Analysis of Gene Expression Directed by a Thymic Locus Control in Transgenic Mice: Mechanisms and Application to Vectors for Gene Therapy

The formation of peripheral T cells from thymocyte progenitors is an intricate developmental process that requires the organized and coordinate expression of multiple genes. Adenosine deaminase (ADA) is an example of a gene that is subject to strong developmental regulation in T-cell precursors and is essential for the subsequent formation of T-cells in humans. We have sought to understand the mechanisms of ADA gene regulation from a basic point of view as well as to employ this to potential vectors for gene therapy.Using transgenic mice have shown that the first intron of the ADA gene contains a powerful locus control region that directs high level gene expression within cortical thymocytes. Based on extensive mutational analysis of the regulatory region and analyses of gene expression that include quantitative gene expression, in situ hybridization, and biochemical characterizations of chromatin structure, we have demonstrated that the intronic locus control region (LCR) consists of a hierarchically structured 2300 base pairs of DNA sequence (Figure 1). The LCR is composed of a series of regulatory elements that include a centrally positioned 300 base pair classical enhancer domain within which there is a critical 30 base pair enhancer core. Within this core, there is a single binding site for the transcription factor c-Myb that is required for activity of the enhancer core, the enhancer, and the intact LCR. Beyond the 300 bp enhancer core on either side the LCR contains novel and puzzling 1 kb non-enhancer sequences that we have termed facilitators. These sequences enable gene copy proportional expression by facilitating the ability of the enhancer to function in chromatin. The effects of the facilitators are evidenced by their ability to allow for insertion-site-independent and gene-copy-proportional expression and they prevent variegated expression among similarly differentiated cell types (Figure 2). Thus, total gene expression does not indicate proper cell type specific expression. The facilitators also allow for the formation of a strong tissue and cell type specific DNAse I hypersensitive site at the enhancer. This suggests that the formation of a discrete organized chromatin structure as a function of developmental differentiation requires extensive DNA sequences, only some of which are of the enhancer type. The capabilities of the facilitators to activate a chromatin domain may also suggest their potential usefulness in vectors for gene therapy of both ADA deficiency and possibly other human genetic diseases. However, the distance and non-enhancer nature of the facilitators suggest that they may act differently than conventional regulatory elements. In support of this, the facilitators obey a strict position and orientation rules with respect to the enhancer.