Crlz-1 Homozygous Null Knockout Mouse Embryos Are Lethally Stopped in Their Early Development.

Although the conditional gene knockout (KO) is a better choice for observing its phenotype in a specific cell, tissue, and/or organ, the simple null gene KO could nevertheless be attempted initially to scan its overall phenotypes at the level of the whole-body system, especially for a new gene such as Crlz-1. Therefore, with a hope to glean phenotypic clues for Crlz-1 at the whole-body system, we attempted to generate its null KO mice. Contrary to our original desire, Crlz-1 homozygous null KO mice were not born. However, in the chasing of their homozygous KO embryos, they were found to be lethally impaired from early development, remaining in a state of small globular mass without ever leading to a body shape, indicating the critical role of Crlz-1 as a Wnt target gene for the proliferation and/or differentiation of cells during early mouse embryonic development.

Crlz-1 Controls Germinal Center Reaction by Relaying a Wnt Signal to the Bcl-6 Expression in Centroblasts during Humoral Immune Responses.

Crlz-1 was expressed along with Wnt3a in the rapidly proliferating centroblasts within the dark zone of germinal center (GC) during humoral immune responses. Significantly, Crlz-1 relayed a Wnt/ß-catenin signal to the expression of Bcl-6, the master regulator of centroblasts, by mobilizing the cytoplasmic CBFß into the nucleus to allow Runx/CBFß heterodimerization and its subsequent binding to the Bcl-6 promoter. The knockdown of Crlz-1 or ß-catenin, as well as inhibition of Wnt signaling in the centroblasts, led to the decreased expression of Bcl-6 and, thereby, the altered expression of its various target genes, resulting in their diminished proliferation. Consistently, the administration of Wnt inhibitors into the immunized mice impaired or abolished GC reaction, with concomitant decreases of Crlz-1 and Bcl-6 expression and, thus, centroblastic proliferation. Our observation that Wnt/ß-catenin signaling via Crlz-1 regulates GC reaction would suggest developmental strategies for vaccine adjuvants and cancer therapeutics because both immune efficacy and accidental lymphoma depend on GC reaction. Our studies of Crlz-1 were performed using human cell lines, mice, and their primary cells.

Charged Amino Acid-rich Leucine Zipper-1 (Crlz-1) as a Target of Wnt Signaling Pathway Controls Pre-B Cell Proliferation by Affecting Runx/CBFß-targeted VpreB and λ5 Genes.

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 CBFß (core binding factor ß) into the nucleus to allow Runx (runt-related transcription factor)/CBFß heterodimerization. Runx/CBFß 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 ß-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/ß-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFß heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.