PEBP2 and c-myb sites crucial for lambda 5 core enhancer activity in pre-Bcells

The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the p roliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B-lam bda5, located 5' of the gene. Here we analyze the lambda5 enhancer core, b( lambda5), which in earlier experiments was demonstrated to retain 50% of th e enhancer activity, and show that this activity is restricted to pre-B cel ls. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is e ssential for enhancer activity: mutation within this site dramatically redu ces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds ba cterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA) . Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5) element, implying that if PEBP2 proteins a re responsible for the stage-specific expression, they have to be non-activ ating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located j ust upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambd a5) element is also crucial for enhancer activity, since mutating the myb s ite reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is downregulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific e xpression of the lambda5 gene.