title: T to C substitution at -175 or -173 of the gamma-globin promoter affects GATA-1 and Oct-1 binding in vitro differently but can independently reproduce the hereditary persistence of fetal hemoglobin phenotype in transgenic mice.
abstract: The T to C substitution at position -175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma-to beta-globin gene with the natural chromosome arrangement but with the -175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the mu'LCRAgamma(-173)psibetadeltabeta construct, in which the -175 T to C Agamma gene was substituted with the -173 T to C Agamma gene. In vitro experiments proved that the -175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the -173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the -175 mutation or the -173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences gamma-globin gene expression in normal adult erythrocytes. Both the -173 and -175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the gamma-globin gene in adult erythrocytes.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
authors.forename: Li-Ren
authors.initials: LR
authors.lastname: Liu
authors.forename: Zhan-Wen
authors.initials: ZW
authors.lastname: Du
authors.forename: Hua-Lu
authors.initials: HL
authors.lastname: Zhao
authors.forename: Xiao-Ling
authors.initials: XL
authors.lastname: Liu
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Huang
authors.forename: Jie
authors.initials: J
authors.lastname: Shen
authors.forename: Li-Mei
authors.initials: LM
authors.lastname: Ju
authors.forename: Fu-De
authors.initials: FD
authors.lastname: Fang
authors.forename: Jun-Wu
authors.initials: JW
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D003360:Cosmids / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D017353:Gene Deletion; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D050811:Octamer Transcription Factor-1; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; C494166:Pou2f1 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D012313:RNA; D005914:Globins; D004247:DNA