Identification of a NF-kB site in the negative regulatory element(e-NRAII) of human e-globin gene and its binding protein NF-kB p50 in the nuclei of K562 cells

Group of Globin Gene Expression and Regulation, State Key Labortory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-kB consensus sequence resides in the negative regulatory element (-3028bp ~ -2902bp, termed e-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp ~ -2986bp) containing the NF-kB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-kB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-kB motif) could not, suggesting that the binding protein is a member of NF-kB/Rel family. Western blot assay demonstrated that the molecular weight of NF-kB protein in the nuclei of K562 cells is 50ku. We suggested that NF-kB p50 may play an important role in the regulation of human e-globin gene expression.