Identification of a NF-kappaB site in the negative regulatory element (epsilon-NRAII) of human epsilon-globin gene and its binding protein NF-kappaB p50 in the nuclei of K562 cells.
The developmental control of the human epsilon-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-kappaB consensus sequence resides in the negative regulatory element (-3028bp approximately -2902bp, termed e-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp approximately -2986bp) containing the NF-kappaB 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-kappaB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-kappaB motif) could not, suggesting that the binding protein is a member of NF-kappaB/Rel family. Western blot assay demonstrated that the molecular weight of NF-kappaB protein in the nuclei of K562 cells is 50ku. We suggested that NF-kappaB p50 may play an important role in the regulation of human epsilon-globin gene expression.
Hou
Chun Hui
CH
State Key Labortory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Huang
Jian
J
Qian
Ruo Lan
RL
D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008970:Molecular Weight; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D016328:NF-kappa B; D009687:Nuclear Proteins; D020569:Proto-Oncogene Proteins c-rel; D005914:Globins; D004247:DNA