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Molecular and Cellular Biology, June 1999, p. 4182-4190, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification of Id2 as a Globin Regulatory Protein by Representational Difference Analysis of K562 Cells Induced To Express gamma -Globin with a Fungal Compound

Melissa L. Holmes,1,dagger John D. Haley,2 Loretta Cerruti,1 Wen-lai Zhou,1 Helen Zogos,1 David E. Smith,2 John M. Cunningham,3 and Stephen M. Jane1,*

Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Australia1; Oncogene Science, Inc., Uniondale, New York2; and St. Jude Children's Research Hospital, Memphis, Tennessee3

Received 10 February 1999/Returned for modification 8 March 1999/Accepted 8 March 1999

A fungus-derived compound (OSI-2040) which induces fetal globin expression in the absence of erythroid cell differentiation was identified in a high-throughput drug discovery program. We utilized this compound to isolate gamma -globin regulatory genes that are differentially expressed in OSI-2040-induced and uninduced cells in the human erythroleukemia cell line K562. Representational difference analysis (RDA) of cDNA revealed several genes that were significantly up- or down-regulated in OSI-2040-induced cells. One gene whose expression was markedly enhanced was the gene for the helix-loop-helix (HLH) transcription factor Id2. Southern analysis of RDA amplicons demonstrated progressive enrichment of Id2 with each successive subtraction of uninduced cDNA from induced cDNA. Northern analysis of OSI-2040-induced K562 cells confirmed that Id2 expression was directly up-regulated coordinately with gamma -globin. Analysis of other inducers of fetal globin demonstrated up-regulation of Id2 with sodium butyrate but not with hemin. Retrovirus-mediated overexpression of Id2 in K562 cells reproduced the enhancement of endogenous globin expression observed with OSI-2040 induction. Functional assays demonstrated that an E-box element in hypersensitivity site 2 is required for Id2-dependent enhancement of gamma -promoter activity. Protein binding studies suggest that alterations in E-box site occupancy by basic HLH proteins may influence this activity, thus expanding the potential role of these factors in globin gene regulation.

* Corresponding author. Mailing address: Bone Marrow Research Laboratory, c/o Royal Melbourne Hospital Post Office, Parkville, VIC, Australia 3050. Phone: 61-3-934 28641. Fax: 61-3-934 28634. E-mail: jane{at}wehi.edu.au.

dagger Present address: Department of Hematology Research, Prince of Wales Hospital, Randwick, NSW, Australia 2031.

Molecular and Cellular Biology, June 1999, p. 4182-4190, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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