T-cell receptor and antibody genes are assembled by the process of V(D)J recombination, a highly developmentally regulated process. Developmental coordination of gene rearrangements is necessary to produce subsets of lymphocytes with specific functions, and abnormalities in the process may underlie various disease states. Subtypes of TCR gamma/delta cells are distributed to different tissues and evidence suggests they play various roles in general and specific immunity to infectious agents. This application addresses the role of cis-acting elements, trans-acting factors and gene arrangement in regulating the rearrangement and expression of murine T-cell receptor gamma genes. The C-gamma-l cluster consists of four V-gamma genes, which are rearranged in a highly developmentally ordered sequence. At the extremes, V-gamma-3 is rearranged at a high level in the early fetal thymus, while V-gamma-2 is not. At the later stages in fetal development and on into the adult stage, a switch occurs where V-gamma-3 rearrangement is suppressed and V-gamma-2 becomes the predominant rearrangement. Subtypes of gamma/delta cells defined by usage of different V genes are produced according to this rearrangement pattern and emigrate to different peripheral tissues. Work in the last project period has led to the application that two distinct processes underlie the developmental switch in V-gamma recombination. With the use of transgenic recombination constructs, we showed that reciprocally exchanging the promoter regions of V-gamma-2 and V-gamma-3 reversed the pattern of rearrangement in the adult, but not the early fetal, thymus. Conversely, reciprocally exchanging the entire V-gamma-2 and V-gamma-3 genes in the cluster reversed the rearrangement pattern in the fetal thymus but not in the adult thymus. These data suggest that V-gamma rearrangement is controlled by the promoter regions in the adult, and by the location or order of the genes in the early fetal period. Related studies demonstrated the function of two enhancer-like elements, a previously described downstream enhancer, 3'EC-gamma-1, and a novel upstream element, HsA, located between V-gamma5 and V-gamma-2. Deletion of both elements from a transgenic expression construct prevented gene expression, but either element alone was able to support gene expression. We propose to employ transgenesis, gene targeting, factor analysis and related methods to determine how the various promoter and enhancer elements cooperate to generate the fetal and adult patterns of V-gamma gene rearrangement and expression.
The Specific Aims are:
Specific Aim 1. To assess local chromatin organization across the TCR-gamma locus in adult and fetal stage thymocytes, in order to detect differentially regulated regions or domains. RSS breaks, hyperacetylation and methylation will be assessed.
Specific Aim 2. To determine the role of V-gamma promoter regions in determining the adult pattern of V-gamma gene rearrangement. We will identify cis acting-sites in the promoters that regulate rearrangement, and factors that bind them. Gene targeting and transgenesis will be employed to distinguish competitive from autonomous models of regulation.
Specific Aim 3. To dissect the role of gene order, domains, and flanking cis-acting regulatory sequences in determining the fetal thymic V-gamma gene rearrangement pattern. Gene targeting and transgenesis will be employed.
Specific Aim 4. To determine the roles of HsA and the 3'Ecv1 in TCR-gamma gene rearrangement and expression using mice in which each element, or both simultaneously, are deleted by gene targeting. Factors that regulate the elements will be assessed.
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