A T>G TRANSVERSION AT NT -567 UPSTREAM OF HBG2 IN A GATA-1 BINDING MOTIF IS ASSOCIATED WITH ELEVATED Hb F

Center of Excellence in Sickle Cell Disease, Division of Hematology/Oncology, Department of Medicine, Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA 02118; Department of Pediatrics, Tufts Medical Center, Boston, MA 02111; MGC-Department of Cell Biology and Genetics, Erasmus MC, Faculty of Medicine and Health Sciences, Rotterdam, The Netherlands; Center for Comparative Genomics and Bioinformatics, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802

^* To whom correspondence should be addressed. Email: david.chui{at}bmc.org.

	Abstract

Increased fetal hemoglobin (Hb F; ₂₂) production in adult can ameliorate clinical severity of sickle cell disease and -thalassemia major. Thus, understanding the regulation of -globin gene expression and its silencing in adult has potential therapeutic implications. We studied the father and one of his sons in an Iranian American family who had elevated Hb F levels and found a novel T>G transversion at NT (nucleotide) -567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between NT -675 and -526. In addition, the T>G mutation at the GATA motif increases the promoter activity by 2 to 3 fold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of -globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing -globin gene expression in adult. The T>G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of -globin gene expression in adult.