Diseases such as AIDS and leukemia caused by retroviruses have intensified the need to understand the mechanisms of retrovirus replication. Our primary objective is to understand how retroviral cDNAs are integrated into the genome of infected cells. Because of their similarities to retroviruses, long terminal repeat (LTR)-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. We are particularly interested in Tf1 because of its strong preference for integrating into pol II promoters. This choice of target sites is similar to the strong preferences human immunodeficiency virus 1 (HIV-1) and murine leukemina virus (MLV) have for integration into pol II transcription units. Currently, it is not clear how these viruses recognize their target sites and perform integration. We therefore study the integration of Tf1 as a model system with which we hope to uncover mechanisms general to the selection of integration sites. Such an understanding of the mechanisms responsible for targeted integration could lead to new approaches for blocking the replication of HIV-1. A key goal of our research this year was to identify the mechanism that directs integration to regions upstream of ORFs. To study insertion patterns in specific genes, a target plasmid assay was developed. Integration into plasmids containing various genes occurred upstream of the ORFs in insertion windows of approximately 150 nt. Deletion analyses of the plasmids indicated that the target positions were the only sequence features required for integration. Separate plasmids containing smaller sequences demonstrated that the windows of integration themselves were sufficient to direct integration. Our results indicated that transcription factors bound at their promoters, mediate integration. This model was supported by the finding that a functional binding site for the transcription factor Aft1p played a critical role in the targeting of Tf1 integration to the two major insertion sites in the fbp1 promoter. In addition, we found that Atf1p is required for integration in the fbp1 promoter and that Atf1p interacts with integrase. These data provide strong support for the role of Atf1p in directing integration to target sites. Another project conducted this year was to identify the function of the chromodomain present in the C-termini of some retrotransposon integrases. This chromodomain is related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant (CHD-del). The mutations, V1290A, Y1292A, W1305A, and CHD-del, substantially reduced transposition activity in vivo. Blotting and recombination assays showed that some of these mutants had little or no reduction in the levels of IN or cDNA imported into the nucleus. Chromatin immunoprecipitation assays revealed that CHD-del caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. The integration of Tf1 occurs primarily into pol II promoters. Although we currently beliieve this preference is the result of a mechanism that actively targets Tf1, it is possible that the insertion bias is due to greater accessibility at the promoter sequences. We tested this possibility by studying in S. pombe cells the integration pattern of Hermes, a cut and paste transposon that was isolated from the house fly. Since hermes propagates in a host that is evolutionary distant from S. pombe, it is unlikely that a mechanism exists that would actively position insertion sites. Any integration of hermes in S. pombe would likely occur at positions that were accessible to the transposase. In addition, unbiased activity of a transposon in S. pombe could be widely adapted as a tool for random mutagenesis. Since no methods currently exist for transposon mutagenesis of S. pombe, a method for insertional mutagenesis would be a significant contribution to the field. The transposase of hermes was expressed in S. pombe by fusing its gene to the promoter of nmt1. To measure transposition activity the cells that expressed the transposase also contained a plasmid encoded a copy of neo flanked by the terminal inverted repeats (TIRs) of hermes. The ability of the transposase to cut out neo with the TIRs and insert this DNA into the pombe genome was tested. Twenty six independent strains that became G418 resistant were analyzed and each strain was found to have acquired a copy of hermes. Analysis of these inserted copies revealed that 54% of them disrupted ORFs. These results indicate that the insertion of hermes did not discriminate between coding and noncoding sequences. This is in strong contrast to the integration of Tf1 where virtually none of the inserts occur in ORFs. These data indicate that hermes can readily be used as a tool for the random disruption of pombe genes.
Showing the most recent 10 out of 24 publications
