Full-size recombinant ORF1p-L1 and RT domain of ORF2p-L1: Protein expression, purification and characterization.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

BackgroundThe long interspersed nuclear element 1 (LINE1) retrotransposon has been identified as a specific substrate for fat mass and obesity-related gene (FTO), which facilitates the removal of N6-methyladenosine modifications from its targeted RNAs.MethodsThis study examined the dynamic interaction between FTO and LINE1 in yak tissues and muscle satellite cells, utilizing RT-qPCR, RNA immunoprecipitation (RIP), immunofluorescence staining, and techniques involving overexpression and interference of FTO and LINE1 to elucidate the relationship between FTO and LINE1 in yak tissues and muscle satellite cells.ResultsCloning and analysis of the FTO coding sequence in Jiulong yak revealed a conserved protein structure across various Bos breeds, with notable homology observed with domestic yak, domestic cattle, and Java bison. Comprehensive examination of FTO and LINE1 gene expression patterns in Jiulong yaks revealed consistent trends across tissues in both sexes. FTO mRNA levels were markedly elevated in the heart and kidney, while LINE1 RNA was predominantly expressed in the heart. Immunoprecipitation confirmed the direct interaction between the FTO protein and LINE1 RNA in yak tissues and muscle satellite cells. The FTO–LINE1 axis was confirmed by a significant decrease in LINE1 RNA enrichment following its expression interference in yak muscle satellite cells. Overexpression of FTO substantially reduced the expression of recombinant myogenic factor 5 (MYF5). However, FTO interference had no discernible effect on MYF5 and myoblast determination protein 1 (MYOD1) mRNA levels. Immunofluorescence analysis revealed no alterations in Ki-67 protein expression following FTO interference or overexpression. However, phalloidin staining demonstrated enhancement in the myotube fusion rate of yak muscle satellite cells upon LINE1 interference.ConclusionThis comprehensive mapping of the FTO and LINE1 mRNA expression patterns establishes a direct interaction between the FTO protein and LINE1 RNA in yak. The findings suggest that FTO overexpression promotes muscle satellite cells differentiation, whereas LINE1 negatively regulates myotube fusion. The study provides fundamental insights into the role of the FTO–LINE1 axis in determining the fate of muscle satellite cells in yak, laying a solid theoretical foundation for future investigations.

Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA transposons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Retrotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as "hot" LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3´ transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3´ transduction events stemming from a particular hot LINE-1.

Long interspersed nuclear element-1 (LINE-1) is the only active autonomous transposon in the human genome. Its transposition frequently induces host genome instability, leading to a variety of genetic diseases, including cancers. The host factors play important roles in inhibiting LINE-1 retrotransposition. As an important component of the immune system, the host factor SLFN14 has antiviral activity. Our laboratory shows that SLFN14 possesses potent inhibitory activity against LINE-1 retrotransposition. To explore the potential mechanism of SLFN14 inhibition, we analyzed its effects on transcription, translation, reverse transcription and insertion in the LINE-1 replication cycle. We confirmed that SLFN14 could suppress the LINE-1 mRNA level by affecting its transcription and degradation, thereby diminishing the protein and cDNA levels of LINE-1, which eventually block the LINE-1 retrotransposition. Further, by mapping the active domains of SLFN14, we found its inhibitory activity on LINE-1 being closely related to its endoribonuclease and ribosome binding domains. These results demonstrate the mechanism of SLFN14 in regulating LINE-1 replication, which further provide new insights for improving the regulation network of host factors for controlling genomic instability caused by LINE-1 replication.

OBJECTIVETo investigate whether Sjögren’s syndrome (SS) with anti-HTLV-I antibodies is aetiopathologically distinguishable from SS without these antibodies, the study compared prevalence of autoantibodies in serum samples of SS patients with...

The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.

Long Interspersed Nuclear Elements 1 (LINE1): The chimeric transcript L1-MET and its involvement in cancer

