Molecular Signatures of retrotransposon Ty3 insertion into the RNA Polymerase III transcription initiation site (Integrase)
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ABSTRACT: The Gypsy-like element Ty3 inserts proximal to the transcription start sites of genes transcribed by RNA polymerase 3 (RNAP3). In this study, a random-barcode Ty3 was used to count Ty3 insertions at specific sites. Surprisingly, saturation transposition of the yeast genome showed that tDNAs even within isoacceptor families are targeted at widely different frequencies. Ectopic expression of Ty3 integrase showed that it localizes to integration targets independent of other Ty3 proteins. Binding of integrase, RNAP3 and factor Brf1 at individual targets did not differ to the same extent as integration. Metadata analysis showed that histone modification H3K4Ac correlated positively with insertion frequency. Targeting frequency could be reconstituted on high copy plasmids containing only 75 bp of 5’ flanking sequence plus the tDNA target. Weighting of insertions according to frequency identified an A/T-rich sequence followed by C as the site of gene-proximal strand transfer. This site lies immediately adjacent to the adenines of the RNAP3 transcription start site motif (CAA). Recent structures of DNA in RNAP3 initiation complexes show that in the initiation complex the transcription start site is sharply bent at the position adjacent to the gene-proximal Ty3 strand transfer. We propose that Ty3 integration occurs in two steps: in the first, host Brf1 engages integrase; in the second, integrase exploits YR flexibility, and that together, these steps determine the wide range of Ty3 targeting frequencies.
Project description:The Gypsy-like element Ty3 inserts proximal to the transcription start sites of genes transcribed by RNA polymerase 3 (RNAP3). In this study, a random-barcode Ty3 was used to count Ty3 insertions at specific sites. Surprisingly, saturation transposition of the yeast genome showed that tDNAs even within isoacceptor families are targeted at widely different frequencies. Ectopic expression of Ty3 integrase showed that it localizes to integration targets independent of other Ty3 proteins. Binding of integrase, RNAP3 and factor Brf1 at individual targets did not differ to the same extent as integration. Metadata analysis showed that histone modification H3K4Ac correlated positively with insertion frequency. Targeting frequency could be reconstituted on high copy plasmids containing only 75 bp of 5’ flanking sequence plus the tDNA target. Weighting of insertions according to frequency identified an A/T-rich sequence followed by C as the site of gene-proximal strand transfer. This site lies immediately adjacent to the adenines of the RNAP3 transcription start site motif (CAA). Recent structures of DNA in RNAP3 initiation complexes show that in the initiation complex the transcription start site is sharply bent at the position adjacent to the gene-proximal Ty3 strand transfer. We propose that Ty3 integration occurs in two steps: in the first, host Brf1 engages integrase; in the second, integrase exploits YR flexibility, and that together, these steps determine the wide range of Ty3 targeting frequencies.
Project description:The Gypsy-like element Ty3 inserts proximal to the transcription start sites of genes transcribed by RNA polymerase 3 (RNAP3). In this study, a random-barcode Ty3 was used to count Ty3 insertions at specific sites. Surprisingly, saturation transposition of the yeast genome showed that tDNAs even within isoacceptor families are targeted at widely different frequencies. Ectopic expression of Ty3 integrase showed that it localizes to integration targets independent of other Ty3 proteins. Binding of integrase, RNAP3 and factor Brf1 at individual targets did not differ to the same extent as integration. Metadata analysis showed that histone modification H3K4Ac correlated positively with insertion frequency. Targeting frequency could be reconstituted on high copy plasmids containing only 75 bp of 5’ flanking sequence plus the tDNA target. Weighting of insertions according to frequency identified an A/T-rich sequence followed by C as the site of gene-proximal strand transfer. This site lies immediately adjacent to the adenines of the RNAP3 transcription start site motif (CAA). Recent structures of DNA in RNAP3 initiation complexes show that in the initiation complex the transcription start site is sharply bent at the position adjacent to the gene-proximal Ty3 strand transfer. We propose that Ty3 integration occurs in two steps: in the first, host Brf1 engages integrase; in the second, integrase exploits YR flexibility, and that together, these steps determine the wide range of Ty3 targeting frequencies.
