The Functional Study Of Cbx4 In Zebrafish Hematopoietic Development And Disease Transformation

Background:In vertebrates,hematopoietic system development is an orderly and dynamic process.In this complex process,the early development of hematopoietic stem cells（HSCs）is the most critical and important.When HSC or precursor cells are abnormal,monoclonal amplified cells proliferate and accumulate in bone marrow and other hematopoietic tissues,infiltrate other tissues and organs,inhibit normal hematopoietic function,and then cause leukemia.In recent years,the pathogenesis of leukemia has been thoroughly studied.It has been found that in addition to cytogenetic changes,epigenetic modification abnormalities also play a key role in the occurrence and development of leukemia.Polycomb group complex（PcG）is an important epigenetic regulatory complex that exerts transcriptional inhibition.It silences target genes mainly by trimethylation of H3K9 and H3K27,and participates in important physiological processes such as embryonic development,cell aging and apoptosis.As a malignant clonal disease of hematopoietic stem cells,leukemia is often accompanied by abnormal expression or mutation of PcG gene.CBX4 is one of the core proteins of PcG complex.It is the only member of the CBX family with enzymatic activity and acts as a small ubiquitin-like modifier E3 ligase（small ubiquitin-like modifier E3 ligase）.The special role of CBX4 as E3 enzyme has been confirmed in hepatocellular carcinoma,breast cancer and osteosarcoma.However,there are no reports about the relationship between cbx4 gene and leukemia.NCBI data showed that cbx4 gene was highly expressed in tissues related to human hematopoiesis and embryonic development,such as placenta,bone marrow and spleen.At the same time,QPCR data showed that the expression of cbx4 was up-regulated in clinical samples of myeloid leukemia compared with normal people.Therefore,we speculated that the overexpression of cbx4 might affect the hematopoietic development and play a role in the occurrence of leukemia.Method:We constructed the hsp:cbx4-eGFP plasmid and introduced it into zebrafish embryos by microinjection technology.Overexpression of cbx4 was obtained by heat shock treatment.The expression of marker genes of hematopoietic development in different lineages of hsp:cbx4-eGFP zebrafish was detected by chemical staining and in situ hybridization.A cbx4 zebrafish mutant was constructed by CRISPER/CAS9 technology to explore whether the transient knockdown of cbx4 by CRISPR/CAS9 could save the blood phenotype of hsp:cbx4-eGFP zebrafish.At the same time,the blood cell composition of peripheral blood and kidney of F3 generation hsp:cbx4-eGFP adult fish exposed to continuous heat shock for 6-Month was determined by cell staining.Finally,we explored the mechanism of cbx4 influencing blood phenotype from the perspective of epigenetics through Western Blot and drug treatment experiments.Results:We found that in zebrafish with over-expression of cbx4 of 3dpf at embryonic stage,myeloid progenitors and mononuclear macrophages increased,and the abnormal blood phenotype of hsp:cbx4-eGFP zebrafish could be saved by knocking down cbx4 instantaneously through CRISPR/CAS9.In 6-Month hsp:cbx4-eGFP peripheral blood,myeloid cells increased mainly as monocyte macrophages,while no significant changes were found in kidney marrow.Based on the above results,we speculate that the effect of cbx4 on hematopoietic development may be related to its epigenetic modification.Western Blot results showed that the levels of H3K9me3 and H2AK119ub in zebrafish overexpressing cbx4 were higher than sibling levels at 3dpf,and the phenotype of the increase of myeloid and mononuclear macrophage cells in zebrafish overexpressing cbx4 was inhibited after the treatment of zebrafish embryos with methylation and ubiquitination inhibitors.Conclusions:Overexpression of cbx4 gene in zebrafish has abnormal phenotype of increasing myeloid and mononuclear macrophages in embryonic and adult stages.The zebrafish model may become a new model for the study of chronic myelomonocytic leukemia（CMML）and drug screening.