Physiological activation of the IgH 3′ enhancer in B lineage cells is not blocked by Pax‐5

Abstract The mouse 3′ enhancer contains a high‐affinity binding site for the paired box protein Pax‐5. Here, we demonstrate by genomic footprinting that the rat 3′ enhancer contains a low‐affinity binding site for Pax‐5, which is occupied in activated splenic B cells. Thus, binding of Pax‐5 to the IgH 3′ enhancer appears to be evolutionarily conserved in rodents. Analysis of Pax‐5 expression in primary B cells demonstrates that Pax‐5 remains expressed after 4 days of lipopolysaccharide (LPS) induction, but is down‐regulated in 5‐day stimulated cells. Similarly, the expression of Pax‐5 is down‐regulated in vivo in activated large splenocytes, in contrast to small resting cells. Multimerization of the high‐affinity Pax‐5 binding site linked to a heterologous reporter gene demonstrates that Pax‐5 can function as a transcriptional activator. In contrast, Pax‐5 overexpressed in cell lines represses both the mouse and the rat 3′ enhancer. Surprinsingly, cross‐linking of the IgM receptor in BAL‐17 cells containing a stably integrated 3′ enhancer‐dependent β globin reporter gene demonstrates that induction of 3′ enhancer activity is not blocked by Pax‐5. Moreover, stimulation of 3′ enhancer β globin‐transgenic splenocytes demonstrate that Pax‐5 cannot repress activation of the 3′ enhancer upon LPS induction or CD40 receptor stimulation. Hence, activation of the IgH 3′ enhancer occurs independently of changes in Pax‐5 gene expression. This indicates that previous studies conducted in vitro may be an oversimplification of the function of Pax‐5 and 3′ enhancer activity.