Diseases such as AIDS and leukemia caused by retroviruses have intensified the need to understand the mechanisms of retrovirus replication. One of our objectives 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 its integration exhibits a strong preference for pol II promoters. This choice of target sites is similar to the strong integration preferences human immunodeficiency virus 1 (HIV-1) and murine leukemina virus (MLV) have for pol II transcription units. Currently, it is not clear how these viruses recognize their target sites. 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. An understanding of the mechanisms responsible for targeted integration could lead to new approaches for antiviral therapies and to improvements in the application of viral vectors in gene therapy. The extraordinary capacity of DNA sequencing can create ultra dense maps of integration that are being used to study the mechanisms that position integration. Unfortunately, the great increase in the numbers of insertion sites detected comes with the cost of not knowing which positions are rare targets and which sustain high numbers of insertions. To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions. We sequenced 1 million insertions of retrotransposon Tf1 in the genome of S. pombe and obtained the first profile of integration with frequencies for each individual position. Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration. The serial number system is a general method that can be applied to determine precise integration maps for retroviruses and gene therapy vectors. Transposable elements constitute a substantial fraction of the eukaryotic genome and as a result, have a complex relationship with their host that is both adversarial and dependent. To minimize damage to cellular genes TEs possess mechanisms that target integration to sequences of low importance. However, the retrotransposon Tf1 of Schizosaccharomyces pombe integrates with a surprising bias for promoter sequences of stress response genes. The clustering of integration in specific promoters suggests Tf1 possesses a targeting mechanism that is important for evolutionary adaptation to changes in environment. We found this year that Sap1, an essential DNA binding protein, plays an important role in Tf1 integration. A mutation in Sap1 resulted in a 10-fold drop in Tf1 transposition and measures of transposon intermediates supports the argument that the defect occurred in the process of integration. Published ChIP-Seq data of Sap1 binding combined with high-density maps of Tf1 integration that measure independent insertions at single nucleotide positions show that 73.4% of all integration occurred at genomic sequences bound by Sap1. This represents high selectivity since Sap1 binds just 6.8% of the genome. A genome-wide analysis of promoter sequences revealed that Sap1 binding and amounts of integration correlate strongly. More importantly, an alignment of the DNA binding motif of Sap1 revealed integration clustered on both sides of the motif and showed high levels specifically at positions +19 and -9. These data indicate that Sap1 contributes to the efficiency and position of Tf1 integration. Transposable elements (TEs) are common constituents of centromeres. However, it is not known what causes this relationship. Schizosaccharomyces japonicus contains 10 families of Long Terminal Repeat (LTR)-retrotransposons and these elements cluster in centromeres and telomeres. In the related yeast, Schizosaccharomyces pombe LTR-retrotransposons Tf1 and Tf2 are distributed in the promoter regions of RNA pol II transcribed genes. Sequence analysis of TEs indicates that Tj1 of S. japonicus is related to Tf1 and Tf2, and uses the same mechanism of self-primed reverse transcription. Thus, we wondered why these related retrotransposons localized in different regions of the genome. To characterize the integration behavior of Tj1 we expressed it in S. pombe. We found Tj1 was active and capable of generating de novo integration in the chromosomes of S. pombe. The expression of Tj1 is similar to Type C retroviruses in that a stop codon at the end of Gag must be present for efficient integration. 17 inserts were sequenced, 13 occurred within 12 bp upstream of tRNA genes and 3 occurred at other RNA pol III transcribed genes. The link between Tj1 integration and RNA pol III transcription is reminiscent of Ty3, an LTR-retrotransposon of Saccharomyces cerevisiae that interacts with TFIIIB and integrates upstream of tRNA genes. The integration of Tj1 upstream of tRNA genes and the centromeric clustering of tRNA genes in S. japonicus demonstrate that the clustering of this TE in centromere sequences is due to a unique pattern of integration. With the introduction of new deep sequencing technology it is now possible to sequence many millions of transposon insertions in a single experiment. We tested whether Illumina sequencing could be used to generate a dense profile of transposon insertions that would reveal which genes are required for cell division. For this experiment we used a haploid strain of S. pombe and Hermes, a DNA transposon from the housefly. In previous work we found that the Hermes transposon was highly active in S. pombe and that a large fraction of the insertions occurred in ORFs. We predicted that in actively growing cultures, Hermes insertions would not be tolerated in essential ORFs. We induced Hermes transposition in a large culture S. pombe that was grown for 80 generations. With ligation mediated PCR and Illumina sequencing we were able to sequence 360,513 independent insertion events. On average, this represented one insertion for every 29 bp of the S. pombe genome. An analysis of integration density revealed that the ORFs largely separated into two classes, one with high numbers of insertions and another with much lower numbers. In collaboration with a group that deleted each of the genes of S. pombe, we found the ORFs with low numbers of Hermes insertion corresponded to the essential genes. The ORFs with higher integration densities were in genes classified as nonessential. These results validated integration profiling as a new method for identifying genes with essential function. Importantly, by applying specific conditions of selection during growth, this method can be adopted to identify genes that contribute to a wide variety of functions. Recently we have pursued the use of Hermes integration profiles to identify the complement of genes that contribute to heterochromatin. Since heterochromatin is a complex process that silences genes and because S. pombe is an excellent model for the study of heterochromatin, we generated independent libraries containing 1 million integration events. The density of these libraries is substantially greater then previously generated and average one insertion per 10 bp of the S. pombe genome. This year our analysis of these integration sites generated a list of approximately 150 genes that may contribute to hetrochromatin formation. Importantly, many of these candidates are novel and our study of mutations in several of these genes indicates they do play a role in hetrochromati
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