Activation of globin gene expression by cDNAs from induced K562 cells. Evidence for involvement of ferritin in globin gene expression.
We have isolated non-globin cDNA clones specific for erythroid differentiation from K562 human erythroleukemia cells and have identified those that may regulate globin gene transcription. A cDNA library was constructed from K562 cells induced by hemin for production of embryonic and fetal hemoglobins and screened against cDNA from uninduced K562 cells. Full-length clones specific for induced K562 cells were ligated into a eukaryotic expression vector and transfected into HeLa cells to allow for production of the corresponding coded polypeptide. The ability to increase epsilon- or gamma-globin promoter activity was identified using cotransfection with a second vector containing a globin gene promoter fused to a reporter gene. Six of the induced K562-specific clones exhibited the ability to increase the levels of the reporter genes, bacterial chloramphenicol acetyltransferase and human growth hormone. Sequencing analyses of these clones indicated that five were homologous to ferritin heavy and light chains and one had no homology with known DNA or protein sequences. The ferritin light chain cDNA had the greatest effect on globin gene promoter activation, increasing the gamma-globin promoter activity by 6-8-fold. The activation of the globin gene promoter in the absence of globin gene translation suggests that ferritin (or iron) may have a direct role in globin gene transcription. The subtractive library cloning strategy has enabled us to isolate cDNA clones that activate specific gene promoter without the requirement of direct DNA binding. This approach may allow further identification of the genes encoding proteins that are involved in the control of erythropoiesis.
Wu
Y J
YJ
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Noguchi
C T
CT
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D005293:Ferritins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D005914:Globins; D004247:DNA; D005293:Ferritins