A Natural DNMT1 Missense Mutation Elevates Fetal Hemoglobin Via Epigenetic De-Repression Of The ?-Globin Gene In ?-Thalassemia

BACKGROUND AND OBJECTIVE?-thalassemia is among the most common human monogenic diseases worldwide and characterized by absent or decreased production of ?-globin chains and subsequently ?/-globin chain imbalance which is caused by over 300 different mutations in the adult ?-globin gene.The highest frequencies of ?-thalassemia occur in southern China,particularly the Guangxi and Guangdong Provinces.The total heterozygote frequency of ?-thalassemia among the populations of Guangxi Zhuang Autonomous Region,where haemoglobinopathies are most endemic in China is 6.43%.It has a wide spectrum of clinical severity,while which is primarily determined by the type of ?-allele,patients with same ?-globin genotype display variable clinical expression,implicating the existence of other genetic modifiers.However,the potential modifiers that affect the ?-thalassaemia phenotype and the underlying mechanisms remained largely unknown,which are worth further investigation.Elevated levels of fetal hemoglobin(HbF)has shown to ameliorate phenotypic severity of ?-thalassemia.Several transcription factors,such as KLF1.BCL11A.MYB.have been revealed as master regulator with increasingly established role in y-globin gene regulation.A few of functional mutations in these transcription factors have been identified as ameliorators of ?-thalassemia.Therefore,elucidating the complex molecular mechanisms underlying developmental globin gene switching,and investigating genetic variants responsible for the reactivation of ?-globin genes are very important for the improvement of the management of ?-thalassaemia.With the continuous understanding of the mechanism underlying the regulation of hemoglobin switching.in addition to these well-studied transcription factors,there has been a growing appreciation of the role of epigenetic signals and their cognate factors in y-globin gene silencing regulation.DNA methylation is the major epigenetic modification that has been closely associated with gene silencing.Methylation levels at specific CpG sites of HBG promoters inversely relate to y-globin gene expression.In mammals,DNA methyl transferases DNMT1 is the major and ubiquitously expressed DNA methyltransferase which displays an important role in maintaining specific DNA metylation pattern in many human tissues.Previous works have supported a role of DNMT1 as a co-repressor to a number of transcription factors in repressing target genes.DNMT1 has been shown to participate in the regulation of human y-globin gene repression by interacting with kinds of well known y-globin regulators,including erythroid transcription factors BCL11A,GATA1,LRF,TR2/4,SP1,MYB,as well as epigenetic co-regulators HDAC1/2.These regulators are indispensable for the recruitment of DNMT1 to the promoter region of HBG gene to repress human y-globin expression via maintenance of DNA methylation.In addition,clinical studies for depleting DNMT1 have been shown to effectively upregulate HbF levels in patients with ?-hemoglobinopathies.Whiles studies have identified epigenetic regulation as being important for the regulation of y-globin gene silencing,the precise mechanisms driving epigenetic regulation of y-globin gene expression involved in ameliorating ?-thalassemia severity have not been comprehensively studied and the mutation spectrum of epigenetic regulators and its impact on phenotypes of ?-thalassemia patients have yet to be determined.In this study,we analyzed a set of genes associated with epigenetic regulation of human hemoglobin gene expression from a cohort of 1142 Chinese ?-thalassemia individuals with a targeted NGS approach to identify genetic modifiers which can ameliorate the clinical severity of?-thalassemia.and investigated the underlying mechanisms.MATERIALS AND METHODS1.Target capture-based next generation sequencing(NGS)assay and statistical analysis.In this study,a total of 49 genes associated with the epigenetic regulation of hemoglobin gene expression from 1142 ?-thalassemia individuals(thalassemia major TM and thalassemia intermedia TI),were analyzed with NGS-based gene sequencing strategies for genetic modifiers responsible for ameliorating ?