Combined Screening of Thymocytes Using Apoptosis-Specific cDNA Array and Promoter Analysis Yields Novel Gene Targets Mediating

We have used pathway-specific cDNA arrays coupled with analysis of gene promoter regions to identify novel genes that may mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the thymus. C57BL/6 mice were injected ip with 50 g/kg TCDD, and 3, 6, or 24 h later, RNA was extracted from the thymus and subjected to microarray analysis. Several members of the TNF and TNFR family were induced following TCDD expo-sure, including receptor/ligand pairs Lt-R/LIGHT, OX40/OX40L and TNF-/TNFR1. In addition, Fas and CD30 were also upregu-lated. Pro-apoptotic bcl-2 gene family members Bax and Hrk, among others, were also induced, as were pro-survival bcl-2 family genes Bcl-x and Bcl-w. Cell-cycle regulator p21Cip1 was also induced. In addition, we analyzed the promoter regions of genes induced by TCDD for the presence of dioxin-responsive elements (DREs). The Fas and LIGHT gene promoters were found to contain DREs as analyzed by Matinspector Web-based search algorithm. Furthermore, binding of the aryl hydrocarbon receptor (AhR) to the DREs present on these genes was confirmed by chromatin immunoprecipitation. Given that several of the genes, including Fas, LIGHT, and CD30 are involved in negative selec-tion of T cells in the thymus, our studies suggest that TCDD-induced upregulation of these genes may enhance negative selec-tion leading to thymic atrophy. Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; Fas; LIGHT; apoptosis; negative selection. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is the prototype for a class of halogenated aromatic hydrocarbons that are common environmental contaminants. The toxic ef-fects of TCDD have been well characterized and include hep-atotoxicity, teratogenicity, fetotoxicity, chloracne, and immun