LINE-1 Promoter Research Articles

Impact of prematurity on LINE-1 promoter methylation.

Aim: Promoter methylation of LINE-1 may be affected by prematurity, but there is little evidence in the literature.Materials & methods: Blood from premature and full-term neonates on days 0, 5, 30 and 90 was analyzed for DNA methylation percentage in a promoter region of the LINE-1, after bisulfite conversion and pyrosequencing.Results: Premature infants, as a whole, showed significantly lower methylation percentage at birth, but this difference diminished over time. However, the subgroup of extremely premature (<28weeks gestational age) had higher methylation percentages, similar to full-term newborns.Conclusion: This research underscores the critical role of prematurity on the methylation pattern of LINE-1. These findings underline the complexity of epigenetic regulation in prematurity and emphasize the need for further studies.

Two-factor authentication underpins the precision of the piRNA pathway

The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice1. piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. 2–5). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. 6). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1–SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation.

LINE-1 retrotransposons contribute to mouse PV interneuron development

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

Abstract LB226: Deciphering lung adenocarcinoma evolution and the role of LINE-1 retrotransposition

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide. Understanding lung cancer evolutionary dynamics can help identify tools to intercept its growth and suggest strategies for treatment. Multiple factors can impact the tumors’ natural history and distinctly affect growth rate. However, research on the evolutionary trajectories of lung cancer across demographic or exposure scenarios remains inadequate. Additionally, the roles of mutational processes and complex genomic alterations on the evolution of lung cancer are still largely unexplored. In the largest genomic study of lung cancer to date, we analyzed deep whole-genome sequencing (~ 81x) and other omics data of 1217 lung cancers from the Sherlock-Lung study. To ensure adequate statistical power for identifying subclone architectures and constructing lung cancer evolutionary histories, we utilized a metric known as NRPCC (number of reads per tumor chromosomal copy) to select 542 lung adenocarcinoma (LUAD) samples for clonal evolution analyses, including 186 and 181 samples from never-smoker subjects of European and Asian ancestry, respectively, and 121 samples from smokers of European ancestry. We found that major driver genes and exogenous mutations contribute to tumor initiation, while copy number gains and endogenous processes appear later in tumor evolution. Tumors harboring EGFR mutations in never-smoker females of European descent show long latency, while tumors with KRAS mutations have shorter latency regardless of ancestry and sex. Notably, tumors harboring the mutational signature ID2 have short latency and aggressive phenotype, accompanied by increased genomic instability, elevated hypoxia scores, high CpG methylator phenotype, low neoantigen burden, and propensity to develop metastasis. We show that LINE-1 retrotransposition-induced mutagenesis contributes to the origin of ID2 mutations. The transcriptional factor ZNF695, a member of the KZFP family, up-regulated in LUAD, appears to contribute to LINE-1 retrotransposition through a dominant-negative effect and LINE-1 promoter demethylation. In a multivariate analysis of genomics, exposures and demographic factors, LUAD latency was most significantly associated with ID2, followed by EGFR mutations, KRAS mutations, and sex, suggesting an independent impact of these factors on LUAD evolution. Our findings underscore the complex interplay of ancestry, sex, exogenous mutagenesis, epigenetic regulation, and LINE-1 retrotransposition in shaping LUAD evolutionary trajectories, paving the way for potential targeted therapeutic interventions. Citation Format: Tongwu Zhang, Wei Zhao, Christopher Wirth, Marcos Díaz-Gay, Jinhu Yin, Phuc H. Hoang, Jian Sang, John McElderry, Alyssa Klein, Azhar Khandekar, Caleb Hartman, Jennifer Rosenbaum, Frank Colon-Matos, Kristine M. Jones, Neil E. Caporaso, Robert Homer, Angela C. Pesatori, Dario Consonni, Lixing Yang, Bin Zhu, Jianxin Shi, Kevin Brown, Nathaniel Rothman, Stephen J. Chanock, Ludmil B. Alexandrov, Jiyeon Choi, Maurizio Cardelli, Qing Lan, Martin A. Nowak, David C. Wedge, Maria Teresa Landi. Deciphering lung adenocarcinoma evolution and the role of LINE-1 retrotransposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB226.

Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G.

Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered 'fingerprints of parent cells', which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30-150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.

