title: Functional Analysis of an (A)γ-Globin Gene Promoter Variant (HBG1: g.-225_-222delAGCA) Underlines Its Role in Increasing Fetal Hemoglobin Levels Under Erythropoietic Stress.
abstract: Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for β-thalassemia (β-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors' binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for β-thal patients.
authors.affiliation: a Laboratory for Molecular Biomedicine , Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Belgrade , Serbia .
authors.forename: Milena
authors.initials: M
authors.lastname: Ugrin
authors.affiliation: a Laboratory for Molecular Biomedicine , Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Belgrade , Serbia .
authors.forename: Maja
authors.initials: M
authors.lastname: Stojiljkovic
authors.affiliation: a Laboratory for Molecular Biomedicine , Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Belgrade , Serbia .
authors.forename: Branka
authors.initials: B
authors.lastname: Zukic
authors.affiliation: a Laboratory for Molecular Biomedicine , Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Belgrade , Serbia .
authors.forename: Kristel
authors.initials: K
authors.lastname: Klaassen
authors.affiliation: b Department of Pharmacy , University of Patras School of Health Sciences , Patras , Greece .
authors.forename: Theodora
authors.initials: T
authors.lastname: Katsila
authors.affiliation: a Laboratory for Molecular Biomedicine , Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Belgrade , Serbia .
authors.forename: Jovana
authors.initials: J
authors.lastname: Vasiljevic
authors.affiliation: c Department of Hematology and Oncology , University Children's Hospital, School of Medicine, University of Belgrade , Belgrade , Serbia.
authors.forename: Lidija
authors.initials: L
authors.lastname: Dokmanovic
authors.affiliation: c Department of Hematology and Oncology , University Children's Hospital, School of Medicine, University of Belgrade , Belgrade , Serbia.
authors.forename: Dragana
authors.initials: D
authors.lastname: Janic
authors.affiliation: b Department of Pharmacy , University of Patras School of Health Sciences , Patras , Greece .
authors.forename: George P
authors.initials: GP
authors.lastname: Patrinos
authors.affiliation: a Laboratory for Molecular Biomedicine , Institute of Molecular Genetics and Genetic Engineering, University of Belgrade , Belgrade , Serbia .
authors.forename: Sonja
authors.initials: S
authors.lastname: Pavlovic
mesh_terms: D000328:Adult; D002460:Cell Line; D015536:Down-Regulation; D004920:Erythropoiesis; D005319:Fetal Hemoglobin / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D017086:beta-Thalassemia / Q000235:genetics; D055545:gamma-Globins / Q000235:genetics*
chemical_list: D055545:gamma-Globins; D005319:Fetal Hemoglobin
keywords: Aγ-globin gene; deletion; erythropoietic stress; hereditary persistence of fetal hemoglobin (HPFH); promoter