Molecular Signatures of retrotransposon Ty3 insertion into the RNA Polymerase III transcription initiation site
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ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500 (Saccharomyces cerevisiae)
SUBMITTER: James Yu
PROVIDER: GSE97894 | GEO | 2018-04-15
SECONDARY ACCESSION(S): GSE97892 GSE97890 GSE97893
REPOSITORIES: GEO
Similar Datasets
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. Overall design: In the S. cerevisiae strain BY4741, the genomic BRF1 gene was modified to express a N-terminal 3X FLAG tagged protein for ChIP-seq. The native BRF1 gene protmoter was retained to maintain wild type gene expression after modification. As a negetive control, untagged wild type BY4741 culture was grown and treated identically to Brf1 and Rpc34 ChIP protocols.
2018-04-15 | GSE97890 | GEO
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. Overall design: The Ty3 Integrase was cloned into Estradiol-inducible plasmid pKP3915 with a N-terminal 3X FLAG sequence to make plasmid pKP4010. For ChIP-seq, pKP4010 or pKP3915 (empty vector control) were transformed into the S. cerevisiae strain BY4741 and selected for on synthetic media lacking Uracil. Prior to harvesting cells for immuoprecipitation, 10 µM β-estradiol was added to cultures to induce integrase expression for approximately six hours.
2018-04-15 | GSE97892 | GEO
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. Overall design: In the S. cerevisiae strain BY4741, the genomic RPC34 gene was modified to express a N-terminal 3X FLAG tagged protein for ChIP-seq. The native RPC34 gene protmoter was retained to maintain wild type gene expression after modification.
2018-04-15 | GSE97893 | GEO
Project description:Molecular Signatures of retrotransposon Ty3 insertion into the RNA Polymerase III transcription initiation site
| PRJNA383147 | ENA
Project description:Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes.
2012-01-01 | S-EPMC3317150 | BioStudies
Project description:Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.
2019-01-01 | S-EPMC6673722 | BioStudies
Project description:Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2-TBP-Bdp1 complex bound to DNA at 2.7?Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.Transcription initiation by RNA polymerase III requires TFIIIB, a complex formed by Brf1/Brf2, TBP and Bdp1. Here, the authors describe the crystal structure of a Brf2-TBP-Bdp1 complex bound to a DNA promoter and characterize the role of Bdp1 in TFIIIB assembly and pre-initiation complex formation.
2017-01-01 | S-EPMC5526994 | BioStudies
Project description:Converting the single-stranded retroviral RNA into integration-competent double-stranded DNA is achieved through a multi-step process mediated by the virus-coded reverse transcriptase (RT). With the exception that it is restricted to an intracellular life cycle, replication of the Saccharomyces cerevisiae long terminal repeat (LTR)-retrotransposon Ty3 genome is guided by equivalent events that, while generally similar, show many unique and subtle differences relative to the retroviral counterparts. Until only recently, our knowledge of RT structure and function was guided by a vast body of literature on the human immunodeficiency virus (HIV) enzyme. Although the recently-solved structure of Ty3 RT in the presence of an RNA/DNA hybrid adds little in terms of novelty to the mechanistic basis underlying DNA polymerase and ribonuclease H activity, it highlights quite remarkable topological differences between retroviral and LTR-retrotransposon RTs. The theme of overall similarity but distinct differences extends to the priming mechanisms used by Ty3 RT to initiate (-) and (+) strand DNA synthesis. The unique structural organization of the retrotransposon enzyme and interaction with its nucleic acid substrates, with emphasis on polypurine tract (PPT)-primed initiation of (+) strand synthesis, is the subject of this review.
2017-01-01 | S-EPMC5371799 | BioStudies
Project description:The magellan transposable element is responsible for a spontaneous 5.7-kb insertion in the maize wx-M allele. This element has the sequence and structural characteristics of a Ty3/gypsy-like retrotransposon. The magellan element is present in all Zea species and Tripsacum andersonii; it is absent, however, in the genomes of all other Tripsacum species analyzed. The genetic distances between magellan elements suggest that this retrotransposon is evolving faster than other Zea nuclear loci. The phylogeny of magellan within Zea and T. andersonii also reveals a pattern of interspecies transfers, resulting in the movement of magellan subfamilies between different species genomes. Interspecific hybridization may be a major mechanism by which this retrotransposon invades and establishes itself in new taxa.
1000-01-01 | S-EPMC45294 | BioStudies
Project description:Here we report that transcriptional effects of the insertion of a retrotransposon can occur simultaneously both upstream and downstream of the insertion site. We have identified an intra-cisternal A particle (IAP) retrotransposon in intron 6 of a gene that we have named Cabp (CDK5 activator binding protein). The presence of the IAP is associated with an aberrant transcript initiating from a cryptic promoter in the IAP, reading out into the adjacent Cabp gene sequence. The expression of this transcript is highly variable among isogenic mice within the C57BL/6J strain and so Cabp(IAP) can be classified as a metastable epiallele. As expected, the presence or absence of the transcript correlates with differential DNA methylation of the 5' LTR of the IAP. More surprisingly, in mice where the retrotransposon is unmethylated and presumably transcriptionally active, we find a number of short Cabp transcripts which initiate at the normal 5' end of the gene but terminate prematurely, just 5' of the retrotransposon. This is the first report of a retrotransposon having both upstream and downstream effects on transcription at the site of insertion and it suggests that alternative polyadenylation may sometimes be caused by a downstream convergent transcription unit.
2004-01-01 | S-EPMC528799 | BioStudies