Study Of A Distant Regulatory Element Required For Expression Of The Embryonic ?-globin Gene

Hemoglobins are a superfamily of heme-containing globular proteins,involved in binding and transporting oxygen.Most normal human hemoglobins,which are expressed at different developmental stages,have indentical a-globin chains and ?-globin chains differ from each other.At the embryonic stage,hemoglobins were consist of ?2?2,?2?2,?2?2,fetal stage was ?2?2 and adult stage were ?2?2 and ?2?2.?-globin chains are a globin like chains that are present primarily in yolk sac-derived during the first 5 to 6 weeks of gestation.In almost all infants older than 3 months of ages and in adults,?-globin chains are not detected and a globin as a replacement.To date,four erythroid-specific DNaseI hypersensitive sites,located 10(HS-10),33(HS-33),40(HS-40),and 48(HS-48)kb upstream of the ?-globin mRNA cap site,have been identified.The regulatory sequence located 40 kb upstream of the ?-cluster(HS-40)has a more significant effect on ?-gene expression.While the mechanism(s)responsible for the co-ordinated expression of these genes is hypothesised to occur at the transcriptional level,little is known about the specific sequences within these genes that may play a role in the regulation of the switching process.Interestingly,?-globin chains are detected in--SEA,--MED and--SPAN deletions,not in other longer deletions.In this study,we foucus on study ?-globin gene expression regulation in--SEA population.We detected ?-globin expression in 6229 individuals carrying the SEA deletion allele,showing highly variable expression levels of ?-globin chains.According to the different types of ?-thalassemia deletions,we found a 5kb fragment,which may contain sequences necessary for regulating ?-globin gene.We sequenced the entire 5K regulatory region from 900 individuals and identified 3 highly linkaged SNPs are significantly associated with?-globin expression.To investigate underlying regulatory mechanism that links to?-globin gene expression,we used the genomic editor method Crispr cas9 to knockout 5K,results shown that partially deletion of this region led to decrease in expression levels of?-globin genes,which indicated 5K was a importamt element for?-globin gene expression.Next,the 3C-qPCR results shown that there were interactions among 5K,HBZ,HBA and HS48,HS40.In addition,we found H3K4mel and H3K27ac were enriched at the 3' of 5K,and strongly decreased enrichment after 3' of 5K KO,which illustrated 3' of 5K might a enchance region.Furthermore,ChIP-qPCR result indicated MAX-MYC heterodimers bound to 3' of 5K to activate expression;the gel shift assay result shown the 2 SNP down-regulated?-globin chaine expression by reduced MAX's binding strength.In this study,we found a downstream regulator elment 5K as enhancer to active?-globin gene expression via long-range interaction with ?-globin gene promoter mediated by MAX-MYC heterodimers.This study contributes to understanding of regulate?-globin gene expression and may provide insight into thalassemia clinical therapy in the future.