The Effect And Mechanisms Of TET3 On Terminal Erythroid Differentiation

Background and purpose:Erythropoiesis is a complex process that requires tight regulation.Erythropoiesis can be subdivided into 3 stages: early erythropoiesis,terminal erythroid differentiation,and reticulocyte maturation.During the past decades,the research on mechanism of erythropoiesis is mostly focusing on cell cytokines and transcription factors,such as erythropoietin and its receptor,GATA1 and KLF1.Recently,DNA methylation,one of the best-characterized epigenetic modifications in mammalian genome,has been proven to play important regulatory roles in the self-renew of hematopoietic stem cell.Although,nearly 30 years' studies have demonstrated that DNA methylation/demethylation is critical for the development of erythroblasts,the stage specific regulatory role and molecular mechanism still remain to be further clarified.Members of TET family have been well established to act as the primary enzyme that mediate DNA demethylation.Our RNA-seq analysis showed that the expression of TET3 is highest among TET family members and the expression level of TET3 is gradually increased with the differentiation,which indicates that TET3 may play an important role on human erythropoiesis.Hence,the present study were proposed to firstly construct the dynamic DNA demethylation profile of the whole process of erythropoiesis using ERRBS analysis,and then perform multi-omics analysis together with functional genomics approaches to investigate the effect and mechanism of TET3 on human erythropoiesis.In addition,to further verify the roles of Tet3,we constructed conditional knockout mice model in which Tet3 was specifically knocked out in erythroid and studied the roles of Tet3 in this in vivo system.Our study led to a better understanding of erythroid cell development and differentiation,and provide the basis for future research endeavors in developing mechanistic understanding of normal and disordered erythropoiesis.Study methods:(1)The method to obtain highly pure populations of human erythroblasts at distinct developmental stages during terminal erythroid differentiation was developed and published previously by our lab.Next-generation bisulfite sequencing using ERRBS assay was performed on highly purified human erythroblasts at distinct developmental stages to study the dynamics of global DNA methylation and demethylation during human terminal erythroid differentiation.(2)CD34+ cells that were isolated from human cord blood were transduced with lentivirus packaged with TET3 sh RNA to knock down TET3,and then were cultured in vitro to differentiate into erythroblasts.Quantitative real-time PCR was conducted to check knockdown efficiency.The process of erythropoiesis,apoptosis and enucleation were detected with flow cytometery.Cell counting was performed to check the proliferation and cytospins was done to observe morphology of cells and nuclei.(3)Fluorescence-activated cell sorting was performed to obtain highly purified populations of erythroblasts at distinct developmental stages of control group and TET3 knockdown group during terminal erythroid differentiation.RNA-seq was performed with RNA extracted from these highly purified populations of erythroblasts in control group and TET3 knockdown group.Quality control was performed on the sequenced reads and low quality reads and reads with adapter sequences were removed from the analyzed data set.The reads were then aligned to the human hg19 reference genome with Tophat2.Raw read counts were extracted from the aligned read BAM file using htseq-count from the HTSeq framework.Differential gene expression was determined using DESeq2 a bioconductor package in R.(4)Based on the RNA-seq analysis,we identified several possible genes that associated with TET3 knockdown phenotype.Among these genes,KLHDC8 B was sorted out since it was reported to be associated with mitosis,suggesting that it might be involved in the generation of bi/multinuclei.Thus,the constructs for KLHDC8 B knock down and over-expression were prepared for the function study and rescue experiment.Quantitative real-time PCR and Western Blot were used to check the knockdown and over-expression efficiency.Flow cytometry and cytospins were used to observe the phenotypes of KLHDC8 B knockdown and ectopic expression of KLHDC8 B after TET3 knockdown.(5)Genomic DNA was extracted from these isolated populations of erythroblasts at distinct developmental stages of control group and TET3 knockdown group during terminal erythroid differentiation.Mass spectrometry was done to compare the 5-m C and 5-hm C levels in control group with those in TET3 knockdown group.The methylation status of KLHDC8 B promoter region after TET3 knockdown was examined using methylation-sensitive restriction enzyme analysis as well as Me DIP followed by real time PCR.ATAC-seq was performed to assess whether TET3 knockdown influenced chromatin accessibility on a genome wide scale.(6)The method to obtain highly pure populations of murine erythroblasts at all distinct developmental stages during terminal erythroid differentiation was developed and published previously.DNA promoter methylation assay(HELP assay)was performed on highly purified murine erythroblasts at distinct developmental stages to study the dynamics of DNA promoter methylation status during murine terminal erythroid differentiation.The expression of Tet family was analysed with the transcriptome database published previously and confirmed by quantitative real-time PCR.Tet3 conditional knockout mouse model(Epor-GFP-Cre Tet3 conditional knockout mice)was generated to verify the roles and mechanisms of Tet3 on erythropoiesis in vivo.Results:(1)We sorted cells at distinct stages of terminal differentiated erythroblasts and performed ERRBS to check the DNA methylation profile.The analysis of ERRBS data reveals that it is cumulative and dynamic changes of global demethylation during human erythroid differentiation.