Immunoglobulin and T cell receptor gene assembly is usually thought of as relatively random process, generating a broad repertoire of cells expressing different specificities, from which """"""""useful"""""""" cells are selected. In the case of gammadelta T cells, however, there is now abundant evidence that the process of TCR gene assembly is highly programmed in ontogeny. The early fetal thymus produces only cells expressing Vbeta/Vdelta1 and Vgamma4/Vdelta1 TCR genes, which then emigrate to epithelium. Later in development, production shifts later to cells expressing Vgamma2 and other Vgamma and Vdelta genes. These cells emigrate to the secondary lymphoid organs. A good deal of evidence indicates that rearrangement of Vgamma2, Vgamma4 and Vdelta1 genes early in development is preprogrammed in the early progenitor cells, and that the shift to other gamma and delta genes later in development is also preprogrammed. These observations suggest that the thymus divides its labor ontogenetically, so that the required gammadelta cells can be produced in an organized fashion.
Our aim i s to elucidate the molecular mechanisms, and eventually the cellular mechanisms, that govern programmed rearrangement of TCR gamma genes. Our first specific aim is to determine the mechanisms regulating Vgamma rearrangement in the thymus. We have produced mice carrying transgenes composed of several Vgamma genes, each harboring frame-shift mutations, as well as the Jgamma1Cgamma1 gene segments, all in their unrearranged configuration. The constructs undergo efficient T cell specific rearrangement in vivo. This system will be extended to identify cis-acting DNA elements that control differential Vgamma gene rearrangement. Such elements will be further characterized in terms of their interactions with other gamma control elements, including enhancers, and promoters, as well as in terms of the factors that bind and control the elements. Ultimately the effect of knocking-out the element will be assessed. Our second specific aim is to determine the general role of enhancer and promoter elements that control transcription in the TCRgamma rearrangement process. A deletion of the previously identified Cgamma1 enhancer on the transgene demonstrated that the construct still undergoes efficient rearrangement and transcription in vivo, strongly suggesting that an additional enhancer element is present in the locus. This enhancer will be localized and characterized. The roles of the new enhancer as well as the previously described enhancer in recombination and transcription of the locus will then be systematically assessed. In addition, promoter deletion mutants will be further characterized to assess the role of transcription in rearrangement.
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