A CENTRAL ROLE FOR A SINGLE C-MYB BINDING-SITE IN A THYMIC LOCUS-CONTROL REGION

Locus control regions (LCRs) are powerful assemblies of cis elements t hat organize the actions of cell-type specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhan cer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhan cer contains a 28-bp core region and local adjacent augmentative cis e lements. We now show that the core contains a single critical c-Myb bi nding site, In both transiently cotransfected human cells and stable c hromatin-integrated yeast cells, c-Myb strongly transactivated reporte r constructs that contained polymerized core sequences, c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was activ e and was localized within discrete nuclear structures. Fetal murine t hymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression . Point mutation of the c-Myb site within the intact 2,3-kb LCR severe ly attenuated enhancer activity in transfections and LCR activity in t ransgenic thymocytes. Within the context of a complex enhancer and LCR , c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site.