3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.     

Reduced EBF expression underlies loss of B‐cell potential of hematopoietic progenitors with age

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age‐related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2–5‐fold, while myeloid potential remains unchanged. This age‐related decrease in B‐cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B‐lineage specifying factors, EBF and Pax5. Notably, retrovirus‐mediated expression of EBF complemented the age‐related loss of B‐cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B‐cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B‐cell fate, without altering their potential to generate myeloid cells.     

Molecular role of the PAX5-ETV6 oncoprotein in promoting B-cell acute lymphoblastic leukemia

PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6⁺ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development.