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Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo (unselected insertions)

ABSTRACT: Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo (unselected insertions)

PROVIDER: PRJEB31086 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR3139780.fastq.gz	Fastqsanger.gz
	ERR3139781.fastq.gz	Fastqsanger.gz
	ERR3139782.fastq.gz	Fastqsanger.gz
	ERR3139783.fastq.gz	Fastqsanger.gz
	ERR3139774.fastq.gz	Fastqsanger.gz

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Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo (unselected insertions)

Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a neomycin-resistance-based retrotransposition cassette, as well as a hygromycin-resistance cassette on the plasmid backbone. For this set of experiments, cells were only selected for transfection (hygromycin) but not for retrotransposition (neomycin). Then we prepared ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of hygromycin-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016). For some libraries, the linker-ligated genomic DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to limit amplification from the plasmid and enrich for retrotransposition-mediated insertion events into the genomic DNA.

2019-04-03 | E-MTAB-7644 | biostudies-arrayexpress

Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo

2019-04-04 | E-MTAB-6933 | biostudies-arrayexpress

Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo

Project description:Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo

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DNA sequencing of Arabidopsis thaliana nrpd1-3 background with novel insertions of LTR retrotransposon ONSEN/COPIA78.

Project description:The intent of the experiment was to infer, from re-sequencing of genomic DNA, the coordinates of neo-insertions from activated ONSEN/COPIA78 LTR retrotrasnposon in Arabidopsis thaliana. For this, we performed Illumina 75 bp pair-end PCR-free DNA genome re-sequencing in several independent lines of RdDM mutant nrpd1-3.

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Arrested replication forks guide retrotransposon integration

Project description:Long Terminal Repeat (LTR) Retrotransposons are an abundant class of genomic parasites that replicate by insertion of new copies into the host genome. LTR retrotransposons prevent mutagenic insertions through diverse targeting mechanisms that avoid coding sequences, but a universal set of principles guiding their target site selection hasn’t been established. Here we show that insertion of the fission yeast LTR retrotransposon Tf1 is guided by the DNA binding protein Sap1, and that the efficiency and location of the targeting depend on the activity of Sap1 as a replication fork barrier. We propose that Sap1 guides insertion of Tf1 by blocking the progression of the replication fork, and that the Tf1 transposon uses features of arrested forks to insert into the host genome. These observations point to a universal mechanism for determination of LTR retrotransposon target site selection.

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Project description:Mammalian genomes have evolutionary harbored numerous mobile elements, a part of which are still active and evoke genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression and disruption of neighboring genes in the host genome. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposons in mammalian genome can be identified at a time. By this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA-integrating genomic regions. Among 147 putative insertions located nearby the promoter regions, 91 were considered to be present discriminatively in the genome either of C3H/He or C57BL/6J mice, suggesting the existence of considerable strain differences. In addition, by comparing genomic DNAs from a radiation-induced myeloid leukemia and its reference normal tissue, we detected 3 genomic regions around which an IAP element was integrated. These results demonstrate for the first time the successful genome-wide survey of a type of retrotransposon in mammalian genome. Keywords: retrotransposon mapping Genomic DNA fragments containing sequences that flank a mouse retrotransposon are purified and hybridized to the microarray. Experiments are comparisions between signal intensities obtained from Cy3-labeled purified DNA fragments (test) and Cy5-labeled whole genomic DNA fragments (reference).

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2019-03-16 | E-MTAB-5884 | biostudies-arrayexpress

Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon

Project description:Eukaryotic genomes harbor hundreds of rRNA genes, many of which are transcriptionally silent. However, little is known about selective regulation of individual rDNA units. In Drosophila melanogaster, some rDNA repeats contain insertions of the R2 retrotransposon, which is capable to be transcribed only as part of pre-rRNA molecules. rDNA units with R2 insertions are usually inactivated, although R2 expression may be beneficial in cells with decreased rDNA copy number. Here we found that R2-inserted rDNA units are enriched with HP1a and H3K9me3 repressive mark, whereas disruption of the heterochromatin components slightly affects their silencing in ovarian germ cells. Surprisingly, we observed a dramatic upregulation of R2-inserted rRNA genes in ovaries lacking Udd (Under-developed) or other subunits (TAF1b and ТAF1c-like) of the SL1-like complex, which is homologues to mammalian Selective factor 1 (SL1) involved in rDNA transcription initiation. Derepression of rRNA genes with R2 insertions was accompanied by a reduction of H3K9me3 and HP1a enrichment. We suggest that the impairment of the SL1-like complex affects a mechanism of selective activation of intact rDNA units which competes with heterochromatin formation. We also propose that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.

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