An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

doi:10.1128/MCB.20.20.7572-7582.2000

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Molecular and Cellular Biology, October 2000, p. 7572-7582, Vol. 20, No. 20
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

David W. O'Neill,¹ Stuti S. Schoetz,² Rocio A. Lopez,² Madalyn Castle,² Lisa Rabinowitz,² Erika Shor,² Dayana Krawchuk,² Mary G. Goll,² Manfred Renz,³ Hans-Peter Seelig,³ Sunmi Han,⁴ Rho H. Seong,⁴ Sang D. Park,⁴ Theodora Agalioti,⁵ Nikhil Munshi,⁵ Dimitrios Thanos,⁵ Hediye Erdjument-Bromage,⁶ Paul Tempst,⁶ and Arthur Bank²^,⁷^,^*

Departments of Pathology,¹ Genetics and Development,² Biochemistry and Molecular Biophysics,⁵ and Medicine,⁷ Columbia University College of Physicians and Surgeons, New York, New York 10032; Institute of Immunology and Molecular Genetics, Karlsruhe D-76133, Germany³; Institute of Molecular Biology and Genetics and Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea⁴; and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021⁶

Received 17 February 2000/Returned for modification 12 April 2000/Accepted 12 July 2000

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type)hematopoietic cells and that specifically binds DNA sequencescontaining long stretches of pyrimidines. Deletion of an intergenicDNA-binding site for this complex from a human $beta$ -globin locusconstruct results in delayed human $gamma$ - to $beta$ -globin switching intransgenic mice, suggesting that the PYR complex acts to facilitatethe switch. We now show that PYR complex DNA-binding activityalso copurifies with subunits of a second type of chromatin-remodelingcomplex, nucleosome-remodeling deacetylase (NuRD), that has beenshown to have both nucleosome-remodeling and histone deacetylaseactivities. Gel supershift assays using antibodies to the ATPase-helicasesubunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 isa component of the PYR complex. In addition, we show that thehematopoietic cell-restricted zinc finger protein Ikaros copurifieswith PYR complex DNA-binding activity and that antibodies to Ikarosalso supershift the complex. We also show that NuRD and SWI/SNFcomponents coimmunopurify with each other as well as with Ikaros.Competition gel shift experiments using partially purified PYRcomplex and recombinant Ikaros protein indicate that Ikaros functionsas a DNA-binding subunit of the PYR complex. Our results suggestthat Ikaros targets two types of chromatin-remodeling factors $---$ activators(SWI/SNF) and repressors (NuRD) $---$ in a single complex (PYR complex)to the $beta$ -globin locus in adult erythroid cells. At the time ofthe switch from fetal to adult globin production, the PYR complexis assembled and may function to repress $gamma$ -globin gene expressionand facilitate $gamma$ - to $beta$ -globinswitching.

^* Corresponding author. Mailing address: Department of Genetics and Development, Hammer Health Sciences Room 1604, 701 West168th Street, New York, NY 10032. Phone: (212) 305-4186. Fax:(212) 923-2090. E-mail: bank{at}cuccfa.ccc.columbia.edu.

Molecular and Cellular Biology, October 2000, p. 7572-7582, Vol. 20, No. 20
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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