Retrotransposons as a Source of DNA Damage in Neurodegeneration

The etiology of aging-associated neurodegenerative diseases (NDs), such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), still remains elusive and no curative treatment is available. Age is the major risk factor for PD and AD, but the molecular link between aging and neurodegeneration is not fully understood. Aging is defined by several hallmarks, some of which partially overlap with pathways implicated in NDs. Recent evidence suggests that aging-associated epigenetic alterations can lead to the derepression of the LINE-1 (Long Interspersed Element-1) family of transposable elements (TEs) and that this derepression might have important implications in the pathogenesis of NDs. Almost half of the human DNA is composed of repetitive sequences derived from TEs and TE mobility participated in shaping the mammalian genomes during evolution. Although most TEs are mutated and no longer mobile, more than 100 LINE-1 elements have retained their full coding potential in humans and are thus retrotransposition competent. Uncontrolled activation of TEs has now been reported in various models of neurodegeneration and in diseased human brain tissues. We will discuss in this review the potential contribution of LINE-1 elements in inducing DNA damage and genomic instability, which are emerging pathological features in NDs. TEs might represent an important molecular link between aging and neurodegeneration, and a potential target for urgently needed novel therapeutic disease-modifying interventions.

Clinically silent LINE 1 insertion in the PNPLA3 gene may impede genotyping of the p.I148M variant

The patatin-like phospholipase domain containing 3 (PNPLA3) gene (viz. its I148M variant) is one of the key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We have identified a novel insertion/deletion variant of 1114 bp, localized in the second intron of the PNPLA3 gene, which corresponds to the 3′ terminal sequence of the long-interspersed element (LINE-1). DNA analysis of 122 NAFLD patients and 167 control subjects as well as RNA analysis of 19 liver biopsies revealed that the novel variant is very common (frequency = 0.41), fully linked to the clinically important I148M variant, and clinically silent. Although the LINE-1 insertion does not seem to have any biological effect, it can impede genotyping of the I148M variant. If insertion prevents the attachment of the diagnostic primer, then the non-insertion allele will be selectively amplified; and thus the frequency of the 148M "risk" allele will be significantly overestimated due to the complete linkage of the LINE-1 insertion and the 148I allele of the PNPLA3 gene. Therefore, our findings underline the importance of careful design and consistent documentation of the methodology, including primer sequences. Critical revisions of the results of some studies that have already been reported may therefore be needed.

Enterovirus Infection Restricts Long Interspersed Element 1 Retrotransposition

Long interspersed element 1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome that can serve as an endogenous upstream activator of cytoplasmic nucleic acid sensing pathways to elicit an antiviral immune response. In this study, we investigated the influence of enteroviral infection on L1 mobility. The results showed that infection with different enteroviruses, both EV-D68 and EV-A71, blocked L1 transposition. We screened diverse viral accessory proteins for L1 activity and identified EV-D68 2A, 3A, 3C, and EV-A71 ORF2p proteins as viral L1 inhibitors. EV-D68 2A suppressed L1 mobility by expression suppression of L1 proteins. Viral proteins 3A and 3C restricted ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins. The newly identified enteroviral protein ORF2p inhibited the expression of L1 ORF1p. Altogether, our findings shed light on the strict modulation of L1 retrotransposons during enterovirus replication.

Resveratrol Blocks Retrotransposition of LINE-1 Through PPAR α and Sirtuin-6

The retroelement long interspersed element-1 (LINE-1 or L1) comprises about 17% of the human genome. L1 retrotransposition is known to cause genomic instability and related disorders, and resveratrol suppresses this retrotransposition; however, the underlying mechanism is still not elucidated. Recent observations showed that low-molecular-weight compounds might induce L1 retrotransposition through unknown mechanisms. This study aimed to determine polyphenol resveratrol (RV)’s effect on L1-RTP (retrotransposition) in somatic cells. Surprisingly, RV completely blocked L1-RTP. Experiments using the PPARα inhibitor GW6471 or siRNA-mediated PPARα depletion showed that RV-mediated L1-RTP’s inhibition depended on peroxisome proliferator-activated receptor α (PPARα). We demonstrated that RV inhibits p38 and CREB phosphorylation, which are involved in MAPK signaling, and the L1-ORF1 protein’s chromatin recruitment. Furthermore, RV increased the expression of sirtuin-6 (SIRT6), which inhibited the activation of L1. The sirtuins family, SIRT1, SIRT6, and SIRT7, but not SIRT3, are involved in RV-mediated inhibition of L1-RTP. Overall, our findings suggest that RV directly modulates PPARα-mediated L1-RTP in somatic cells and that MAPK signaling interacts with SIRT1, SIRT6, and SIRT7 closely and may play a role in preventing human diseases such as cancer.

Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9

Background The autonomous retroelement Long Interspersed Element-1 (LINE-1) mobilizes though a copy and paste mechanism using an RNA intermediate (retrotransposition). Throughout human evolution, around 500,000 LINE-1 sequences have accumulated in the genome. Most of these sequences belong to ancestral LINE-1 subfamilies, including L1PA2-L1PA7, and can no longer mobilize. Only a small fraction of LINE-1 sequences, approximately 80 to 100 copies belonging to the L1Hs subfamily, are complete and still capable of retrotransposition. While silenced in most cells, many questions remain regarding LINE-1 dysregulation in cancer cells. Results Here, we optimized CRISPR Cas9 gRNAs to specifically target the regulatory sequence of the L1Hs 5’UTR promoter. We identified three gRNAs that were more specific to L1Hs, with limited binding to older LINE-1 sequences (L1PA2-L1PA7). We also adapted the C-BERST method (dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging) to identify LINE-1 transcriptional regulators in cancer cells. Our LINE-1 C-BERST screen revealed both known and novel LINE-1 transcriptional regulators, including CTCF, YY1 and DUSP1. Conclusion Our optimization and evaluation of gRNA specificity and application of the C-BERST method creates a tool for studying the regulatory mechanisms of LINE-1 in cancer. Further, we identified the dual specificity protein phosphatase, DUSP1, as a novel regulator of LINE-1 transcription.

