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Silencing of ^A-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the –566 GATA Site ^,

Susanna Harju-Baker,^1,, Flávia C. Costa,^1, Halyna Fedosyuk,¹ Renee Neades,¹ and Kenneth R. Peterson^1,2*

Departments of Biochemistry and Molecular Biology,¹ Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160²

Received 11 October 2007/ Returned for modification 20 November 2007/ Accepted 8 March 2008

Autonomous silencing of -globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the ^A-globin gene was identified between –730 and –378 relative to the mRNA start site. A marked copy of the ^A-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the -globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the –566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low -globin levels, whereas only a weak signal was detected when -globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the ^A-globin –566 or ^G-globin –567 GATA site when -globin expression was low (day 18) but not when -globin was expressed (day 12). These data suggest that during definitive erythropoiesis, -globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the –566/–567 GATA sites of the proximal -globin promoters.

Published ahead of print on 17 March 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Center for Lung Biology, University of Washington School of Medicine, Seattle, WA 98109.

Co-first authors: these authors contributed equally to this work.