ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6

PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.