Binding patterns of RNA-binding proteins to repeat-derived RNA sequences reveal putative functional RNA elements

Recent reports have revealed that repeat-derived sequences embedded in introns or long noncoding RNAs (lncRNAs) are targets of RNA-binding proteins (RBPs) and contribute to biological processes such as RNA splicing or transcriptional regulation. These findings suggest that repeat-derived RNAs are important as scaffolds of RBPs and functional elements. However, the overall functional sequences of the repeat-derived RNAs are not fully understood. Here, we show the putative functional repeat-derived RNAs by analyzing the binding patterns of RBPs based on ENCODE eCLIP data. We mapped all eCLIP reads to repeat sequences and observed that 10.75 % and 7.04 % of reads on average were enriched (at least 2-fold over control) in the repeats in K562 and HepG2 cells, respectively. Using these data, we predicted functional RNA elements on the sense and antisense strands of long interspersed element 1 (LINE1) sequences. Furthermore, we found several new sets of RBPs on fragments derived from other transposable element (TE) families. Some of these fragments show specific and stable secondary structures and are found to be inserted into the introns of genes or lncRNAs. These results suggest that the repeat-derived RNA sequences are strong candidates for the functional RNA elements of endogenous noncoding RNAs.

The Rhox gene cluster suppresses germline LINE1 transposition

Transposable elements (TEs) are mobile sequences that engender widespread mutations and thus are a major hazard that must be silenced. The most abundant active class of TEs in mammalian genomes is long interspersed element class 1 (LINE1). Here, we report that LINE1 transposition is suppressed in the male germline by transcription factors encoded by a rapidly evolving X-linked homeobox gene cluster. LINE1 transposition is repressed by many members of this RHOX transcription factor family, including those with different patterns of expression during spermatogenesis. One family member—RHOX10—suppresses LINE1 transposition during fetal development in vivo when the germline would otherwise be susceptible to LINE1 activation because of epigenetic reprogramming. We provide evidence that RHOX10 suppresses LINE transposition by inducing Piwil2, which encodes a key component in the Piwi-interacting RNA pathway that protects against TEs. The ability of RHOX transcription factors to suppress LINE1 is conserved in humans but is lost in RHOXF2 mutants from several infertile human patients, raising the possibility that loss of RHOXF2 causes human infertility by allowing uncontrolled LINE1 expression in the germline. Together, our results support a model in which the Rhox gene cluster is in an evolutionary arms race with TEs, resulting in expansion of the Rhox gene cluster to suppress TEs in different biological contexts.

Binding patterns of RNA binding proteins to repeat-derived RNA sequences reveal putative functional RNA elements

Recent reports have revealed that repeat-derived sequences embedded in introns or long noncoding RNAs (lncRNAs) are targets of RNA binding proteins (RBPs) and contribute to biological processes such as RNA splicing or transcriptional regulation. These findings suggest that repeat-derived RNAs are important as scaffolds of RBPs and functional elements. However, the overall functional sequences of the repeat-derived RNAs are not fully understood. Here, we show the putative functional repeat-derived RNAs by analyzing the binding patterns of RBPs based on ENCODE eCLIP data. We mapped all eCLIP reads to repeat sequences and observed that 10.75 % and 7.04 % of reads on average were enriched (at least 2-fold over control) in the repeats in K562 and HepG2 cells, respectively. Using these data, we predicted functional RNA elements on the sense and antisense strands of long interspersed element 1 (LINE1) sequences. Furthermore, we found several new sets of RBPs on fragments derived from other transposable element (TE) families. Some of these fragments show specific and stable secondary structures and are found to be inserted into the introns of genes or lncRNAs. These results suggest that the repeat-derived RNA sequences are strong candidates for the functional RNA elements of endogenous noncoding RNAs.

Hepatitis B virus polymerase restricts LINE-1 retrotransposition

Long interspersed element-1 (LINE-1, L1) retrotransposon composes about 17% of the human genome. However, genetic and biochemical interactions between L1 and hepatitis B virus (HBV) remain poorly understood. In this study, we found that HBV restricts L1 mobility without inhibiting the L1 promoter activity. Notably, HBV polymerase (Pol) strongly inhibited L1 retrotransposition in a reverse transcriptase (RT)-independent manner. Indeed, the ribonuclease H (RNase H) domain was essential for inhibition of L1 retrotransposition. L1 ORF1p RNA-binding protein predominantly localized into cytoplasmic RNA granule termed P-body. However, HBV Pol sequestered L1 ORF1p from P-body and colocalized with L1 ORF1p in cytoplasm, when both proteins were co-expressed. Altogether, HBV Pol seems to restrict L1 mobility through a sequestration of L1 ORF1p from P-body. Thus, these results suggest a novel function or activity of HBV Pol in regulation of L1 retrotransposition.