Identification of Id2 as a globin regulatory protein by representational difference analysis of K562 cells induced to express gamma-globin with a fungal compound

A fungus-derived compound (OSI-2040) which induces fetal globin expression in the absence of erythroid cell differentiation was identified in a high-t hroughput drug discovery program. We utilized this compound to isolate gamm a-globin regulatory genes that are differentially expressed in OSI-2040-ind uced and uninduced cells in the human erythroleukemia cell line K562. Repre sentational difference analysis (RDA) of cDNA revealed several genes that w ere significantly up- or down-regulated in OSI-2040-induced cells. One gene whose expression was markedly enhanced was the gene for the helix-loop-hel ix (HLH) transcription factor Id2. Southern analysis of RDA amplicons demon strated progressive enrichment of Id2 with each successive subtraction of u ninduced 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-m ediated overexpression of Id2 in K562 cells reproduced the enhancement of e ndogenous globin expression observed with OSI-2040 induction. Functional as says demonstrated that an E-box element in hypersensitivity site 2 is requi red for Id2-dependent enhancement of gamma-promoter activity. Protein bindi ng studies suggest that alterations in E-box site occupancy by basic HLH pr oteins may influence this activity, thus expanding the potential role of th ese factors in globin gene regulation.