpha and TCR delta enhancers. J Exp Med 1994;179:1913-21.
Paolini J, Willard D, Consler T, Luther M, Krangel MS. The chemokines interleukin-8, monocyte chemoattractant protein-1 and I-309 are monomers at physiologically relevant concentrations. J Immunol 1994;153:2704-17.
Selvan RS, Butterfield JH, Krangel MS. Expression of multiple chemokine genes by a human mast cell leukemia. J Biol Chem 1994;269:13893-8.