Acanthamoeba causes an essentially untreatable form of corneal keratitis in humans. Difficulty in treatment is linked to the ability of Acanthamoeba to differentiate into a drug-resistant cyst. In order to adopt a rational approach to Acanthamoeba therapy, based on antisense oligonucleotides, it is necessary to first understand at the molecular level how Acanthamoeba regulates its lifecycle of growth and differentiation. Our previous work on gene transcription in Acanthamoeba has identified several promoter elements and regulating transcription factors that are required for expression of the TATA box binding protein (TBP) gene. TBP is itself required for all gene transcription in Acanthamoeba. The level of TBP gene expression is regulated by a unique DNA binding protein called TPBF. TPBF can in turn be regulated by phosphorylation. This is a proposal to characterize in detail the regulatory proteins and promoter elements of the TBP gene, and to investigate their role in gene expression during Acanthamoeba differentiation. It is hypothesized that TPBF and a coactivator needed for its activity are inactivated during encystment. In addition, an inhibitor of TBP gene expression may become active during differentiation, thereby turning off TBP gene expression. Specifically, we will construct and assay TBP gene promoters to determine the effects of positive and negative regulatory elements during differentiation. The transcription factor TFBF has been cloned, permitting a complete evaluation of its structure and the significance of its phosphorylation. The mechanism of transcription stimulation by TPBF will be examined by investigating its interaction with a coactivator, APC1, as well as downstream events that occur during initiation. The effect of differentiation on these interactions will be determined in order to test the hypothesis stated above. Last, the coactivator APC1 and the putative cyst-specific negative factor will be purified, and in the longer term cloned in order to determine their mechanisms. The relevance of these studies to disease treatment is direct. They will identify the biochemical pathways that control or permit differentiation. These pathways can be specifically targeted by antisense approaches in order to either kill cells or to prevent differentiation. Here, we wish to accumulate the basic formation and reagents necessary for this or related approaches.