(2)Our quantitative real-time PCR and RNA-seq analysis revealed that TET3 is the predominant TET family member that was expressed in human erythroblasts,indicating that TET3 may play an important role on erythropoiesis.(3)To explore the role of TET3 in erythropoiesis,we employed shRNA mediated knockdown approach in human CD34+ derived from cord blood and cultured in 3-phase medium.The deficiency of TET3 delayed but not blocked the differentiation,reduced cell growth accompanied by increased apoptosis of late stage erythroblasts,impaired enucleation,and increased bi/multinucleated polychromatic and orthochromatic erythroblasts.(4)Since TET3 was found to be highly expressed in poly and ortho erythroblasts,and TET3 knockdown was found to induce significant abnormalities in late stage of erythroblasts.Therefore,in order to study the mechanism of increased bi/mutinucleied cells,we sorted the specific stages of erythroblasts to conduct RNA-seq analysis.After being analysed with Tophat2,HTSeq and DESeq2,principle component analysis(PCA)of RNA-seq results showed that biological replicates of control group and TET3 knockdown group at the same stage of development are tightly clustered separately,and clear separation of the same staged population in control group and TET3 knockdown group,especially poly and ortho,which indicated that the gene expressions of late stage erythroblasts were predomminantly affected by TET3 knockdown.KLHDC8 B was identified as the fifth down-regulated gene.Previous studies showed that mirror image cells in Hodgkin's lymphoma were correlated with decreased KLHDC8 B expression.We hypothesized that the decreased expression of KLHDC8 B may be correlated with the phenotype of bi/muti-nucleated of polychromatin and orthochromatin erythroblasts.(5)Knockdown of KLHDC8 B didn't affect the differentiation and proliferation,but impaired enucleation and increased bi/multinucleated polychromatic and orthochromatic erythroblasts,which was consistent with the effect of TET3 on late stage of erythroblasts.Rescue experiment showed that overexpression of KLHDC8 B partially rescued bi/multi-nucleation defect caused by loss of function of TET3.(6)Mass spectrometry was done to compare the 5-m C and 5-hm C level in control group with those in TET3 knockdown group on pure populations of erythroblasts at distinct developmental during terminal erythroid differentiation.The results showed that TET3 knockdown led to no significant increase in 5-m C levels at the stages of proerythroblasts,early basophilic erythroblasts,late basophilic erythroblasts and polychromatic erythroblasts.However,at the stage of orthochromatic erythroblasts,TET3 knockdown increased global level of 5-m C although there was no significant statistic difference(P=0.053).The global level of 5-hm C could not be determined because the basal level of 5-hm C was very low to be detected.We also checked the methylation level at the promoter of KLHDC8 B but did not detect cytosine methylation at the CCGG sites in the promoter region of KLHDC8 B,implying that the expression of KLHDC8 B is not regulated via the role of TET3 on DNA demethylation.Furthermore,ATAC-seq analysis did not reveal differences in chromatin accessibility at the KLHDC8 B locus,neither.We would like to note that the ATAC-seq was only performed on orthochromatic erythroblasts.Thus,we could not exclude the possibility that TET3 knockdown may affect the chromatin configure of basophilic or polychromatic erythroblasts.Finally,we tried all the available anti-TET3 antibodies,however,all of the antibodies failed to distinguish TET3 in erythroblasts.(7)We sorted the specific stages of mice erythroblasts to do HELP assay.The result showed that global DNA demethylation occurs during murine terminal erythroid differentiation,which is consistent with published data.Our quantitative real-time PCR and RNA-seq analysis of highly purified erythroblast populations revealed that Tet3 is the predominant TET family member that was expressed in murine erythroblasts.Therefore,we performed further study to check the roles of Tet3 on the regulation of mouse erythroid development.(8)Since Tet3 deficiency will lead to embryonic lethality,the current role of Tet3 in the development of mouse erythropoiesis still remain unknown.In the present study,we used Cre/Loxp recombinase system to establish Tet3 conditional knockout mice.Under normal condition,the blood routine of Tet3 conditional konckout mice showed no significant change comparing with the wild type mice.However,under the stress condition that was induced by the treatment with Phenylhydrazine,the hematological parameters of Tet3 conditional knockout mice recovered much slower than that in wild type mice,and the weight of spleen in Tet3 conditional knockout mice was more heavy than that in wild type mice,and orthochromatic erythroblasts of Tet3 conditional knockout mice was increased while reticulocytes and red blood cells were decreased,which prompts that Tet3 may have a significant effect on acute anemia in mice.Conclusions:(1)The DNA demethylation level during human and murine terminal erythroid differentiation are cumulatively changed in a dynamic manner;(2)TET3 is the predominant TET family member that was expressed in both of human and mice erythroblasts,and the expression level is increased during terminal erythropoiesis;(3)The deficiency of TET3 impairs human erythropoiesis on enucleation,morphology and proliferation;(4)Down regulated of KLHDC8 B induced by TET3 knockdown results in increased bi/multinucleated polychromatic and orthochromatic erythroblasts;(5)TET3 knockdown increases the global level of 5-m C of orthochromatic erythroblasts;TET3 knockdown does not affect DNA methylation of KLHDC8 B promoter;further study on the effect of TET3 on chromatin accessibility is needed;(6)Tet3 conditional knockout in the erythroid lineage impairs terminal erythroid differentiation under stress condition.