Stimulation of Interferon $ Gene Transcri tion in Vitro by Purified NF-icB and a Novel TH Protein

The human interferon fi (IFN-$) regulatory element consists of multiple enhanson domains which are targets for transcription factors involved in inducible expression of the promoter. To further characterize the protein-DNA interactions mediating IFN-$ induction, positive regulatory domain (PRD) II binding proteins were purified from phorbol ester induced Jurkat 1-cells and from IFN primed, cycloheximide/polyinosinicpolycytidylic acid treated HeLa S3 cells. From Hela cells, two major proteins of 52 and 45 kilodaltons (kD) copurified with DNA binding activity, whereas from 1cells, four proteins-a major protein of 52 kD and three minor proteins of 82, 67, and 43-47 kD-were purified. Also, an induction specific DNA binding protein was purified from HeLa cells that interacted with the (AAGTGA)4 tetrahexamer sequence and the PRDI domain. This protein is immunologically distinct from IRF-i/ISGF2. Uninduced or Sendai virus induced HeLa extracts were used to examine transcription in vitro using a series of lEN fi promoter deletions. Deletions upstream of the PRDII element increased transcription in the uninduced extract, indicating predominantly negative regulation of the promoter. A 2-4-fold increase in lFN9 promoter transcription was observed in Sendai virus induced extracts, and deletion of PRDI and PRDII elements decreased this induced level of transcription. When purified PRDII and tetrahexamer binding proteins were added to the induced extract, a 4-fold increase in transcription was observed. These experiments demonstrate that it is possible to modulate IFN-j8 transcription in vitro but indicate that additional proteins may be required to fully activate IFN-$ transcription. Introduction Induction of type i IFN3 genes (IFN-a and IFN-/3) occurs as an early response to virus infection in hemopoietic, epithelial, and fibroblastic cells. Activation and repression of interferon fi expression is controlled primarily at the transcriptional level and is mediated by multiple factors interacting with overlapping regulatory promoter domains, located immediately adjacent to the structural gene (for reviews, see Refs. 1 and 2). Three positive regulatory domains and two negative regulatory domains have been defined by mutational analysis of the promoter. The PRDI domain (-77 to -64) functions as a constitutive or virus-inducible enhancer when multimerized, depending on the cell type (3). Tandem repeats of the hexamer sequence AAGTGA, a conserved sequence motif present twice in PRDI, is also a virus inducible element (4). The positive regulatory domain PRDII (-66 to -55) is also essential for virus inducibility; base mutations or deletions which alter the PRDII domain of the natural promoter cause a loss in virus inducibility (5, 6). Multiple copies of this element likewise function as a virus-inducible enhancer (3). Recently, a third positive regulatory domain, PRDIII (-94 to -78), was shown to synergize with PRDI and PRDII domains to increase virus inducibility (7). Two negative regulatory domains, NRDI and NRDII, were identified by mutational analysis and in vivo footprinting (6, 8), but the exact boundaries of these regions have not been determined (see Fig. 1 for the relative location of these regions). The complexity of the induction process suggested by the organization of the IFN-/ promoter is further emphasized by the fact that multiple factors interact with the PRDI and PRDII domains. Three proteins have been characterized that interact with the PRDI domain. IRF-i is involved in positive virus mediated induction of lFN3 and IFN-a gene expression (9-il). Recently, ISGF-2, a protein recognizing the promoters of IFN-stimulated genes, was shown to be identical to IRF-i (12). Two other PRDI binding proteins, IRF-2-a protein structurally related to IRF-i-and PRDI-BF1-an 88 kD zinc finger containing protein-have been identified as transcriptional repressors of IFN-fl expression in cotransfection experiments (13, i4). The PRDII domain interacts with the transcription fac(or NF-KB (15, 16) and with a 300 kD protein termed PRDII-BF1 (1 7). Induction of NF-KB DNA binding activity by virus or double stranded RNA correlates directly with