Characterization of a novel negative regulatory element in the human interleukin 4 promoter.
Interleukin 4 (IL-4) is a multifunctional cytokine that plays an important role in hematopoiesis, tumor cell growth, and cellular immune responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription, and many positive regulatory cis-elements have been identified in the proximal IL-4 promoter region. Relatively little is known about factors that downregulate IL-4 transcription. We performed a detailed deletional analysis of the proximal human IL-4 promoter and studied reporter gene activity in transiently transfected Jurkat T lymphoblasts. In this report, we characterize a novel negative regulatory element (termed P2 NRE) that is adjacent to a binding site for nuclear factor of activated T cells. Mutation of P2 NRE significantly enhanced the activity of a 175 base pair IL-4 promoter construct in transiently transfected Jurkat T lymphoblasts. Using nuclear extracts from Jurkat cells, we identify a candidate factor (termed Rep-1) that binds uniquely to the P2 NRE in DNA-binding assays. Rep-1 is not related to other factors previously shown to interact with the IL-4 promoter, and by UV cross-linking and SDS-PAGE analysis, we found that it migrates with a molecular mass of approximately 150 kDa. Characterizing the molecular mechanisms responsible for downregulating the IL-4 promoter should enhance our understanding of IL-4-gene dysregulation in disease states.
Georas
S
S
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University Asthma and Allergy Center, Baltimore, MD 21224, USA.
Cumberland
J
J
Burke
T
T
Park
E
E
Ono
S
S
Casolaro
V
V
D001665:Binding Sites; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D015847:Interleukin-4 / Q000096:biosynthesis / Q000235:genetics*; D019169:Jurkat Cells; D046911:Macromolecular Substances; D008970:Molecular Weight; D050778:NFATC Transcription Factors; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D015847:Interleukin-4