-thalassemia severity.We implemented a student's t-test of the 1142 ?-thalassemia samples to compare the mean HbF levels between the carrier and non-carrier group of each selected candidate variant which was identified as being statistically significant according to the P-values.The selected significant variants were then entered into a backward stepwise Cox proportional hazards model,together with the previously verified ameliorating factors:HBB mutations(?+),HBA disease-causing mutations,KLF1 functional mutations,rs368698783(G)in HBG1,rs9399137(C),rs4895441(G)in HBS1L-MYB,rs4671393(A),rs766432(T),rs11886868(C)in BCL11A to evaluate the association between these putative ameliorating alleles and the transfusion-free age of the 1142 ?-thalassemia samples.According to the result of Cox regression analysis,subjects with and without the DNMT1(c.2633G>A,p.Ser878Phe)mutation were selected into the subsequent case-control and survival analysis after correction for the genotypes of the ameliorating factors to further estimate the effects of S878F on the clinical severity of ?-thalassemia.A univariate analysis with the Fisher's exact test and the Mann-Whitney U test were performed to compare the quantitative variables between case and control groups.The transfusion-free survival curve analysis was performed in the 66 HbE/?0-thalassemia samples using the Kaplan-Meier log rank test method to assess the risk of receiving systematic transfusion of these subjects with(n=3)or without(n=63)the S878F mutation.Identifying the potential ameliorating factor through a series of statistical analysis.2.Verification and functional prediction of the S878F mutant allele.Through a series of systematically statistical analysis.we identified a novel DNMT1 mutation(c.2633G>A.p.Ser878Phe)that is associated with increased HbF levels and a mild phenotype of ?-thalassemia,in three cases among the 1142?-thalassemia patients.We extract the peripheral blood of the 3 patients and their family members to extract the genetic DNA by using the standard phenol/chloroform method.The thalassemia genotype definition of these samples were described in the previous study by our group.The genotypes of the primary HbF-associated variants(KLF1 mutations,rs368698783)and the novel DNMT1 mutation(S878F)were confirmed by Sanger sequencing.The functional predictions of the S878F mutation were performed by using different bioinformatics methods:1.Evolutionary conservation of amino acid residue alteration was analyzed by comparing the human wild-type DNMT1 protein sequence(NP₀01370)with different species downloaded from the UniProt website(https://www.uniprot.org/);2.The three-dimensional(3D)structure model of the mutant DNMT1 protein were predicted by the homology modeling programs SWISS-MODEL(http://swissmodel.expasy.org)based on crystal structure of wild-type DNMT1 protein(Protein Data Bank ID:3SWR)and visualized by the PyMOL software(Schr(?)dinger.Japan);3.The impact of non-synonymous variants on the protein function was estimated with a composite prediction model composed of PolyPhen-2(http://genetics.bwh.harvard.edu/pph2)and SIFT(http://sift.jcvi.org).3.Functional analysis of the DNMT1 mutation(S878F).(1)We firstly Investigated the effect of DNMT1 on the regulation of y-globin gene expression.We constructed the DNMT1 expression vectors to transfect the K562 cells and estimated the effects of DNMT1 on the y-globin gene expression by using the q-PCR and Western Blot assays.We constructed lentivirus vector with DNMT1 shRNA infecting the lentivirus to HUDEP-2 cells knockdown DNMT1 and estimated the effects of knocking down DNMT1 expressionon the y-globin gene expression by using the q-PCR and Western Blot assays.(2)We performed the Western Blot assay to analyze the effects of S878F mutation on the DNMT1 phosphorylation status by using a customized antibody against the phosphorylated form of Ser878 of DNMT1(pDNMT1S878).(3)We constructed the DNMT1 expression vectors(WT.MT)to transfect the HUDEP-2 cells.Cells were treated with cycloheximide and collected at different time points.DNMT1 was detected by using Western Blot assays and that the effects of S878F on the protein stability of DNMT1 were analyzed by calculating the its protein degradation rate.