Whiteside, Simon Thomas; (1992) Regulation of human beta-interferon gene expression. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Type I Interferons (IFNs) are secreted polypeptides that are produced in many cell lines in response to viral challenge or treatment with synthetic double-stranded RNA, which have been demonstrated to activate a protective anti-viral state in non-infected cells. Although Type I Interferon is a heterogeneous mixture of gene products, a single immunologically distinct β-form has been identified in humans, and the corresponding gene molecularly cloned. Expression of this gene is induced transiently induced at the level of transcriptional activation following viral infection or treatment with synthetic double-stranded RNA. This induction can occur in the absence of de novo protein synthesis, suggesting that responsive cells contain pre-existing factors that are modified upon induction. Genetic analysis of this process has demonstrated that the sequence elements 5' to the gene are responsible for mediating this induction. Closer inspection of these sequences has revealed them to consist of multiple positive and negative genetic elements. One of these. Positive Regulatory Domain I (PRD I) has been shown function as a virally-inducible enhancer element capable of binding to the positively-acting transcription factor IRF-1. In this thesis, it is shown that, in HeLa cells, PRD I can confer inducibility by synthetic double-stranded RNA in the absence of protein synthesis. Since levels of IRF-1 in these cells are undetectable before induction, it would seem that some other factors are responsible for this inducibility. To investigate the existence of such factors, novel activities capable of binding specifically to PRD I have been identified in nuclear extracts prepared from HeLa cells. In addition to the previously identified repressor protein IRF-2, several of these factors are present in uninduced cells. Two of these have properties consistent with a role in negative regulation, while one of the others may be the transcription factor ISGF3γ, an activity involved in the regulation of genes that are induced by Interferons themselves. A fourth activity, induced by treatment with doublestranded RNA in the absence of protein synthesis, is shown to be a proteolytically-processed form of the repressor protein IRF-2. Interestingly, this activity also has properties consistent with a role in negative regulation. In light of these findings, a model for the induction of PRD I-mediated transcription is proposed. Finally, a number of experiments that were performed in an attempt to isolate cDNA clones which encoded these novel activities are also described.

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