Study On The Function Of ASXL1 In Hematopoiesis And The Related Leukemogenic Mechanisms

Myeloid malignancies are clonal diseases caused by hematopoietic stem or progenitor cells with the alterations of self-renewal,proliferations and differentiation.Somatic Addition of Sex Combs Like 1(ASXL1)mutations occur in patients with multiple myeloid malignancies.Mutations affecting ASXL1 most frequently occur as heterozygous frameshift or nonsense mutations,usually in exons 11 or 12 and usually lead to its C-terminal truncation.Asxll deletion and expression of C-terminus truncated ASXL1 results in dysregulated hematopoiesis regulators through the abnormal histone modifications.Notwithstanding the phenotypes,what roles ASXL1 play on hematopoiesis,particularly during embryonic development,is currently unclear.Furthermore,leukemogenic mechanisms of these endogenous ASXL1 mutants have not been fully examined.We want to use zebrafish to solve these questions.1.asxll is expressed in hematopoietic cellsThe expression pattern of asxll at various embryonic developmental stages was detected by WISH and real-time PCR.At 18 to 24hpf,asxl1 was detected in ALM and PLM.Besides,asxl1 can be detected in kidney marrow and more specific in myeloid cells.It indicates that asxll may be participated in zebrafish hematopoiesis.2.Zebrafish asxll mutant has impaired neutrophil differentiationTo examine the role of asxl1 during embryonic hematopoiesis,we generate asxl1 mutant line by CRISPR/Cas9.We observed Neutrophil markers lysozyme C(lyz)and myeloperoxidase(mpx)significantly reduced as shown by WISH as well as qRT-PCR in 3dpf.To support it,SBB staining was showed that SB+ neutrophils were reduced in asxll mutant.Furthermore,May-Grunwald-Giemsa-staining showed that mature neutrophils were decreased in 5dpf asxll mutants.Analysis of hematopoietic cells in kidney marrow,the functional ortholog of bone marrow,via cytological indicated mature neutrophil significant decreased in 6-month and 1-year mutants.3.asxll deficiency leads to myeloid malignancies in adult zebrafishWestern blot analysis with 2dpf embryos showed decreased levels of global H2AK119ub and H3K4me3 but not H3K27me3.After H3K4me3 demethylase inhibitor treatment,Neutrophil markers lyz significantly increased both in siblings and mutants as shown by WISH.4.Truncated ASXL1 can be detected in knock-in cellsTo further know the leukemogenic mechanisms of C-terminal truncated ASXL1 mutants.We use mouse embryonic stem cells constructed the BAP 1-tagged FLAG cell line.And on that basis,expressed endogenous full-length ASXL1-tagged HA and Cterminal truncated ASXL1-tagged HA were constructed.We found that Bap1 expression was increased in Asxl1 mutant.Besides,we also found that truncated ASXL1 can be detected after IP.But full-length ASXL1 can't be detected.Furthermore,truncated ASXL1 can have stable interaction with BAP1.It indicates that Asxl1 mutants lead to disease may because of gain-of-function.