Analysis Of DNA Methylome And Transcriptome Of Bone Marrow Derived Terminal Erythrocytes From β-thalassemia By Whole Genome Bisulfite Sequencing And Single Cell RNA Sequencing

Objectives The pathogenesis of β-thalassemia is mainly caused by the mutation of β-globin gene,which leads to the decrease or lack of normalβ-globin chain synthesis,and the excess α-globin depositing on the erythrocyte membrane,resulting in the damage of erythrocytes and inefficiency of hematopoiesis in the medullary.Most of the pediatric patients withβ-thalassemia major(β-TM)usually died due to complications caused by anemia and long-term blood transfusion.So far,the only clinical curative therapy,allogeneic hematopoietic stem cell transplantation(allo-HSCT)is limited by problems such as donor source and transplantation related complications.Therefore,gene editing therapy came into being.In this study,whole genome bisulfite sequencing(WGBS)and single cell transcriptome sequencing(sc RNA-seq)were applied to analyze differential DNA methylation and transcriptome between β-TM patients before and after HSCT and healthy donors,aiming to provide candidate gene targets for studying the molecular mechanism of erythrocyte differentiation in β-thalassemia.Methods1.The peripheral blood samples of 33 patients with β-TM were collected to analyze the changes of hemoglobin content,the expression of globin genes and erythrocyte specific related genes at different time points after HSCT,at 2-week,1-month,2-month,3-month and 6-month.2.Bone marrow samples were respectively collected from 2 patients withβ-TM before HSCT and 1 month after HSCT and 2 related donors.Ex vivo differentiation protocols now exist to cultivate sufficient number of human erythroblasts to detect their DNA methylome and single cell transcriptome.3.WGBS of erythroblasts was conducted to compare the methylation levels between recipients pre-HSCT and 1-month post-HSCT and donors.Bioinformatics methods were used to analyze the differentially methylated regions(DMRs),the transcription factors binding motif of DMRs,and the GO terms of DMRs related genes between different groups.4.Sc RNA-seq of erythroblasts was performed to the erythroblasts.The library construction and sequencing of terminal erythrocytes were performed by the 10× Genomics system.We applied Monocle to reconstruct cell differentiation trajectory,and combined with the marker genes of terminal erythrocytes reported in previous study to annotate the differentiated erythrocytes.The variation trends of gene expression levels during differentiation among different groups of samples were also analyzed.The association of DMG related genes(DMGs)and differentially expressed genes(DEGs)were analysis at last.Results1.Patients with β-TM showed fluctuations in hematopoietic recovery during the 6 months after HSCT,and their hematopoiesis usually returned to donor level by 6 months after HSCT.Taking the donors as the control,the expression of HBB,KLF1 and BCL11 A at 1 month after HSCT was significantly higher than that at other time points after HSCT,while the expression of HBG at1 month after HSCT was lower than that at other time points after HSCT.2.High purity CD34+ cells from bone marrow were isolated by immunomagnetic beads.The two-stage serum-free culture system can obtain terminal erythrocytes with high amplification and high proportion.During the directed differentiation,the expression of erythrocyte specific related genes was consistent with normal physiological process.3.Genome-wide DNA methylation profiles showed that the frequency and level of DNA methylation in CG context accounted for the majority.DNA methylation levels of different gene functional regions were compared,the highest methylation levels were found in exon and intron regions,and the lowest methylation levels were found in gene body regions near transcription start site.The methylation levels of donors were higher in all gene functional regions than that of patients before HSCT or after HSCT.DMRs were identified between patients pre-HSCT and post-HSCT and related donors.Many transcriptional differences associated with hematopoietic and erythrocyte differentiation were captured in DMRs,particularly in the transcriptional promotor binding region.Finally,GO enrichment analysis was performed for these DMRs related genes.Results show that mitotic cell cycle regulation,cell growth and division regulation,apoptosis regulation,protein localization and maintenance,protein secretion regulation,gene expression regulation,histone H2 A acetylation and other processes were significantly enriched.4.Through cell cycle and GO enrichment analysis of DEGs in different cell stages,it was found that early-Ortho E can still be carried out erythrocyte differentiation,which was different from previous results.We also discovered the biological process of negative regulation of erythrocyte homeostasis at the stage of Ortho E in the patients with β-TM.In the analysis of dynamic changes in the gene expression during terminal erythroid differentiation,IFI27,MGST3 and HEMGN were found to be common DEGs in the groups of the pre-HSCT recipients vs.the post-HSCT recipients and groups of the post-HSCT recipients vs.the related donors.In the association analysis of the DMGs and DEGs,compared with group of the post-HSCT recipients,CST3,USP34,CD164,TNRC6 B,ELF1 and CD44 were upregulated with hypomethylated from the group of pre-HSCT recipients.Conclusion WGBS and sc RNA-seq were conducted in this study.We drew the DNA differential methylation landscape for the terminal erythrocytes of β-TM,and captured transcriptional differences associated with erythroid differentiation,especially in the transcriptional promotor binding regions.The DEGs: IFI27,MGST3 and HEMGN and the DMGs: CST3,USP34,CD164,TNRC6 B,ELF1 and CD44 were found to be related with erythroid differentiation in thalassemia.These DMGs and DEGs may provide genetic targets for studying the molecular mechanism of erythrocyte differentiation inβ-TM.