An RSNP Reactives Gamma-globin Expression By LYAR-mediated Demethylation

Background and aims?-Thalassemia is caused by a series of different genetic variation.According to their clinical phenotypes,it can be divided into thalassemia minor,thalassemia intermedia and thalassemia major.Thalassemia major is ? ?-thalassemia with very serious clinical phenotype,mainly for a serious imbalance in the ratio of the a chain and non-a chain,severe ineffective hematopoiesis and survival dependent on RBC transfusion.The average monthly blood transfusions within these patients who had iron overload,these iron deposits in their gut and need iron chelator therapy to relieve this threat1.?-thalassemia abnormal hematopoiesis mainly in expansion of early erythroid progenitor cells?proerythroblasts and primary earlier stage?and ineffective hematopoiesis2.Number of erythroid precursor cells in bone marrow of patients with?-thalassemia is five to six times than normal person,with increased basophilic erythroblasts and polychromatophilic erythroblasts and decreased orthochromatic erythroblasts 3,4.Moreover,?-thalassemia red blood cells in bone marrow to contain electron-dense alpha-globin inclusion beginning at early polychromatophilic stages,and increase in volume and frequency of occurrence polymers come along with the red blood cells mature 5.These polymers can be enriched in the erythrocyte membrane and erythrocyte membrane damaged.The expression of fetal globin gene to some extent alleviate the a-chain and non-a chain ratio imbalance to relieve severe?-Mediterranean anemia in patients.HbF is also called fetal hemoglobin,which is a combination of two ?peptide chains and two a peptide chains together.Gamma-globin chains were encoding by the gene of HBG1 and HBG2 at the 16th chromosome.HbF mainly express in the fetal period,which is the mainly components of hemoglobin that transports oxygen.HbF express gradually decreased before and after birth,and then the adult hemoglobin?HBA?gradually begin to express.The adult hemoglobin express reach the peak after 1-2 years birth and sustain throughout their life.The gene mutation within the regulatory region of ?-globin gene cluster inhibits ? to ? switching,resulting in HbF increased throughout their life,these people were called with hereditary persistence of fetal hemoglobin?HPFH?.Interestingly,?-thalassemia mutations accompany with HPFH showed less disease severity in a few cases and even asymptomatic 6,7.These clinical observations suggest reverse the y to 3-globin switch may be an effective way for ?-thalassemia treatment.In addition,the individual has only HPFH mutations are asymptomatic that demonstrate high expression of HbF won't result in adverse consequences.In thalassemia patients with no history of blood transfusion,HbF levels were negatively correlated with the clinical severity of the disease 7.Thus,induction of HbF expression is important to alleviate severe clinical symptoms in patients with ?-Mediterranean.The presence of HbF expression levels show individual differences,it is subject to many gene regulatory element.According to previous research,three important quantitative trait loci associated with HbF has been determined.The single nucleotide polymorphism C/T variation on the 158bp upstream of HBG2 of chromosome 11p15 8,polymorphism between HBS1L-MYB gene on chromosome 6q23 9 as well as BCLlla gene on 2p16 chromosome 10,which can regulate the HbF levels and alleviate the severity of ?-thalassemia 11.Further studies showed BCL11a is a major inhibitory transcription factor against y-globin gene,which can inhibit the expression of HbF.In sickle cell transgenic mouse model,inactivation BCL11a with sustained high levels of expression of a whole-cell HbF,corrected the defect sickle cell anemia and pathological symptom 12.BCL11a possibly silence ?-globin gene through a combination of other transcription factor,for example,with SOX6,or directly effect by binding to different regions of the ?-globin gene cluster 13.Indeed,some HPFH mutations in the ?-globin gene cluster enhance the y-globin gene transcription,the reason is the mutation prevents the binding of BCL11a 14.The m+62 orthologue deletion mice showed normal frequencies of B-cell progenitors in the fetal liver and mature B lymphocytes in the adult peripheral blood,but in erythroid progenitor cells a significant reduction in BCL11a level 15.Recently reported shows that erythroid transcription factor KLF1 is a ?-globin gene to ?-globin gene switch key regulatory factor 16.In human and mouse erythroid progenitor cells knockdown KLF1 can significantly reduce the BCL11a expression levels and increase the ratio of ?-globin gene expression levels to ?-globin gene expression levels,suggesting KLF1 through direct activation of ?-globin gene expression and indirect inhibition y-globin gene expression to regulate globin gene switch 17.There are some common transcription factor regulating the expression levels of HbF.Common DNA polymorphism and rare mutation reducing the expression of MYB gene show the improving of the HbF levels in erythroid progenitor cell18.The variants in the HBS1L-MYB intergenic region along with the variants at the BCL11A locus have additive effects suggest that targeting both of these pathways together could yield more powerful effects than targeting other pathway alone19.The latest report,the Leukemia and Lymphoma relevant factor LRF encoding by the ZBTB7A gene is a ZBTB transcription factor,which is binding to DNA by C-terminal C2H2 type zinc fingers,but it is also possible via its N-terminal BTB domain Recruitment transcriptional repressor protein complex 20.LRF is an inhibitory transcription factor against ?