A comprehensive approach to expression of L1 loci

Deininger, Prescott L.; Morales, Maria E.; White, Travis B.; Baddoo, Melody; Hedges, Dale J.; Servant, Géraldine; Srivastav, Sudesh; Smither, Madison E.; Concha, Monica; deHaro, Dawn; Flemington, Erik K.; Belancio, Victoria P.

doi:10.1093/nar/gkw1067

Cited by 97 publications

(158 citation statements)

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“…There are several hundred potentially active L1 loci with intact open reading frames that have been characterized in modern humans [6,49], and these sequences are highly identical to one another at both the nucleotide and amino acid level. The specific repertoire of L1 loci that is expressed varies among individuals, and by cell or tissue type [20,22,50,51]. To evaluate the potential of our mAbs to detect proteins originating from distinct copies of L1Hs, we mapped the specific epitopes recognized by each antibody and evaluated the conservation of each epitope among L1Hs loci.…”

Section: Orf2 Antibodies Identify Non-overlapping Epitopesmentioning

confidence: 99%

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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Background: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results:We report mass spectrometry based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically-acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions:Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth. References

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Section: Orf2 Antibodies Identify Non-overlapping Epitopesmentioning

confidence: 99%

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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“…2D). Note, however, the qRT-PCR experiment would not discriminate between authentic endogenous L1 transcripts and those cotranscribed within genes (44), and should thus be interpreted with caution (Discussion).…”

Section: Developing An L1 Transgene Regulated By the Endogenous Mouse L1mentioning

confidence: 99%

Intact piRNA pathway prevents L1 mobilization in male meiosis

Newkirk

Lee

Grandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1 −/− testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1 −/− germ cells compared with the wildtype. Analysis of adult Mov10l1 −/− germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1 −/− phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.LINE-1 reporter transgene | meiotic arrest | PIWI-interacting RNA | retrotransposition | spermatogenesis T he bulk of mammalian genomes are made up of transposable elements, the majority of which are retrotransposons (1). Retrotransposons are classified into long-interspersed elements (LINEs), short-interspersed elements (SINEs), and LTR retrotransposons, which collectively account for 43% and 37% of the human and mouse genomes, respectively (2). Retrotransposons amplify in the genome through an RNA intermediate, a process termed retrotransposition. LINEs are autonomous elements. An intact, full-length LINE-1 (L1) encodes two ORFs (i.e., ORF1 and ORF2); both are required for L1 mobilization (3). SINEs are nonautonomous elements and rely on L1's proteins for propagation in the genome (4). LTR retrotransposons are autonomous but appear to be inactivated in the human genome (5). Retrotransposition endangers the integrity of both somatic and germline genomes through insertional mutagenesis. In somatic tissues, both elevated L1 expression and retrotransposition have been strongly associated with many types of human cancers (6, 7). In a few cases, specific retrotransposition events (i.e., insertions) have been determined to drive t...

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“…Deininger et al (Deininger et al 2017) used the 5'RACE technique to specifically amplify LINE-1 transcripts beginning with the L1 5'UTR in HEK293 cells. PacBio reads exceeding 1000 letters in length were sequenced from the first 1237 bases of the amplified transcripts.…”

Section: L1-em Estimates Agree With L1 5'race Long Read Sequencing Inmentioning

confidence: 99%

“…Recent evidence both at the level of RNA (Philippe et al 2016;Deininger et al 2017) and at reinsertion (Tubio et al 2014) indicate that a small number of loci are responsible for most of the LINE-1 activity, further highlighting the need for locus specific LINE-1 RNA quantification.…”

Section: Introductionmentioning

confidence: 99%

“…Philippe et al (Philippe et al 2016) measured transcripts that overrun the 3' end of LINE-1, but our results indicate that the volume of this "run-on" transcription varies between loci (see results, figure 4.) Deininger et al (Deininger et al 2017) identified transcribed loci by looking at the subset of LINE-1 reads that align uniquely, but this accounts for only a small fraction of the reads aligning to the young L1HS elements. Other algorithms do exist that use EM to quantify transposable element RNA sequences (eg Jin et al 2015;Yang et al 2019), but none account for passive co-transcription.…”

Section: Introductionmentioning

confidence: 99%

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L1EM: A tool for accurate locus specific LINE-1 RNA quantification

McKerrow

Fenyö

2019

Preprint

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Motivation: LINE-1 elements are retrotransposons that are capable of copying their sequence to new genomic loci. LINE-1 derepression is associated with a number of disease states, and has the potential to cause significant cellular damage. Because LINE-1 elements are repetitive, it is difficult to quantify RNA at specific LINE-1 loci and to separate transcripts with protein coding capability from other sources of LINE-1 RNA. Results:We provide a tool, L1-EM that uses the expectation maximization algorithm to quantify LINE-1 RNA at each genomic locus, separating transcripts that are capable of generating retrotransposition from those that are not. We show the accuracy of L1-EM on simulated data and against long read sequencing from HEK cells.Availability: L1-EM is written in python. The source code along with the necessary annotations are available at https://github.com/FenyoLab/L1EM and distributed under GPLv3.

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A comprehensive approach to expression of L1 loci

Cited by 97 publications

References 53 publications

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Intact piRNA pathway prevents L1 mobilization in male meiosis

L1EM: A tool for accurate locus specific LINE-1 RNA quantification

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