In vivo footprinting and mutational analysis of the proximal CD19 promoter reveal important roles for an SP1/Egr-1 binding site and a novel site termed the PyG box.

Abstract CD19 expression begins at the pro-B cell stage of B cell development. As such it serves as a good prototype for B cell-specific genes whose expression begins shortly after lineage commitment. To understand the molecular mechanisms controlling CD19 gene expression, we isolated and functionally characterized the CD19 promoter using in vivo footprinting, gel shift assays, and transfection studies. Reporter constructs spanning portions of the promoter identified a region between -85 and -200 that produced high levels of reporter gene activity in lymphoid cells. In vivo footprinting identified protected regions over the known high affinity B cell lineage-specific activator protein (BSAP) site, the low affinity BSAP site, a SP1/Egr-1 site termed the CD19 GC box, and two novel sites named the AT box and PyG box. Phorbol ester treatment of a pre-B cell line up-regulated CD19 expression, induced Egr-1, and enhanced the footprint over the GC box. Gel shift assays demonstrated SP1 and Egr-1 binding to the CD19 GC box, while unknown nuclear proteins bound the PyG and AT boxes. Mutations in the AT box or in the BSAP sites did not affect CD19 reporter construct activity, while a mutation of the GC box reduced it modestly, and a PyG box mutation reduced it dramatically. BSAP failed to trans-activate CD19 promoter constructs in B cells or non-B cells, suggesting that cis elements such as the PyG and GC boxes are also necessary for high level CD19 promoter expression.