Abstract B012: Transcriptional activation of mouse LINE-1 promoter by transcription factor YY1

Abstract Long interspersed element type 1 (LINE-1) are highly abundant non-LTR retrotransposons in mammalian genomes. LINE-1 hypomethylation and overexpression has been considered as a hallmark of many human cancers. Moreover, active movement of LINE-1 results in insertional mutagenesis and may contribute to cancer initiation or progression. Transcription is the initial and critical regulatory step for active LINE-1 movement. Therefore, investigating LINE-1 transcriptional regulation is important for our understanding of LINE-1’s role in cancer. In human LINE-1, an intact YY1 transcription factor binding motif is required for accurate transcription initiation. Two subfamilies of mouse LINE-1, Tf_I and Gf_I, have predicted YY1 binding motifs in their promoter region but their role in mouse LINE-1 transcription has not been characterized. In this study, we aim to investigate the role of these putative YY1 motifs in Tf_I and Gf_I subfamilies. Promoter sequences carrying the wild-type or mutant YY1 motifs were cloned into a dual-luciferase reporter system. Reporter assays showed transcription inactivation upon mutation of the YY1 motif for the Tf_I subfamily. The electrophoretic mobility shift assay (EMSA) demonstrated specific interaction of the Tf_I promoter sequence with the YY1 protein. Knockdown of YY1 protein level in cells reduced Tf_I promoter activity by 2-fold compared to the wild-type control. These results indicate that YY1 is a transcriptional activator of mouse LINE-1 Tf_I subfamily. On the other hand, our data suggest that the predicted YY1 binding site in the promoter of Gf_I subfamily is not functional due to one nucleotide deviation from the consensus YY1 motif. Indeed, restoring it to the conserved YY1 motif sequence enhanced the promoter activity by 6-fold. In EMSA, YY1 protein did not interact with the putative YY1 motif of the Gf_I subfamily but did so when the motif was restored to consensus YY1 sequence. Findings from our study increase our understanding of the transcriptional regulation of different mouse LINE-1 subfamilies, highlight the differing roles of YY1 in regulating human and mouse L1 transcription, and have important implications in the role of LINE-1s during cancer development. Citation Format: Karani Saha, Grace Nielsen, Wenfeng An. Transcriptional activation of mouse LINE-1 promoter by transcription factor YY1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B012.

A frequent SNP in TRIM5α strongly enhances the innate immune response against LINE-1 elements.

The intracellular restriction factor TRIM5α inhibits endogenous LINE-1 retroelements. It induces innate immune signaling cascades upon sensing of cytoplasmic LINE-1 complexes, thereby underlining its importance for protecting the human genome from harmful retrotransposition events. Here, we show that a frequent SNP within the RING domain of TRIM5α, resulting in the variant H43Y, blocks LINE-1 retrotransposition with higher efficiency compared to TRIM5α WT. Upon sensing of LINE-1 complexes in the cytoplasm, TRIM5α H43Y activates both NF-κB and AP-1 signaling pathways more potently than TRIM5α WT, triggering a strong block of the LINE-1 promoter. Interestingly, the H43Y allele lost its antiviral function suggesting that its enhanced activity against endogenous LINE-1 elements is the driving force behind its maintenance within the population. Thus, our study suggests that the H43Y variant of the restriction factor and sensor TRIM5α persists within the human population since it preserves our genome from uncontrolled LINE-1 retrotransposition with higher efficiency.

Abstract 1005: LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Abstract Background: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients, and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. Results: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival - OS: 28.1% for LINE-1 methylation&amp;lt;35% versus 69.1% for ≥55%; p&amp;lt;0.0001). When LINE-1 methylation was dichotomized as &amp;lt;55% vs. ≥55%, interaction with HPV16 emerged: compared with hypermethylated HPV-16 positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR: 4.83, 95% CI: 2.24-10.38) and progression (HR: 4.54, 95% CI: 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53≥50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53≥50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. Conclusions: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations, and lead to altered gene expression in OPSCC. Citation Format: Mariateresa Casarotto, Valentina Lupato, Giorgio Giurato, Roberto Guerrieri, Sandro Sulfaro, Annamaria Salvati, Elisa D'Angelo, Carlo Furlan, Anna Menegaldo, Lorena Baboci, Barbara Montico, Irene Turturici, Riccardo Dolcetti, Salvatore Romeo, Vittorio Baggio, Stefania Corrado, Gianluca Businello, Maria Guido, Alessandro Weisz, Vittorio Giacomarra, Giovanni Franchin, Agostino Steffan, Luca Sigalotti, Emanuela Vaccher, Paolo Boscolo-Rizzo, Jerry Polesel, Giuseppe Fanetti, Elisabetta Fratta. LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1005.

Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy.

