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

2000; Taylor & Francis; Volume: 20; Issue: 20 Linguagem: Inglês

10.1128/mcb.20.20.7572-7582.2000

1098-5549

David O’Neill, Stuti Schoetz, Rocío López, M Castle, Lisa Rabinowitz, Erika Shor, Dayana Krawchuk, Mary Goll, Manfred Renz, Hans-Peter Seelig, Sunmi Han, Rho Hyun Seong, Sang D. Park, Theodora Agalioti, Nikhil Munshi, Dimitrios Thanos, Hediye Erdjument‐Bromage, Paul Tempst, Arthur Bank,

Chromatin Remodeling and Cancer

AbstractWe 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 sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human β-globin locus construct results in delayed human γ- to β-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors—activators (SWI/SNF) and repressors (NuRD)—in a single complex (PYR complex) to the β-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress γ-globin gene expression and facilitate γ- to β-globin switching. ACKNOWLEDGMENTSThis work was supported by PHS grants DK25274 and HL28381 from the National Institutes of Health, a grant from the Ahepa Cooley's Anemia Foundation, and NCI grant P30 CA08748. D.W.O. was supported by NIH Clinical Investigator Award DK02260.We thank the National Cell Culture Center for large-scale MEL cell cultures; Eugene Leung for help with the large-scale preparation of MEL nuclear extracts; Lynne Lacomis, Mary Lui, Anita Grewal, and Scott Geromanos for help with MS analysis; Matthias Mann for the PeptideSearch and SequenceTag programs; Christina Starke and Jason Garyu for technical assistance; Una Terrie Collins for assistance in the preparation of the manuscript; and Stephen Smale, Bradley Cobb, Gerald Crabtree, Katia Georgopoulos, Ganjam Kalpana, and Michael Bustin for generously supplying antibodies.