(4)We constructed the DNMT1 expression vectors(WT,MT)to transfect the HUDEP-2 cells.The effects of S878F on the methyltransferase activity of DNMT1 was analyzed using a high sensitivity and specificity assay by measuring the binding ability of DNMT1 protein to its hemimethylated trapping DNA substrate.(5)Analysis of the effects of S878F on the protein-protein interaction of DNMT1 with its protein partners by co-immunoprecipitation(Co-IP)assay.4.Analysis of effects of S878F on the y-globin gene expression in primary erythroid cells from HbE/?0-thalassemia patients.(1)Heparinized peripheral blood samples from HbE/?0-thalassemia patients with(n=2)or without(n=2)S878F mutation were collected to select CD34+cells by magnetic cell column separation using immunomagnetic separation magnetic beads that bind human CD34.CD34+cells were cultured by a two-phase liquid culture procedure and harvested at different cultural periods during the differentiation stage.(2)Analysis of the effects of S878F on the enrichment of DNMT1 protein at the HBG promoter by chromatin Immunoprecipitation(ChIP)assay(3)Genomic DNA was extracted from the cultured progenitor erythroid cells and then bisulfite-modified.A nested PCR reaction was used to specifically amplify the region in the CpG island of the HBG gene to analyze the effects of S878F on the HBG promoter methylation levels.(4)Quantitative measurement of HBG mRNA expression by qPCR and HbF production by Western Blot and flow cytometiy assays;Analysis of hemoglobin by high-performance liquid chromatography(HPLC).(5)The effects of S878F on the erythroid maturation were estimated by analyzing the expression of cell surface markers by flow cytometry assays and Cell morphology by Wright-Giemsa staining assays.RESULTS1.Identification of a novel missense mutation in the DNMT1 gene ameliorates HbE/?-thalassemia.Among the samples from 1142 consecutive ?-thalassemia patients recruited to identify the potential genetic modifiers of ?-thalassemia,379 non-synonymous variants in 45 genes were identified by NGS assay,which including 94 rare(MAF?.01)non-synonymous variants in 33 candidate genes.Sixteen variants in 11 genes were found to be statistically significant(P<.05)by the student's/-test.After a Bonferroni adjustment,only 3 variants[DNMT1(c.2633G>A,p.Ser878Phe);IKZF2(c.278A>G,p.Asn93Ser);CHD3(c.1443C>G,p.His481Gln)](P<.00053)remained to be significant.These variants were then assessed by a Cox regression model to evaluate their impact on the transfusion-free age of the 1142 ?-thalassemia samples.Our results suggest that a novel DNMT1 missense heterozygous mutation(c.2633G>A,p.Ser878Phe)was associated with attenuated clinical severity of?-thalassemia(HR=0.237,P=.014).The effect of this mutation was weaker than that of KLF1 mutations(HR=0.216,P<.001),which have been previously identified in our cohort34 as the most critical ameliorators of the ?-thalassemia phenotype,but was stronger than those of HBB mutations(?+)(HR=0.395.P<.001).HBG1:rs368698783(HR=0.535,P<.001),HBS1L-MYB:rs9399137(HR=0.704,P<.001),HBA disease-causing mutations(HR=0.796,P<.001),and BCL11A:rs766432(HR=0.812.P<.001).The N93S mutation in IKZF2(P=.975).H481Q mutation in CHD3(P=.940),rs4671393(P=.667).rs4895441(P=.352)and rs11886868(P=.150)were removed from the model based on the P value.The novel DNMT1 mutation was identified in three cases among the 1142 ?-thalassemia patients.Correction for the genotypes of other critical ameliorators which were identified by Cox regression analysis,a univariate analysis revealed that the S878F mutation can act as a modifier of ?-thalassemia,with associated elevation in the levels of hemoglobin(8.4 ± 1.3 vs 6.5 ± 1.9 g/dl,P=.028)and HbF(3.2 ± 0.5 vs 1.1±0.7 g/dl,P<.0001).This mutation was associated with increased mean age at onset of anemia(148.0 ± 48.5 vs 31.6±57.9,P=.001)and the first transfusion age(log-rank test P=.027),as well as reduced transfusion requirements(P=.004)and reduced risk of developing ?