A Calmodulin Interacting Protein Iqcg Is Required for Definitive Hematopoiesis in Zebrafish

Abstract 764

We previously identified a novel fusion gene NUP98-IQCG in a bi-phenotypic acute T-lymphoid/myeloid leukemia patient. However, the function of IQCG has never been demonstrated. By taking advantage of the zebrafish as a model organism, we investigated the role of IQCG in hematopoietic development and found that the definitive hematopoiesis was severely impaired in the iqcg deficient embryos. The hematopoietic stem cell (HSC) population as well as the number of multilineage differentiated hematopoietic cells was reduced. Moreover, the number of phosphorelated histone3 positive HSC was decreased, which indicated a compromised proliferation in iqcg deficient HSC.

Since IQCG protein contains an IQ calmodulin binding motif (IQ motif), we next examined if IQCG protein can interact with calmodulin. Co-immunoprecipitation assay results showed that both human and zebrafish IQCG proteins have the ability to precipitate calmodulin. Deletion of IQ motif disrupted this interaction. The isothermal titration calorimetry results indicated that the IQ motif of IQCG protein have a lower association constant with calcium-loaded calmodulin than apo-calmodulin. We next crystallized the complex of calmodulin and IQ motif of IQCG in two circumstances-with and without calcium. Determination of these two crystal structures indicated different interaction details between them. These results suggest that IQCG may be involved in calcium signaling in HSC.

Previous studies reported that calmodulin dependent protein kinase IV (CaMKIV) involves in HSC maintenance and survival. So we wondered if CaMKIV acts downstream of IQCG. Indeed, whole-mount immunofluorescence data showed that the level of phosphorelated CaMKIV markedly reduced in iqcg morphants compared with controls. Reactivation of CaMKIV by co-injection with constitutively activated CaMKIV (CACaMKIV) mRNA rescued the HSC number in iqcg deficient embryos. Furthermore, embryos injected with camkIV morpholino phenocopied the iqcg morphants.

In summary, we demonstrate an important role of the novel NUP98 partner gene IQCG in definitive hematopoietic development. IQCG may control the HSC proliferation through interacting with calmodulin and acting upstream of CaMKIV, which suggests a novel pathway in definitive hematopoietic development.