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Molecular and Cellular Biology, July 2008, p. 4386-4393, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.00071-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A T-to-G Transversion at Nucleotide –567 Upstream of HBG2 in a GATA-1 Binding Motif Is Associated with Elevated Hemoglobin F

Zhiyi Chen,^1, Hong-Yuan Luo,^1, Raveen K. Basran,¹ Tien-Huei Hsu,¹ Daniel W. H. Mang,¹ Lalana Nuntakarn,¹ Cathy G. Rosenfield,³ George P. Patrinos,⁴ Ross C. Hardison,⁵ Martin H. Steinberg,^1,2 and David H. K. Chui^1,2*

Center of Excellence in Sickle Cell Disease, Division of Hematology/Oncology, Department of Medicine,¹ Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118,² Department of Pediatrics, Tufts Medical Center, Boston, Massachusetts 02111,³ MGC-Department of Cell Biology and Genetics, Erasmus MC, Faculty of Medicine and Health Sciences, Rotterdam, The Netherlands,⁴ Center for Comparative Genomics and Bioinformatics, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802⁵

Received 14 January 2008/ Returned for modification 10 February 2008/ Accepted 5 April 2008

Increased fetal hemoglobin (Hb F; ₂₂) production in adults can ameliorate the clinical severity of sickle cell disease and β-thalassemia major. Thus, understanding the regulation of -globin gene expression and its silencing in adults has potential therapeutic implications. We studied a father and son in an Iranian-American family who had elevated Hb F levels and found a novel T-to-G transversion at nucleotide (nt) –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 the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt –675 and –526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by two- to threefold 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 adults. The T-to-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 adults.

Published ahead of print on 28 April 2008.

Z.C. and H.-Y.L. contributed equally to this work.