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-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.
D015152:Blotting, Northern; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005914:Globins / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D020014:K562 Cells; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D012190:Retroviridae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors; D014161:Transduction, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation