Regulation And Function Of Retroelements

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Identification of an integrase-independent pathway of retrotransposition Despite the central role of integration in the propagation of retroviruses, important questions remain about residual insertion that occurs in the absence of IN activity. Mutations in the catalytic residues of HIV-1 integrase (IN) produce residual infectious titer, typically with a 3 to 4-log decrease. However, in continuous cultures of HIV-1 lacking IN activity, insertion efficiency can be as high as 0.2-0.8% of wild type virus. These results indicate that retroviruses possess a secondary, IN-independent pathway, that incorporates viral DNA into the host genome. Since IN-independent infections could compromise the treatment of HIV-1 patients with IN inhibitors, it is important to identify the nature of this pathway. Long terminal repeat (LTR) retrotransposons are important models of retroviruses due to structural and mechanistic similarities. We found that the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe retains 5% insertion activity in the absence of IN. Genome-wide insertion profiles of Tf1 lacking IN (Tf1-INfs) were significantly different from that of Tf1 expressing active IN. DNA logo analysis showed that the sequences downstream of the Tf1-INfs insertion sites had a prominent bias of ATAAC and upstream flanks showed a preference of CAA. Interestingly, the downstream logo matches that of the primer binding site (PBS), an 11 bp sequence retained after reverse transcription on the 3end of the +strand cDNA. The CAA matches the last three bp of the poly purine tract (PPT), that is retained on the 3 end of the -strand cDNA. The PBS and PPT preferences suggested that these single-stranded sequences contributed to insertion through homologous recombination (HR). Transposition assays revealed the insertions occurred through Rad52-dependent single-strand annealing (SSA), as Rad51 was dispensable. The rad52-R45A mutation, which specifically abolishes SSA activity of Rad52, significantly reduced the frequency of Tf1-INfs insertions and resulted in dissociation of Rad52 from Tf1 cDNA. These data indicate that Rad52 plays a critical role in IN independent insertions by binding to the ends of the cDNA causing recombination with sequences similar to PBS and PPT. S. pombe contains 13 pre-existing copies of Tf2, an LTR-retrotransposon with PBS and PPT sequence identical to Tf1. We tested whether Tf2 was a significant site of IN independent insertion by re-analyzing raw sequence reads from Tf1-INfs insertions that otherwise would be discarded because they map to multiple positions. Indeed, we found that 70% of the insertion activity mapped to the PBS of Tf2. The insertion sequence of individual strains expressing Tf1-INfs revealed insertions occurred at existing Tf2s and resulted in tandem elements. Importantly, tandem copies of Tf2 regularly form in wild isolates of S. pombe. Our efforts to determine whether IN independent events occur naturally showed cultures with continuing expression of WT Tf1 produced insertions that were predominantly IN independent. These data demonstrate that Tf1 possesses two efficient insertion pathways, one relies on IN and the other is IN independent but requires Rad52. Significantly, we found in previously published data of HIV-1 IN independent insertions that five of 69 sites had strong similarity to the HIV-1 PBS. Together, these results indicate that homology dependent SSA provides a significant pathway of IN independent insertion. Retrotransposon insertions associated with risk of neurologic and psychiatric diseases Genetic variation can directly cause or increase susceptibility to neurologic and psychiatric diseases. Genome-wide association studies (GWAS) have been very useful in identifying single nucleotide polymorphisms (SNPs) associated with common and complex human diseases. However, identifying causal genetic variants remains challenging because most disorders are influenced by many loci and SNPs do not typically provide adequate resolution to pinpoint causal genes. In addition, GWAS often do not directly identify structural variants such as TE insertions which can have substantial impact on gene expression. TE activity contributes to disease with reports of more than 124 cases of TE insertions that result in human genetic diseases, and elevated transposition in brain is observed in neurologic diseases. GWAS SNPs can serve as proxies for genomic insertions and deletions because these structural variants are more likely to alter gene expression than SNPs. Importantly, polymorphic TEs map next to disease-associated SNPs more frequently than expected by chance. Therefore, we hypothesized that polymorphic TEs highly associated with GWAS SNPs of neurologic and psychiatric diseases are potential causative variants of those diseases. We took advantage of the 17,000 polymorphic TEs mapped by the 1,000 Genomes Project to determine what role TEs may have in neurologic and psychiatric diseases. For our analysis we reviewed GWAS of neurologic and psychiatric diseases including amyotrophic lateral sclerosis, migraine, schizophrenia, Parkinson's disease, Alzheimer's disease, and identified 753 Trait Associated SNPs (TASs) with P < 106. We calculated and ranked the levels of genetic association between TASs and cataloged polymorphic TEs. This generated 76 candidate TEs that are highly associated with a TAS. From these variants, we identified causal candidate TEs by searching for 1) TEs inserted in regulatory chromatin active in brain tissues as determined by Hidden Markov modeling of data from the NIH Roadmap Epigenomics Consortium, and 2) TEs associated with altered gene expression in brain tissues using RNA-seq data from the Genotype-Tissue Expression (GTEx) project to identify expression Quantitative Trait Loci (eQTLs). We further validated association level of TAS and candidate TEs by genotyping polymorphic TE insertions using genomic DNA from a 30-trio reference panel of CEPH Utah residents with ancestry from northern and western Europe (CEU) HapMap samples from the 1000 Genome Project. In summary, we found 10 polymorphic TE insertions that are important causal candidates of neurologic and psychiatric disorders. Among them, lead TE insertions were tested for regulatory effects in human neural stem cells by luciferase reporter assay, and 5 of 6 tested caused significant changes in the activity of a minimal promoter. Future studies include testing the direct impact of the causal candidate TEs by CRISPR/Cas genome editing of human iPSCs.