IMPORTANCE OF THE 2 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 str ongly in many cell lines following interferon-gamma treatment. We repo rt the cloning and characterization of the full-length human INDO prom oter. This promoter is 1,245 base pairs long and includes two interfer on-stimulated response elements (ISRE) separated by an approximately 1 -kilobase sequence. The presence of these two ISREs is critical for ma ximum INDO promoter activity (50-fold induction). When the ISREs are p resent in two separate fragments cloned upstream of the chloramphenico l acetyltransferase (CAT) reporter vector, the INDO promoter activity drops significantly (7-fold induction). 5' end deletions of the wild t ype promoter sequence indicate that removal of the ISRE (ISRE1) at pos ition -1126 reduces the induction level to approximately 25-fold. This activity does not change appreciably when the promoter is deleted dow n to position -241. Furthermore, site-directed mat-agenesis of ISRE1 a lso decreases the promoter activity in a similar way. When ISRE1 is ke pt intact, deletion of the second ISRE (ISRE2) at position -111 leads to only 11-fold induction of the promoter. A similar result is obtaine d when substitution mutations are introduced in ISRE2. Deletion of a 7 48-base pair sequence between the two ISREs only shows a slight decrea se in the INDO promoter activity. These data indicate that the two ISR E sequences are required for the fall transcriptional induction of the interferon-gamma-inducible human INDO gene, INDO activity is not. ind uced 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 repre ssor or lack of a transcription factor, This lack of induction could b e correlated with a truncated or unstable IRF-1, However, the levels o f IRF-2, JAK2, and STAT 91 were similar in both ME180 and HepG2 cells.