-globin gene.In the adult stage,it is bound to the y-globin gene holding chromosome with compact nucleosome structure and then silence y-globin gene expression.LRF silence the fetal globin gene independent of BCL11a,which can be suppressed fetal globin gene by NURD complex 21.These studies provide a theoretical basis for reactivating HbF subsequently.Epigenetics includes DNA methylation,histone modifications,RNA interference and other modifications.Cytosine methylation of DNA usually associated with transcriptional silencing 22.The changes of DNA methylation affect the activation or inhibition of complex binding to the gene,thereby affecting gene expression 23.A recent test proved DNA methylation changes in the globin genes affect globin gene switch conversion 24.Increased histone acetylation has long been considered to be decompressed with chromatin and activation of gene expression.Over the years,a number of histone deacetylase inhibitors have been discovered or synthesized.In some model organisms,butyric acid and its derivatives has been found to induce silencing of embryonic and fetal globin gene expression 25.Elevated levels of HbF expression is related to the improving of ?-globin gene histone acetylation 26.MicroRNA is a group of small short non-coding regulatory RNAs,which can be through mRNA degradation or translation inhibition of protein to regulate target gene expression.MiRNA also involved in y-globin gene regulation.MiR-486-3p binding to the 3'UTR BCL11a directly inhibits expression of the gene,and then reactivates the expression of ?-globin gene 27.In human trisomy 13,the sustained expression of HbF levels and embryonic hemoglobin levels,this delayed the switch of y-globin gene to?-globin gene.Further studies showed that high level expression of miR-15a,-16-1 is suppressed the gene expression of MYB,and MYB play an important role in the silencing of fetal globin gene and embryonic globin gene.Thus,epigenetics may be an important pathway to reactivate HbF levels and thus reduce the severity symptoms of ?-thalassemia patients 28.Recent studies,histone arginine methyltransferase PRMT5 add a symmetry methylation to third arginine of Histone H4?H4R3me2?,H4R3me2 mark recruit the methyltransferase DNMT3A,DNMT3A makes the CG site of the y-globin gene promoter methylate,thereby inhibiting the expression of?-globin gene 29,while PRMT5 attach itself to LYAR bounding to y-globin gene promoter and plays an important role in the inhibition of y globin gene expression 30.The present study was to investigate whether the changes of HBG1-rs368698783 SNP locus on LYAR?combination sequence can affect the binding of LYAR,thereby affecting the recruitment PRMT5 complex,and ultimately affect the DNA methylation levels of y-globin gene promoter and y-globin gene the expression.This studys provide new ideas and new ways for reactivating HbF and provide precise targets for ?-thalassemia treatment.Materials and Methods1.Definition of genotypeHBB genotype categories are defined as follows:??0?:HBB:c.126₁29delCTTT,HBB:c.52A>T,HBB:c.316-197C>T,HBB:c.216₂17insA,HBB:c.92+l G>T,HBB:c.130G>T,HBB:c.84₈5insC,HBB:c.91A>G,HBB:c.A5₄6insC,HBB:c.165₁77delTATGGGCAACCCT,HBB:c.315+1G>A,HBB:c.287₂88insA,HBB:c.113G>A,HBB:c.93-1G>C;?p+?:HBBS:c.-78A>G,HBB:c.79G>A,HBB:c.-79A>G,HBB:c.315+5G>C,HB:c.-140C>T,HBB:c.-81 A>C,and HBB:c.92+5G>C.2.CD235-positive cell sorting of the bone marrow of ?-thalassemia patientsWe extract the bone marrow of nine cases patients with ?0/?0 thalassemia.We used anticoagulant heparin and fivefold dilute with 1XPBS.Mononuclear cells were separated using Ficoll and sorting nucleated red blood cells with CD235 positive beads by manual splitter.3.Detection the levels of DNA methylationWe extracted DNA with phenol chloroform method and using bisulfite treated DNA,and purified DNA by column.We use nested PCR amplify target fragment,then recycling PCR product by gel extraction.The product is connected on the carrier and followed by picking clone,the expansion of bacteria and sequencing.The sequencing results show that methylated cytosine is C and unmethylated cytosine is T.4.The dual luciferase reporter assayTarget fragment PCR primers were designed with restriction sites,it was amplified by high-fidelity enzymes and purified PCR product.The target fragment and PGL3 carrier with HS2 enhancer and SV40 promoter were double digested followed by Gel Extraction.The product is connected on the carrier and transferred to plasmids,and plasmids were extracted.K562 cells were transfected by the 4D nuclear transfection system and using the the dual luciferase reporter assay system to detect the fluorescence.5.The electrophoretic mobility shift assay and antibody supershift assayAccording to the literature synthesis the probe which was binding by LYAR?wild-type thermal probes,wild-type and mutant cold probe?,and the probe annealed into short double-stranded DNA sequence.we extract the fresh nucleoprotein with nucleoprotein Extraction Kit.In the cold competition experiments,wild-type or mutant cold probe 10 times,30 times and 90 times o'ver wild-type heater probe by using specialized EMSA kit.To verify whether the binding protein complex with or without LYAR protein,we add a LYAR antibody or control double distilled water and play the supershift assay.6.Chromatin immunoprecipitation experiments?CHIP?After sorting CD235-positive cells of the bone marrow of beta-thalassemia patients,,about 107 erythroblast were cross-linked with 1%formaldehyde at room temperature for 10min and the reaction was quenched by adding glycine.Nuclei were sonicated,followed by antibody enrichment,decrosslinking and DNA purification.We use a probe to quantitative the proportion of HBG2-G and HBG1-A at position +25 of HBG gene.7.Statistical treatmentAll statistical tests were performed using the t-test,P<0.05 was considered significant difference between the two sets of data.Organize and process data using IBM SPSS Statistacs 20 software.we first plotted with GraphPad Prism 5 software,then modified with Adobe Illustrator CS4 software.Main conclusion1.The G/A mutation on HBG1-rs368698783 of ?0/?0 thalassemia patients reduce the methylation levels of CGs near HBG-promoter.2.While the G/A mutation on HBG1-rs3686987839 the LYAR protein became less enriched in the y-globin gene,further effecting the LYAR,PRMT5,H4R3me2 and DNMT3A pathway,so that reduce the methylation levels of y-globin gene promoter,thereby opening up the gene expression.