Roles Of Promoter DNA Methylation And MicroRNA In Regulating EKLF Expression And HbF Induction

Erythroid Kruppel-like factor(EKLF/KLFl)is the founding member of Kruppel-like factors(KLFs).As a master regulator of erythropoiesis,EKLF is involved in a wide variety of molecular process including ?-like globin gene expression,cell cycle,and synthesis of heme,cytoskeleton,and cell membrane.Recently,EKLF has emerged as one of the key regulators of the y-to ?-globin gene switch.Haploinsufficiency of EKLF due to genetic variants results in significant increases in fetal hemoglobin(HbF)levels.Thus,attenuating EKLF activity may be an effective approach to raise HbF levels in patients with ?-thalassemia.To understand the regulation of EKLF via promoter DNA methylation,we conducted bisulfite sequencing and quantitative RT-PCR on different human tissues and glycophorin A-positive(GPA+)and GPA-cells isolated from bone marrow.In vitro,5-aza-2'-deoxycytidine(5-Aza-CdR)treatment,luciferase assay and electrophoretic mobility shift assay(EMSA)were performed.Additionally,luciferase assay identified miR-326 targeting EKLF 3'UTR.For further functional analysis,K562 cells and CD34+cells with increased or knock-down miR-326 were prepared.Reticulocytes from peripheral blood collected from 13 normal controls and 23 ?-thalassemia major patients,were used for quantitative RT-PCR analysis.We analyzed a panel of normal human tissues and documented that the EKLF promoter is almost completely devoid of methylation in erythroid cells but is heavily methylated in other non-erythrod cells and tissues,in association with highly specific endogenous expression of EKLF in erythroid cells.Demethylation of the EKLF promoter led to elevating EKLF expression in non-erythroid cells.We further uncovered that EKLF promoter methylation reduces the binding affinity of transcription factors GATA1 and MYB,which in turn silence EKLF expression.Additionally,our work identified miR-326 as a potential microRNA targeting EKLF 3'UTR.Ectopic expression of miR-326 in K562 cells or CD34+hematopoietic progenitor cells led to decreased expression of EKLF protein,increase of y-globin mRNA and reduction of ?-globin and of BCL11A mRNA.In view of our observations in a cohort of 36 random Chinese subjects(13 normal controls and 23 ?-thalassemia major patients),we uncovered increased expression of miR-326 in reticulocytes appears to positively correlate with HbF level in?-thalassemia major patients.The present study documents two previously unknown regulatory mechanisms that control EKLF expression in erythroid cells.Hypomethylation of the EKLF promoter has a functional significance in the establishment and maintenance of tissue-specific gene expression,while miR-326 is involved in the regulation of stress-induced fetal hemoglobin synthesis in the condition of ?-thalassemia by silencing EKLF expression.This study offers a new strategy for activating HbF by attenuation of EKLF in individuals with ?-thalassemia or sickle cell disease.