Project description:Distinct integration patterns of different retroviruses have puzzled virologists for over 20 years. The viral integrase (IN), as part of the intasome complex, docks onto the target DNA (tDNA) and catalyzes the insertion of the viral genome into the host chromatin. We identified retroviral IN amino acids directly contacting tDNA bases and affecting the local integration site sequence biases. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms INS119G and INR231G retarget viral integration away from gene dense regions, without affecting the interaction with the lentiviral tethering cofactor LEDGF/p75 (PSIP1). Precisely these variants were associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors. LEDGF/p75 (PSIP1) ChIP-Seq using A300-848 antibody (recognizes p75 isoform) and input control in primary CD4+ T-cells
Project description:Distinct integration patterns of different retroviruses have puzzled virologists for over 20 years. The viral integrase (IN), as part of the intasome complex, docks onto the target DNA (tDNA) and catalyzes the insertion of the viral genome into the host chromatin. We identified retroviral IN amino acids directly contacting tDNA bases and affecting the local integration site sequence biases. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms INS119G and INR231G retarget viral integration away from gene dense regions, without affecting the interaction with the lentiviral tethering cofactor LEDGF/p75 (PSIP1). Precisely these variants were associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.
Project description:Integrative and conjugative elements (ICEs) are found in many bacterial species and are mediators of horizontal gene transfer. Tn916, an ICE found in several Gram-positive species, can integrate into many sites in the host chromosome, in contrast to the many ICEs that preferentially integrate into a single site. The consensus integration motif for Tn916, based on analyses of approximately 200 independent insertions, is an ~16 bp AT-rich sequence. Here, we describe the identification and mapping of approximately 105 independent Tn916 insertions in the Bacillus subtilis chromosome. The insertions were distributed between 1554 chromosomal sites, and approximately 99% of the insertions were in 303 sites and 65% were in only ten sites. One region, between ykuC and ykyB (kre), was a 'hot spot' for integration with ~22% of the insertions in that single location. In almost all of the top 99% of sites, Tn916 was found with similar frequencies in both orientations relative to the chromosome and relative to the direction of transcription, with a few notable exceptions. Using the sequences of all insertion regions, we determined a consensus motif which is similar to that previously identified for Clostridium difficile. The insertion sites are largely AT-rich, and some sites overlap with regions bound by the nucleoid-associated protein Rok, a functional analog of H-NS of Gram-negative bacteria. Rok functions as a negative regulator of at least some horizontally acquired genes. We found that the presence or absence of Rok had little or no effect on insertion site specificity of Tn916.
Project description:The goal of these experiments was to define the targets of Ty3 transposition in Saccharomyces cerevisiae. Ty3 is a retroviruslike element that is found at the transcription initiation site of chromosomal tRNA genes.
Project description:The goal of these experiments was to define the targets of Ty3 transposition in Saccharomyces cerevisiae. Ty3 is a retroviruslike element that is found at the transcription initiation site of chromosomal tRNA genes. A Ty3 that can be induced by growth in galactose-containing medium and which was marked by an insertion of HIS3 downstream of the second open reading frame of the element (POL3) was induced to undergo transposition by plating cells onto galactose containing medium and replica-plating onto medium selective for cells that had undergone transposition. These cells were collected, DNA was extracted, and inverse PCR was performed using primers inside the Ty3 element in order to generate a library of insertion sites flanked by Illumina sequence-compatible primers.
Project description:The two obstacles that impede a wider application of genetically modified cells expressing therapeutic transgenes for ex vivo gene therapy are the immune mediated rejection of the transplanted cells, combined with their potential to cause iatrogenic oncogenesis. In this study we describe a new cellular vehicle for this form of therapy,; termed the cord lining epithelial cell (CLEC). CLECs are derived from the human amnion and incorporate many of the immunoregulatory functions associated with the fetal/maternal interface. We show that CLECs can be safely transfected by phage ÏC31 integrase to accomplish site-specific integration of a therapeutic human transgene. We also show that transplanted CLECs are not oncogenic in vivo and can be maintained in immunocompetent mice where acute xeno-rejection rapidly destroys other human cell types. Finally, we demonstrate the utility of CLECs for ex vivo gene therapy by delivering human coagulation factor 8 to mice with Hemophilia A. Experiment Overall Design: The transcriptome datasets of human umbilical cord lining epithelial cells were compared before(CLEC) and 1 month after(CLEC-GFP) phage integrase mediated integration of EGFP cDNA into the genome. Transcriptome datasets were generated in singles and genes differentiallty expressed in cells before and after phage integrase treatment were analysed. Genes differentially expressed by at least 2 fold as compared to untreated CLEC were considered to be significantly dysregulated. List of genes with significant dysregulation were used for further analysis and to used to determine if genomic integration events had resulted in any potential geno-toxicity.