Genomic organization and characterization of the promoter for the E2A gene

Although E2A gene products are ubiquitously expressed, E2A-deficient mice display selective abnormalities in lymphocyte development, suggesting a certain requirement of the E2A gene products during lymphocyte development. To gain insights into the mechanisms of E2A transcriptional regulation, we isolated the genomic clones which are composed of four exons and one noncoding exon and span approximately 16 kb. The promoter region of E2A gene lacks a TATA box, and primer extension analysis showed several transcription initiation sites, a feature that characterizes TATA-less promoters. The transient transfection assay using the 5'-flanking region (positions -2994/+62) revealed that both positive (-357/-158) and negative (-831/-358) regulatory segments control E2A transcription in B-cell (WEHI-231) and T-cell (DO11.10) lines. However, contribution of a certain segment to promoter activity was different between lymphocytes and fibroblasts (NIH-3T3). Sequential deletion analysis of the constructs spanning the positive regulatory segments showed that the segment -257/-238 played a critical role in the basal promoter activity of the E2A gene, although other segments within -337 to -158 also appeared to be involved. Mutational analysis using the -257/-238 fragment failed to demonstrate a single cis-element responsible for the basal promoter activity, suggesting that E2A promoter requires the interaction of multiple regulatory elements. Electrophoretic mobility shift assay (EMSA) demonstrated a highly specific complex comprised of a positive regulatory segment (-267/-238) and putative transcription factor(s), which might be necessary for the basal promoter activity of the E2A gene.