Nuclear Proteins ThatBindtheHuman-y-Globin GenePromoter: Alterations inBinding Produced byPoint Mutations Associated withHereditary Persistence ofFetal Hemoglobin

Themolecular mechanisms responsible forthehumanfetal-to-adult hemoglobin switch havenotyetbeen elucidated. Pointmutations identified inthepromoter regions ofy-globin genesfromindividuals with nondeletion hereditary persistence offetal hemoglobin (HPFH) may markcis-acting sequencesimportant for this switch, andthetrans-acting factors whichinteract withthese sequencesmay beintegral parts inthepuzzle ofy-globin generegulation. Wehaveusedgelretardation andfootprinting strategies todefine nuclear proteins whichbindtothenormal -y-globin promoter andtodetermine theeffect ofHPFHmutations on thebinding of a subset ofthese proteins. We haveidentified five proteins inhumanerythroleukemia cells (K562andHEL) whichbindtotheproximal promoter region ofthenormal -y-globin gene.Onefactor, yCAAT,binds the duplicated CCAAT boxsequences;the-117HPFH mutation increases theaffinity ofinteraction between yCAATandits cognate site. Twoproteins, -yCAC1 and-yCAC2, bindtheCACCC sequence.Theseproteins require divalent cations forbinding. The-175HPFHmutation interferes withthebinding ofafourth protein, yOBP,whichbinds an octamersequence(ATGCAAAT)inthenormal'y-globin promoter. TheHPFH phenotype ofthe-175mutation indicates that theoctamer-binding protein may play anegative regulatory role inthissetting. A fifth protein, EF'ya, binds tosequenceswhichoverlap theoctamer-binding site. The erythroid-specific distribution ofEFyaanditsclose approximation toan apparent repressor-binding site