EXTINCTION OF THE TUMOR-NECROSIS-FACTOR LOCUS, AND OF GENES ENCODING THE LIPOPOLYSACCHARIDE SIGNALING PATHWAY

The tumor necrosis factor (TNF-alpha or TNF) gene is activated by both lipopolysaccharide (LPS) and cycloheximide in RAW 264.7 macrophages, whereas neither stimulus activates the gene in 3T3 fibroblasts. Moreov er, the pattern of CG methylation within the TNF gene is readily disti nguishable in DNA derived from cells of these two types. These finding s would suggest that the TNF gene has been rendered inaccessible to tr anscription in the 3T3 cell environment. When RAW 264.7 cells are fuse d with 3T3 cells, an immortal pentaploid hybrid results. In the hybrid cell, all three TNF genes contributed by the RAW 264.7 cell parent be come highly methylated according to the pattern observed in the 3T3 ce ll parent. Permanently transfected chloramphenicol acetyl transferase (CAT) reporter constructs, bearing 2.2 kb of upstream sequence (includ ing the entire TNF promoter and 5'-untranslated region [UTR]) as well as 1.0 kb of downstream sequence (including the entire TNF 3'-UTR and termination sequence), are accessible in both RAW 264.7 cells and 3T3 cells, but are silenced in transition from the RAW 264.7 cell to the h ybrid cell environment. Moreover, the endotoxin signaling pathway is a brogated, as assessed by transient transfection of hybrid cells with L PS-responsive CAT reporter constructs. It would therefore appear that the fusion of 3T3 cells and RAW 264.7 cells activates a system that si lences the TNF gene, as well as the LPS signaling pathway. This system may operate to determine TNF gene accessibility and LPS responsivenes s in the course of cell differentiation. The DNA sequences targeted wi thin the TNF gene are included in the CAT reporter construct; therefor e, the silencing element has been circumscribed to a region of DNA 3.2 kb in length.