-thalassemia major(P=.012).2.The functional prediction of the DNMT1 mutation(S878F).The variant that results in a substitution of phenylalanine for the serine residue at position 878 in the Bromo Adjacent Homology-1(BAH1)domain of DNMT1 was further confirmed by Sanger sequencing.Multiple alignment analysis of the human DNMT1 protein with those of different species indicated that this missense variant(c.2633G>A.p.S878F)occurred at a highly conserved codon and that the Ser878 residue has been identified as a phosphorylation site.Furthermore,the S878F mutation was predicted to impair the function of the DNMT1 protein as estimated by PolyPhen-2 and SIFT.The homologous modeling revealed that the-OH(hydroxyl)of the WT Ser878 residue is predicted to bind to the Gln1293 and Pro 1320 residues of the catalytic domain to form hydrogen bonds,which would not be able to form in the mutated protein.3.The S878F mutation affects the function of DNMT1 protein.(1)Western blots using a specific antibody against the phosphorylated form of Ser878 of DNMT1(pDNMT1S878)confirmed that phosphorylation at Ser878 is erythroid-specific and that the S878F mutant protein has reduced levels of phosphorylation in erythroid cells(HUDEP-2 and primary erythroid cells)(2)Protein stability assay revealed that the mutant protein had a faster degradation rate compared to that of wild type DNMT1.(3)DNA methyltransferase enzymatic activity assay revealed significantly decreased binding ability of MT(S878F)to its hemimethylated trapping DNA substrate compared to that of the WT DNMT1.(4)The coimmunoprecipitation(Co-IP)assays showed that S878F mutation specifically decreased the interactions of DNMT1 with the important regulators of y-globin.including BCL11A.GATA1 and HDAC1/2.4.S878F mutation induces demethylation-mediated ?-globin gene expression in primary erythroid cells from HbE/?0-thalassemia patients.(1)(ChIP-qPCR)assays using CD34+-derived erythroid progenitor cells suggest that the accumulation of mutant DNMT1 at the y-globin promoter was significantly reduced for CD34+ cells with S878F mutation.(2)Results of the DNA methylation analysis revealed that compared to the CD34+cells with wild type DNMT1.the CD34+ cells with S878F mutation exhibited different degrees of hypomethylation at the specific CpG sites within the HBG promoter.(3)Results of q-PCR revealed that the ?/?+? mRNA levels were significantly elevated:Western Blot and flow cytometry assays revealed markedly increased y-globin gene expression at the protein level in the CD34+-derived erythroid progenitor cells with S878F mutation compared to the wild-type controls.(4)Giemsa staining and flow cytometry confirmed that there were no significant changes in the erythroid differentiation kinetics in CD34+ cells from HbE/?0-thalassemia patients with S878F versus WT DNMT1.DISCUSSION1.This study,We investigated the variants in a set of genes associated with the epigenetic regulation of normal hemoglobin gene expression,and analyzed the association of variants in the epigenetic regulators with ?-thalassemia phenotypes in a large-scale population.We identified a novel DNMT1 mutation as a genetic modifier of ?-thalassemia and described the involvement of this DNMT1 germline mutation in a novel epigenetic mechanism de-repressing y-globin expression,which in turn ameliorates the clinical severity of ?-thalassemia.This study is the first to link a natural DNMT1 mutation with epigenetic dysregulation of Hb switching.2.We reported the functional characterization of this DNMT1 mutation by a set of in vitro assays.We found that the S878F mutation abrogates the phosphorylation of DNMT1 at position 878 resulting in the impairment of both stability and catalytic activity of DNMT1 and that the S878F mutation specifically prevents the interactions of DNMT with BCL11A.GATA1 and HDAC1/2.We further determined that the S878F mutation then reduces the recruitment of DNMTI into HBG promoters.thereby bringing about hypomethylation-mediated reactivation of the ?-globin gene in erythroid cells from hemoglobin E(HbE)/?-thalassemia individuals.Our findings suggested an important role for BAH 1 domain of DNMT1 in maintaining DNA methylation and furthered understanding of the roles of distinct epigenetic alterations in the pathogenesis of ?-hemoglobinopathies.In addition,recapitulating this mutation in primary erythroid cells by Cas9 or base editor technology may be potential approach to treat ?-hemoglobinopathies.