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J. Biol. Chem., Vol. 279, Issue 53, 55024-55033, December 31, 2004

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Sequences Downstream of the Erythroid Promoter Are Required for High Level Expression of the Human -Spectrin Gene^*

Ellice Y. Wong, Jolinta Lin, Bernard G. Forget¶, David M. Bodine, and Patrick G. Gallagher||

From the Department of Pediatrics and ^¶Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8021 and Hematopoiesis Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892

-Spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the -spectrin gene. Previously, we demonstrated that the core promoter of the human -spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3' of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the -spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3' of the -spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased -spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.

Received for publication, August 4, 2004 , and in revised form, September 21, 2004.

^* This work was supported in part by a grant from the NHLBI, National Institutes of Health Grant HL65448. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY138967.

^|| To whom correspondence should be addressed: Dept. of Pediatrics, Yale University School of Medicine, 333 Cedar Street, P. O. Box 208064, New Haven, CT 06520-8064. Tel.: 203-688-2896; Fax: 203-785-6974; E-mail: patrick.gallagher{at}yale.edu.

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