Analysis of B lymphocyte activation and differentiation by expression profiling
B cell development is severely impaired in the absence of SLP65 adaptor protein. SLP65-deficient B cells are arrested at the large cycling pre-B cell stage in the bone marrow and transitional B cell stages in the spleen of mice. The zinc-finger transcription factor EGR1 is an immediate early gene induced upon Ag-binding and BCR engagement in B cells. EGR1 promotes B cell differentiation and maturation at the progression from pro-B to pre-B and into the mature B cell stages. The main hypothesis of this study was that EGR1 expression is regulated through a SLP65-dependent signaling chain and therefore, EGR1 overexpression in SLP65-/- mice could restore or at least partially rescue developmental blocks caused by the absence of SLP65 in B cells. The results revealed that both EGRxSLP65-/- and SLP65-/- B cells appear to be arrested at the same developmental (large pre-B cell) stage, suggesting that in the absence of SLP65, EGR1 activity alone is not sufficient to rescue the developmental block in the bone marrow. However, independent of this developmental arrest EGR1 overexpression mediates downregulation of IL-2R alpha chain in B cells. Further analysis showed that although EGR1 could not completely rescue the developmental arrest at the transitional B cells stages in EGRxSLP65-/- mice, splenic B cells from EGRxSLP65-/- mice have a more mature phenotype compared to splenic B cells from SLP65-/- animals. In order to determine the underlying molecular basis of these EGR1-mediated phenotypic changes in EGRxSLP65-/- B cells as well as to identify additional EGR1 target genes in B cells, gene expression profiling experiments were performed. By microarray hybridizations and several validation methods at least nine genes were found to be differentially regulated in EGRxSLP65-/- B cells. Inspection of the promoter sequences suggests that seven genes: Ly-6A/E, TAF10, SLFN1, SLFN2, CalA, CCR2 and Oct1 might be potential EGR1 target genes in B cells. Ly-6D and Ly-6E are upregulated in both EGRxSLP65-/- and SLP65-/- B cells, since the cells are T1 and T2 B cells. We also could show that IRF1 is downregulated upon BCR cross-linking in SLP65-/- B cells. In addition, we found that the SLP65-/- B cells express low levels of TACI and the expression levels of TACI is drastically reduced in EGRxSLP65-/- B cells. Due to the block at the large pre-B cell stage SLP65-/- mice develop pre-B ALL leukemia in 5-10% of cases. Consistent with these data we found pre-B ALL formation in about 6.78% of SLP65-/- mice. Surprisingly, the pre-B ALL incidence was twice as high (13.33%) in EGRxSLP65-/- mice. Searching for possible candidate genes which might be involved in pre-B ALL formation, we found that LEF1 and IRF1 might be associated with tumor formation in pre-B cells. Our results strongly suggest that EGR1 might play a role in pre-B ALL progression by directly regulating LEF1 in pre-B cells.
B Cell Differentiation, microarrays, EGR1 target genes, pre-B ALL
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