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Molecular and Cellular Biology, June 2006, p. 4368-4377, Vol. 26, No. 11
0270-7306/06/$08.00+0     doi:10.1128/MCB.02216-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice{dagger}

Andre M. Pilon,1 Douglas G. Nilson,1 Dewang Zhou,2 Jose Sangerman,3 Tim M. Townes,2 David M. Bodine,1 and Patrick G. Gallagher3*

Hematopoiesis Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-4442,1 Department of Biochemistry and Molecular Genetics, School of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294,2 Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-80213

Received 16 November 2005/ Returned for modification 6 December 2005/ Accepted 6 March 2006

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the ß-globin promoter, where it establishes local chromatin structure permitting ß-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the ß-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and ß-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

* Corresponding author. Mailing address: Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208064, New Haven, CT 06520-8064. Phone: (203) 688-2896. Fax: (203) 785-6974. E-mail: patrick.gallagher{at}yale.edu.

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

Molecular and Cellular Biology, June 2006, p. 4368-4377, Vol. 26, No. 11
0270-7306/06/$08.00+0     doi:10.1128/MCB.02216-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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