Importance of the Two Interferon-stimulated Response Element (ISRE) Sequences in the Regulation of the Human Indoleamine 2,3-Dioxygenase Gene

Indoleamine 2,3-dioxygenase (INDO) is the rate-limiting enzyme in the catabolism of the essential amino acid L -tryptophan. It is induced strongly in many cell lines following interferon-γ treatment. We report the cloning and characterization of the full-length human INDO promoter. This promoter is 1,245 base pairs long and includes two interferon-stimulated response elements (ISRE) separated by an approximately 1-kilobase sequence. The presence of these two ISREs is critical for maximum INDO promoter activity (50-fold induction). When the ISREs are present in two separate fragments cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter vector, the INDO promoter activity drops significantly (7-fold induction). 5′ end deletions of the wild type promoter sequence indicate that removal of the ISRE (ISRE1) at position −1126 reduces the induction level to approximately 25-fold. This activity does not change appreciably when the promoter is deleted down to position −241. Furthermore, site-directed mutagenesis of ISRE1 also decreases the promoter activity in a similar way. When ISRE1 is kept intact, deletion of the second ISRE (ISRE2) at position −111 leads to only 11-fold induction of the promoter. A similar result is obtained when substitution mutations are introduced in ISRE2. Deletion of a 748-base pair sequence between the two ISREs only shows a slight decrease in the INDO promoter activity. These data indicate that the two ISRE sequences are required for the full transcriptional induction of the interferon-γ-inducible human INDO gene. INDO activity is not induced in the hepatic cell line HepG2. An analysis of INDO-CAT activity in this cell line indicated that the lack of INDO activity was at the transcriptional level and could reflect either the presence of a repressor or lack of a transcription factor. This lack of induction could be correlated with a truncated or unstable IRF-1. However, the levels of IRF-2, JAK2, and STAT 91 were similar in both ME180 and HepG2 cells.