The T cell receptor alpha gene: A molecular model of T cell-specific gene expression.

Abstract

In the studies described in this thesis, I have used the human T cell receptor (TCR) $\alpha$ chain gene as a model system to investigate the molecular mechanisms mediating lineage-specific gene expression in T lymphocytes. The approach used was to initially identify and characterize the important cis-acting transcription regulatory elements of the human TCR $\alpha$ gene and then to clone the cognate nuclear proteins that bind to these elements. A potent transcriptional enhancer located 4.5 kb 3$\sp\prime$ to the C$\alpha$ gene segment of the human TCR $\alpha$ gene was identified using transient transfection assays. This enhancer was shown to be essential for high level transcription of the human TCR $\alpha$ gene and to be active only in TCR $\alpha$/$\beta\sp+$ T cells. DNA sequence analyses, DNase1 footprinting, electrophoretic mobility shift assays, deletion analyses, as well as in vitro mutagenesis were used to characterize this enhancer. These studies demonstrated that: (i) The TCR $\alpha$ enhancer contains at least 5 nuclear protein binding sites, T$\alpha$1-T$\alpha$5, that together interact with multiple ubiquitous and T cell-specific nuclear proteins. (ii) Full enhancer activity is retained in a 116 bp restriction enzyme fragment containing only the T$\alpha$1 and T$\alpha$2 binding sites. The T$\alpha$3 and T$\alpha$4 sites appear to provide a degree of redundancy to enhancer function at least in mature T cells. (iii) The T cell specificity of this enhancer is mediated by several distinct molecular mechanisms. (iv) The T$\alpha$2 site can function as both a T cell specific activator and repressor of transcription depending on its neighboring DNA context. This finding suggests a new model of developmentally-regulated gene expression. (v) Finally, we have also demonstrated that the ets-1 proto-oncogene can bind to the T$\alpha$2 site of the human TCR $\alpha$ enhancer in a sequence-specific fashion. This finding allowed the definition of a novel class of DNA binding proteins, the Ets family, whose members appear to play a critical role in regulating the differentiation of the T cell lineage. Thus, the results described in this thesis have laid a foundation for understanding the molecular mechanisms that regulate lineage-specific gene expression during T cell development.