Physiological activation of the IgH 3' enhancer in B lineage cells is not blocked by Pax-5.
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 beta globin reporter gene demonstrates that induction of 3' enhancer activity is not blocked by Pax-5. Moreover, stimulation of 3' enhancer beta 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.
Andersson
T
T
Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
Neurath
M F
MF
Grant
P A
PA
Pettersson
S
S
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D023201:CD40 Ligand; D002478:Cells, Cultured; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005803:Genes, Immunoglobulin; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000502:physiology; D051379:Mice; D009687:Nuclear Proteins / Q000502:physiology*; D051757:PAX5 Transcription Factor; D051381:Rats; D011947:Receptors, Antigen, B-Cell / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors; D015533:Transcriptional Activation; D015854:Up-Regulation
D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D011947:Receptors, Antigen, B-Cell; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D023201:CD40 Ligand