Cell type- and stage-specific expression of the CD20/B1 antigen correlates with the activity of a diverged octamer DNA motif present in its promoter

Abstract

The CD20(B1) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation. Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements. In this study we identified a sequence element present in the most proximal region located between bases -214 and -201, TTCTTCTAATTAA, which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells. This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells. Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site. Cross competition experiments with an octamer sequence from the Ig heavy chain promoter, the BAT box, and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins suggesting that the BAT box binding proteins were Oct-1 and Oct-2. Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2. The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence. Despite this lower affinity, a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells. The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line, PB-697, via phorbol esters. The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct, in contrast to the wild type, was poorly induced by phorbol esters. Together these results suggest that the BAT box binding proteins are important in the B cell specific expression of CD20 and perhaps CD21.