It has long been accepted that immunoglobulins (Igs) can only be expressed in mature B lymphocytes and plasma cells. However, recently several groups reported that Igs could also be produced by non-lymphoid lineage cells 
, including human cancer cells 
, soft tissue tumor cells 
, neurons and glial cells of the central and peripheral nervous system 
, ocular epithelial and ganglion cells 
, mouse testicular spermatogenic cells and epididymal epithelial cells 
and mouse lactating mammary gland epithelial cells 
. Most of the research has thus far focused on Ig expression in cancer cells. The Recombination activating gene (RAG) has also been found expressed in cancer cells both at the mRNA and the protein levels and it is assumed to play a significant role in the synthesis of Igs by these cancer cells 
. However, the regulatory mechanism of RAG expression in cancer cells has not yet been determined.
The variable regions of Ig genes are composed of one variable (V), one diversity (D), and one joining (J) gene segment, the arrangement of which results from V(D)J recombination 
. RAG endonuclease is required for the initiation of the cleavage phase of V(D)J recombination 
. RAG consists of two adjacent genes, RAG1 and RAG2, that synergistically induce V(D)J recombination 
. Previous studies have shown that mice deficient in either RAG1 or RAG2 failed to initiate V(D)J rearrangement 
. RAG1 and RAG2 proteins together were found to be sufficient to cleave recombination substrates in cell free systems 
. In murine B cell development RAG expression occurs in two waves and is regulated by a network of transcription factors, including E2A, Ikaros, Pax5β, Foxo1, Foxp1, and NF-κB 
. The first wave results in the rearrangement of the immunoglobulin heavy chain in pro-B cells. And the second wave of RAG expression leads to the assembly of immunoglobulin light chain in pre-B cells.
In addition to the RAG1 and RAG2 promoters, the RAG gene has also other regulatory elements, such as the proximal enhancer (Ep), the distal enhancer (Ed) and the RAG enhancer (Erag) 
. It is thought that the aforementioned transcription factors regulate RAG expression by binding to their corresponding regulatory sequences in B cells. Erag is the strongest enhancer regulating RAG expression. Targeted deletion of Erag in the mouse germline resulted in a 5-fold to 10-fold decrease in RAG expression and a partial block at the pro-B to pre-B transition 
. E2A, Ikaros, Foxo1, Foxp1 and NF-κB were all shown to activate RAG expression by binding to Erag in murine B cells 
. Pax5β was reported to activate RAG2 promoter in immature B cells 
. Whether these transcription factors are also expressed in cancer cells and whether they have regulatory functions in the expression of RAG in such cells is worthy of investigation.
In this study, we first analyzed the protein and mRNA expressions of those transcription factors that have been found to be essential for RAG activation in B cells, including E2A (E47 and E12), FOXO1, FOXP1, Ikaros, NF-κB, and PAX5β, in four cancer cell lines. We then studied the localization of a number of these transcription factors (E2A, FOXP1, NF-κB and FOXO1) by immunofluorescence (IF). We found that E2A, FOXO1 and FOXP1 were expressed in cancer cells and localized to the nuclei of these cells. Over-expression of these three transcription factors significantly increased RAG expression. Functional inactivation of the genes of any of these three transcription factors by RNA interference decreased RAG expression. In vivo chromatin immunoprecipitation (ChIP) assay showed that the histone H3 of Erag was acetylated and that E2A, FOXO1, FOXP1 were bound to Erag in these cancer cells. These results indicate that transcription factors E2A, FOXO1 and FOXP1 activate the expression of RAG, which is critical for V(D)J recombination, in cancer.