Tissue-specific expression of the T cell receptor alpha-chain (TCR alpha) is dependent upon functional gene rearrangement within the alpha/delta gene locus and the activation of a distal enhancer element. Both of these events are regulated to occur only at a precise time in T cell development. The alpha enhancer has been mapped to a 116 base pair DNA fragment that interacts with three different proteins: two are T cell- -specific (TCF- 1 and TCF-2) and the third binds to a cAMP response element (CRE). This protein-enhancer complex can activate the variable region (V- alpha) promoter at distances as great as 69 kb, depending upon the exact combination of variable, joining, and constant regions during rearrangement. In addition to the minimal sequence required for this activation (116 bp), flanking sequences function as """"""""silencers"""""""" to restrict enhancer activity to only the appropriate stage of T cell ontogeny. Thus, TCR alpha gene expression is controlled, in part, by a combination of positive and negative factors that act together to regulate the alpha enhancer. This control element is especially interesting since the TCR alpha locus is the site of frequent chromosomal translocations that are associated with leukemia. The alpha enhancer may play a critical role in this process by activating the expression of oncogenes that are translocated into this locus. In this proposal, we wish to study the mechanism of enhancer function by developing in vitro systems that reproduce T cell-specific transcription and chromatin structure. At present, enhancer-dependent transcription on naked DNA does not occur in vitro unless the element is in close proximity to the promoter. In an effort to achieve correct regulation, two in vitro reconstitution systems will be used to test the effects of nuclear structure on enhancer activity. In the first system, the role of chromatin formation on enhancer function will be examined by assembling reporter genes linked to the V-alpha promoter and TCR alpha enhancer into nucleosomes using Xenopus oocyte extracts. The function of purified TCF- 1 and TCF-2 in enhancer activation will be assessed in these experiments. The second system will explore the transcriptional regulation of the entire TCR alpha locus by incorporating this DNA into """"""""synthetic"""""""" nuclei using Xenopus egg extracts. This will be used to determine whether the alpha enhancer has greater activity in a more complex nuclear environment and to identify other cis-acting control regions within the TCR locus that may regulate a gene expression. Reconstitution in the presence of proteins from different lymphoid stages may enable one to approximate the nuclear structure of the TCR a locus as it exists during specific times in T cell development. In this way, we can reproduce the T cell-specific regulation of the TCR a gene within a large chromosomal domain and analyze the function of distal control regions and other parameters of nuclear organization. By understanding how the TCR alpha enhancer functions in its normal chromosomal context, we hope to determine how it contributes to neoplasia by altering the transcription of translocated oncogenes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA054418-05
Application #
3731299
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
005436803
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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