IntroductionThe long interspersed nuclear element-1 (LINE1) gene is a retrotransposon whose methylation status appears to play a role in spermatogenesis, the outcome of assisted reproductive techniques (ART), and even in recurrent pregnancy loss (RPL). Advanced paternal age appears associated with altered sperm parameters, RPL, poor ART outcomes, and compromised offspring health. The methylation status of LINE1 has been reported to be affected by age. The latest meta-analysis on the LINE1 methylation pattern in spermatozoa found no significant differences in methylation levels between infertile patients and fertile controls. However, to the best of our knowledge, no updated meta-analysis on this topic has been published recently. Furthermore, no comprehensive meta-regression analysis was performed to investigate the association between sperm LINE1 methylation pattern and age.ObjectivesTo provide an updated and comprehensive systematic review and meta-analysis on sperm LINE1 gene methylation degree in patients with abnormal sperm parameters compared to men with normal sperm parameters and to probe the association between sperm LINE1 methylation status and age and/or sperm concentration.MethodsThis meta-analysis was registered in PROSPERO (registration n. CRD42023397056). It was performed according to the MOOSE guidelines for Meta-analyses and Systematic Reviews of Observational Studies and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P). Only original articles evaluating LINE1 gene methylation in spermatozoa from patients with infertility or abnormalities in one or more sperm parameters compared to fertile or normozoospermic men were included.ResultsOf 192 abstracts evaluated for eligibility, only 5 studies were included in the quantitative synthesis, involving a total of 340 patients and 150 controls. Our analysis showed no significant difference in LINE1 gene methylation degree in patients with infertility and/or abnormal sperm parameters compared to fertile controls and/or men with normal sperm parameters, although there was significant heterogeneity across studies. No significant evidence of publication bias was found, and no study was sensitive enough to alter the results. In meta-regression analysis, we found that the results were independent of both ages and sperm concentration. A sub-analysis examining patients and controls separately was also conducted and we found a trend for a positive correlation between LINE1 methylation and sperm concentration in the control group only.ConclusionsThe results of this systematic review and meta-analysis do not suggest a determining role of sperm LINE1 gene methylation degree in patients with infertility and/or abnormal sperm parameters. Therefore, we do not suggest including LINE1 in the genetic panel of prospective studies aimed at identifying the most representative and cost-effective genes to be analyzed in couples undergoing ART cycles.

Long interspersed nuclear element-1 (LINE-1) methylation status, representing global DNA methylation levels, is associated with patient prognosis in several types of cancer. This study was designed to examine the prognostic significance of LINE-1 methylation in patients with hepatocellular carcinoma (HCC) and the possible mechanisms related to oncogene activation. Seventy-five HCC patients who underwent hepatectomy between 2006 and 2012 were enrolled in this study. Quantitative pyrosequencing was performed to quantify the methylation level of three CpG sites in the LINE-1 promoter. Clinicopathological variables and prognosis were compared between LINE-1 hypo- and hypermethylation groups. LINE-1-inserted c-MET (L1-MET) gene expression and its correlation with LINE-1 methylation levels also were analyzed. LINE-1 was significantly hypomethylated in tumor tissues compared with nontumor tissues (48.3 ± 12.2 % vs. 68.2 ± 2.0 %, respectively, p < 0.0001). LINE-1 hypomethylation was not associated with any clinicopathological factors in HCC patients, except sex (p < 0.05). However, patients with LINE-1 hypomethylation exhibited significantly poorer outcome, and multivariate analysis revealed that LINE-1 hypomethylation was an independent risk factor for overall survival (hazard ratio (HR) = 6.1, p = 0.031) and disease-free survival (HR = 2.34, p = 0.045). L1-MET expression was significantly higher in tumor tissues (p < 0.01). L1-MET expression levels were inversely correlated with LINE-1 methylation levels, and positively correlated with c-MET expression (p < 0.05). Furthermore, higher c-MET protein expression was observed in the LINE-1 hypomethylated tumor tissues compared with hypermethylated tumor tissues (p = 0.032). LINE-1 hypomethylation is significantly associated with poor prognosis in patients with HCC, possibly due to activation of c-MET expression.

AhR ligands reactivate LINE-1 retrotransposon in triple-negative breast cancer cells MDA-MB-231 and non-tumorigenic mammary epithelial cells NMuMG

