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Keywords:

  • B lymphocyte differentiation;
  • 3′ enhancer;
  • Immunoglobulin gene expression;
  • Pax-5;
  • Transcription factors

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.