Open AccessIdentification of Id2 as a Globin Regulatory Protein by Representational Difference Analysis of K562 Cells Induced To Express γ-Globin with a Fungal Compound
Author(s) -
Melissa L. Holmes,
John D. Haley,
Loretta Cerruti,
Wen Lai Zhou,
Helen Zogos,
David Eugene Smith,
John M. Cunningham,
Stephen M. Jane
Publication year - 1999
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.19.6.4182
Subject(s) - biology , globin , k562 cells , microbiology and biotechnology , hypersensitive site , gene , gene expression , regulation of gene expression , complementary dna , genetics , promoter
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.