The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFβ (core binding factor β) into the nucleus to allow Runx (runt-related transcription factor)/CBFβ heterodimerization. Runx/CBFβ then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or β-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/β-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFβ heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.

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The promoter of pre-B cell stage-specific Crlz1 gene, whose protein translocates the cytoplasmic core binding factor β (CBFβ) into the nucleus and thereby allows its heterodimerization with Runx, has a very strong activity, which is about 25% of cytomegalovirus (CMV) promoter activity and comparable to the EF-1α promoter activity. Its transcription start site was mapped at 155 nt upstream of translation initiation codon. 5′-truncation analysis of charged amino acids rich leucine zipper 1 (Crlz1) promoter revealed that one distal region from -612 to -536 and one proximal region from -198 to -100 as numbered from the transcription start site were critical for the promoter activity. The 3′-truncation analysis of the promoter revealed that the basal promoter sequence around the transcription start site, which should be necessary for the assembly of transcription initiation complex and the start of RNA polymerase II, was also essential, although not sufficient by itself. When transcription factor binding sites within those two critical regions were searched by in vivo footprinting, one distal LEF-1 and multiple proximal Ets consensus-like sites were found to be footprinted. Indeed, the protein causing a footprint over the distal region was found to be LEF-1, and the ones causing three footprints over the proximal region were found to be such Ets family members as Fli-1 and GABP, as verified by EMSA and ChIP analyses. Furthermore, those LEF-1 and Ets sites were shown to drive additively a strong transcription of Crlz1 gene.

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