Vascular calcification (VC) is a critical vascular pathological event, contributing to the rise in both the prevalence and fatality of cardiovascular diseases. However, the lack of effective therapeutic strategies for VC is attributed primarily to the incomplete understanding of its underlying molecular mechanisms. In this study, we discovered that long interspersed nuclear element 1 (LINE1) was significantly upregulated in the calcified arteries of both human individuals and mouse models. Mechanistically, silencing LINE1 expression or inhibiting its activity with adding nucleoside reverse transcriptase inhibitors (NRTIs, a class of validated LINE1 inhibitors) effectively prevented the osteogenic reprogramming of vascular smooth muscle cells (VSMCs). Moreover, NRTIs treatment substantially mitigated VC in chronic kidney disease (CKD)-induced and vitamin D3-overloaded VC mouse models. RNA sequencing analysis revealed that LINE1 depletion (via small interfering RNA) or NRTIs intervention downregulated the cGAS-STING signaling pathway and its associated inflammatory genes in VSMCs. Functional validation revealed that stimulation of the cGAS‒STING pathway exacerbated VC, whereas its pharmacological inhibition alleviated VC. Notably, we identified LINE1-derived cDNA as a direct activator of the cGAS‒STING pathway, demonstrating that LINE1 inhibition suppresses VC by blocking cGAS‒STING activation and subsequent inflammatory responses. Clinically, a cross-sectional study involving 1,785 participants revealed that patients receiving NRTIs therapy presented a significantly lower incidence of VC and reduced calcification scores. Multivariate logistic regression analysis further confirmed that NRTIs use is an independent protective factor against VC incidence and progression. Collectively, these findings establish LINE1 as promising therapeutic targets for VC and highlight NRTIs as potential candidates for developing novel strategies against VC.

LINE-1 specific nuclear organization in mice olfactory sensory neurons

A large fraction of eukaryotic genomes is made up of long interspersed nuclear elements (LINEs). Due to their capability to create novel copies via error-prone reverse transcription, they generate multiple families and reach high copy numbers. Although mammalian LINEs have been well described, plant LINEs have been only poorly investigated. Here, we present a systematic cross-species survey of LINEs in higher plant genomes shedding light on plant LINE evolution as well as diversity, and facilitating their annotation in genome projects. Applying a Hidden Markov Model (HMM)-based analysis, 59 390 intact LINE reverse transcriptases (RTs) were extracted from 23 plant genomes. These fall in only two out of 28 LINE clades (L1 and RTE) known in eukaryotes. While plant RTE LINEs are highly homogenous and mostly constitute only a single family per genome, plant L1 LINEs are extremely diverse and form numerous families. Despite their heterogeneity, all members across the 23 species fall into only seven L1 subclades, some of them defined here. Exemplarily focusing on the L1 LINEs of a basal reference plant genome (Beta vulgaris), we show that the subclade classification level does not only reflect RT sequence similarity, but also mirrors structural aspects of complete LINE retrotransposons, like element size, position and type of encoded enzymatic domains. Our comprehensive catalogue of plant LINE RTs serves the classification of highly diverse plant LINEs, while the provided subclade-specific HMMs facilitate their annotation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens formed from the incomplete combustion of organic materials. Exposure to PAHs can be linked to at least 9 different cancer types, including lung and breast cancer. PAH are genotoxic and can form DNA adducts which can either be repaired or lead to mutations. DNA repair can lead to aberrant DNA methylation, however, few studies have investigated the link between PAH exposure and DNA methylation modifications. Here, we have used a mouse model to investigate the epigenetic consequences of PAH exposure. Mice (n=3/group) were treated by oral gavage with 0, 25, 50 and 75 mg/kg bodyweight/day of benzo[a]pyrene (BaP) for 28 days. Lung DNA from these mice was used to prepare Reduced Representation Bisulphite Sequencing (RRBS) libraries which were then sequenced using Illumina HiSeq2500to an average depth of 25x. For each mouse, DNA methylation was averaged over 500 bp tiled windows and differentially methylated regions (DMRs) were identified by comparing the controls (untreated) with all BaP-exposed mice (treated) irrespective of dose. In the treated vs untreated we identified 1815 windows (&amp;gt;25% methylation difference, p&amp;lt;0.05). Of these, we found that DMRs were under-represented in 5' untranslated regions (UTR), promoter, long interspersed nuclear elements (LINEs) regions and CpG islands compared with the genomic distribution of windows. DMRs were found to occur significantly more frequently in transcription termination regions. In addition, we found significantly more hypomethylation events in exon, intergenic, intronic and promoter regions, long terminal repeats (LTR) and short interspersed nuclear elements (SINE) and more hypermethylation in LINEs. Pyrosequencing validation on a selection of these DMRs is ongoing, using DNA from the same mice used in RRBS, plus mice exposed to BaP and other PAHs in lung and other tissues. In summary, these results show that PAHs have an effect on DNA methylation, and that these changes happen mostly within the gene body or intergenically, with significantly less differences observed at traditional gene expression regulators such as promoters. This implies that the DNA methylation changes effected by BaP exposure may serve another purpose other than gene expression regulation. Citation Format: Francesca Galea, Paul A. White, Volker M. Arlt, Paolo Vineis, James M. Flanagan. The epigenetic effects of benzo[a]pyrene exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4327.