PEBP2 and c-myb sites crucial for λ5 core enhancer activity in pre-B cells

Abstract

The λ5 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 proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the λ5 gene. Amongst these is an enhancer, Bλ5, located 5′ of the gene. Here we analyze the λ5 enhancer core, bλ5, which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within bλ5, PEBP2λ5, which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2λ5 site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2λ5 element. PEBP2 proteins in mature B cells also bind to the PEBP2λ5 element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed mybλ5 located just upstream of the PEBP2λ5 site in the core enhancer. The mybλ5 element is also crucial for enhancer activity, since mutating the myb site 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 down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the λ5 gene.