Differential M~6A RNA Landscapes Across Hematopoiesis Reveal A Role For IGF2BP2 In Preserving Hematopoietic Stem Cell Function

In the life cycle of the organism,hematopoietic stem cells(HSCs)reside at the top of the blood system.HSCs maintain blood system homeostasis by balancing self-renewal and differentiation.This process is tightly and finely regulated.The unbalance of blood system homeostasis can lead to various blood diseases such as leukemia,bone marrow failure,myelodysplastic syndrome,which also cause several major human diseases.Therefore,it is of great significance to understand regulation mechanism of blood system homeostasis.N~6-methyladenosine(m~6A)is the most abundant reversible methylation modification on eukaryotic m RNA,it regulate multiple aspects of m RNA metabolism including m RNA stabilization,degradation,translation and localization.Recent studies have shown that m~6A modification plays an important role in normal hematopoietic development,the maintenance of hematopoietic stem cell and leukemia development.Therefore,the m~6A modification dynamics under physiological and pathological blood system have become important scientific issues to be solved urgently.However,conventional m~6A-seq cannot be applied to rare cell populations such as hematopoietic stem/progenitor cells,which restricts the research of m~6A modification in many fields such as stem cells,development,and immunity.We first established a high-sensitivity,low-input m~6A-seq method called SLIM-seq(super-low input m~6A-seq).16 different hematopoietic stem cells,progenitor cells and mature blood cells from mice were sorted by flow cytometry.We performed SLIM-seq of16 cell types,obtained 8599 high-confidence m~6A modification m RNAs through bioinformatics analysis compared with the corresponding RNA-seq data,and drawn the m~6A modification m RNA landscape of the blood system.We found significantly higher global m~6A levels in LT-HSC,but as it differentiate into the erythroid and myeloid populations,the level of m~6A modification will decrease.However,the lymphoid population still retains high m~6A modification.We found that m~6A has high cell-type identity.In view of the strict hierarchical differentiation pattern of the blood system,we tried to analyze the origin of m~6A modification.Interestingly,it was found that most of the m~6A modification patterns in various cells except(short-term hematopoietic stem cells,ST-HSC)were inherited from the upstream stages of their development.We further analyzed the correlation between m~6A modification and m RNA expression level,and found that most m~6A modification in the commited progenitor cell populations such as MEP and Mk P regulate m RNA degradation.Interestingly,a significant portion of m~6A modifications(～25%)in LT-HSC play a role in stabilizing m RNA.Pay attention to this phenomenon,we further explored the molecular mechanism and believe that m~6A reading proteins may play a decisive role in m~6A modification m RNA.By analyzing and comparing the expression levels of these reading proteins,we finally focused on IGF2BP2 and found that it was highly expressed in LT-HSCs and maintained the stability of m~6A modification m RNA in LT-HSC.In terms of physiological function,a series of in vivo and in vitro experiments were carried out using Igf2bp2 knockout mice.It was found that the number of LT-HSC in Igf2bp2 knockout mice was reduced,the colony forming ability was weakened,the apoptosis was significantly increased,the quiescent phase was lost,and the long-term hematopoietic reconstruction ability was severely impaired,thus confirming that IGF2BP2 has an important role in maintaining the function of hematopoietic stem cells.Furthermore,we found that Bmi1 in LT-HSC is an important downstream target of Igf2bp2;Loss of Igf2bp2 promotes Bmi1 m RNA degradation,thereby relieving the inhibition of mitochondrial related gene expression by Bmi1,upregulating mitochondrial activity,and ultimately causing hematopoietic stem cell function depletion.In summary,this study systematically described the m~6A modification of different cell populations in the mouse blood system,revealed its dynamics,discovered the important role of IGF2BP2 in the functional homeostasis of hematopoietic stem cells and clarified its function mechanism.It will help us deeply understand the homeostasis of the blood system and the regulation of hematopoietic stem cell functions.In addition,the research has broken through the technical bottleneck in the field,and established SLIM-seq,a low input m~6A sequencing technology.It will greatly assist the related research of RNA m~6A modification in stem cells,development,immunity and other fields.