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

Abstract

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/b-catenin signal to the expression of Bcl-6, the master regulator of centroblasts, by mobilizing the cytoplasmic CBFb into the nucleus to allow Runx/CBFb heterodimerization and its subsequent binding to the Bcl-6 promoter. The knockdown of Crlz-1 or b-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/b-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.

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

“…The embryonic lethality of Crlz-1 homozygous null KO mice, and thus the failure of obtaining their pups, requires that its conditional KO mice should be pursued in the future to define its physiological functions in any specific cells, tissues, and/or organs during late embryonic development as well as after birth. The embryonic lethality could be explained by considering Wnt-targeted Crlz-1 expression regulation and its protein function in human and/or mouse cells, as reported in our previous papers [ 8 , 9 , 10 , 12 ], as well as the transcriptional derepression activity of Sas10 as a Crlz-1 homologue in yeast [ 2 ] and the role of Crlz-1 as Utp-3 component of the rRNA-processing SSU complex, as reported by other researchers [ 1 , 17 , 18 ]. All of these potential explanations might not be mutually exclusive and together could be responsible for the developmentally stopped embryonic lethal phenotype of Crlz-1 homozygous null KO mice.…”

“…Another potential reason for the embryonic lethality might be related to the role of Crlz-1 as Utp-3 component of small subunit processome (SSU) during the rRNA processing. However, Utp-3 does not appear to be constitutively required as a house-keeping protein for the rRNA processing, as judged by the fact that it is highly expressed mainly in rapidly proliferating cells such as pre-B and GC centroblast cells [ 9 , 10 ], but not ubiquitously in all cells. Based on this information, it is speculated that Crlz-1 should be highly expressed in the proliferating embryonic cells to accelerate the supply of more translating ribosomes by participating as Utp-3 component of SSU, thereby speeding up the timely production of necessary amounts of proteins to match their rapid proliferation.…”

“…Based on the Crlz-1 KO construction map on the mouse chromosome 5 ( Figure 1 ), which was informed by UC Davis KOMP Repository, eight primers, as indicated by arrows on the map, were designed to genotype KO pups and embryos by PCR, which was performed in essentially the same way as reported previously [ 9 , 10 ]. Two 5′-homology arm forward primers (HF1; ATA TCC TTG GCC TAG CGT TCC, HF2; AAC TTC CAC TCT GGC AAA CGG), which are paired with the LacZ reverse primer (Lac-R; GTC TGT CCT AGC TTC CTC ACT G) of the targeting reporter-selection cassette (ZEN-UB1, KOMP), generate the PCR product sizes of 448 and 671 bp, respectively.…”

“…Notably, the promoter of Crlz-1 turned out to be bound by LEF-1 (lymphoid enhancer factor-1) with β-catenin in pre-B cells, which led to the discovery that this gene is a target of the canonical Wnt (wingless-related MMTV integration site) signaling pathway [ 8 , 9 ]. Furthermore, Crlz-1 , as induced by the Wnt/β-catenin signaling pathway, was found to play a critical role in the proliferating B cells such as pre-B [ 9 ] and germinal center (GC) centroblast cells [ 10 ]. Interestingly, it was also found to be expressed in the proliferating spermatogonia and Sertoli cells, as well as differentiating spermatids in the testis, indicating some roles during its development and/or spermatogenesis [ 11 ].…”

“…Crlz-1 was demonstrated to be localized within the nucleus [ 12 ], as anticipated by both its transcriptional derepression activity as Sas10 in yeast [ 2 ] and a constituent of SSU as Utp3 during rRNA processing [ 1 ]. Mechanistically, in accordance with its original cloning by CBFβ as a bait of yeast two-hybrid screen [ 3 ], Crlz-1 was demonstrated to work by mobilizing the cytoplasmic CBFβ into nucleus to allow its heterodimerization with Runx (runt-related transcription factor), and thereby to form a higher affinity form of Runx/CBFβ heterodimer in both pre-B and GC centroblast cells [ 9 , 10 , 12 ]. Consequently, in the case of pre-B cells, Runx/CBFβ turned on the expression of VpreB and λ5 genes, leading to the assembly of pre-BCR (B cell receptor) and its signaling to proliferate pre-B cells for their clonal diversity during early B cell development [ 9 ], whereas, in the case of GC centroblast cells, Runx/CBFβ turned on the expression of the GC master Bcl-6 gene, leading to the regulation of its many downstream target genes to proliferate centroblast cells without their differentiation into plasma cells during humoral immune GC reactions, which are essential for antibody affinity maturation, class switch recombination, and memory B cell generation [ 10 ].…”