The placenta has a unique hypomethylated genome. Due to this feature of the placenta, there is a potential possibility of using regulatory elements derived from retroviruses and retrotransposons, which are suppressed by DNA methylation in the adult body. In addition, there is an abnormal increase in the level of methylation of the LINE-1 retrotransposon in the chorionic trophoblast in spontaneous abortions with both normal karyotype and aneuploidy on different chromosomes, which may be associated with impaired gene transcription using LINE-1 regulatory elements. To date, 988 genes that can be expressed from alternative LINE-1 promoters have been identified. Using the STRING tool, genes (NUP153 and YWHAB) were selected, the products of which have significant functional relationships with proteins highly expressed in the placenta and involved in trophoblast differentiation. This study aimed to analyze the expression of the NUP153 and YWHAB genes, highly active in the placenta, from canonical and alternative LINE-1 promoters in the germinal part of the placenta of spontaneous and induced abortions. Gene expression analysis was performed using real-time PCR in chorionic villi and extraembryonic mesoderm of induced abortions (n = 10), adult lymphocytes (n = 10), spontaneous abortions with normal karyotype (n = 10), and with the most frequent aneuploidies in the first trimester of pregnancy (trisomy 16 (n = 8) and monosomy X (n = 6)). The LINE-1 methylation index was assessed in the chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. The level of expression of both genes from canonical promoters was higher in blood lymphocytes than in placental tissues (p < 0.05). However, the expression level of the NUP153 gene from the alternative LINE-1 promoter was 17 times higher in chorionic villi and 23 times higher in extraembryonic mesoderm than in lymphocytes (p < 0.05). The expression level of NUP153 and YWHAB from canonical promoters was higher in the group of spontaneous abortions with monosomy X compared to all other groups (p <0.05). The LINE-1 methylation index negatively correlated with the level of gene expression from both canonical (NUP153 - R = -0.59, YWHAB - R = -0.52, p < 0.05) and alternative LINE-1 promoters (NUP153 - R = -0.46, YWHAB - R = -0.66, p < 0.05). Thus, the observed increase in the LINE-1 methylation index in the placenta of spontaneous abortions is associated with the level of expression of the NUP153 and YWHAB genes not only from alternative but also from canonical promoters, which can subsequently lead to negative consequences for normal embryogenesis.

LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Background and purposeCurrently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation.ResultsIn this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival—OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24–10.38) and progression (HR 4.54, 95% CI 2.18–9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine–phosphate–guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences.ConclusionsEvaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Associations between DNA methylation and genotoxicity among lead-exposed workers in China

Studies have shown that lead (Pb) exposure caused genotoxicity, however, the underlying mechanisms remain unclear. A mechanism may be via DNA methylation which is one of the most widely studied epigenetic regulations for cellular activities. Whether this is involved in Pb-induced genotoxicity has rarely been studied. Our study aimed to examine whether DNA methylation was associated with Pb exposure and genotoxicity, and to explore its potential mediating roles. A total of 250 Pb-exposed workers were enrolled. Blood lead levels (BLLs) and genotoxic biomarkers (Micronuclei and Comet) were analyzed. Methylation levels at CpG sites of LINE1 and Alu and promoter region of P53, BRCA1, TRIM36 and OGG1 were measured by pyrosequencing. Generalized linear model (GLM) combined with restricted cubic splines (RCS) were used to analyze relationships between Pb exposure, DNA methylation and genotoxicity. Mediation effect was used to explore mediating roles of DNA methylation. The distribution of BLLs was right-skewed and showed wide ranges from 23.7 to 636.2 μg/L with median (P25, P75) being 218.4 (106.1, 313.9) μg/L among all workers. Micronuclei frequencies showed Poisson distribution [1.94 ± 1.88‰] and Comet tail intensity showed normal distribution [1.69 ± 0.93%]. GLM combined with RCS showed that Alu methylation was negatively associated with BLLs, while P53 and OGG1 methylation were positively associated with BLLs. Micronuclei were negatively associated with Alu and TRIM36 methylation but positively with P53 methylation. Comet was positively associated with P53 and BRCA1 methylation. Mediation effect showed that Alu methylation mediated 7% effects on association between Pb exposure and micronuclei, whereas, P53 methylation mediated 14% and BRCA1 mediated 9% effects on association between Pb exposure and Comet. Our data show that Pb exposure induced changes of global and gene-specific DNA methylation which mediated Pb-induced genotoxicity.

Abstract 1462: Oncogenic activation of FOXR2 driven by somatic acquisition of a LINE-1 promoter in pediatric high-grade glioma

Abstract Long Interspersed Element-1 (LINE-1 or L1) is the only autonomously active mobile genetic element in the human genome. It has been shown to mobilize (i.e., retrotranspose) in cancers, causing de novo somatic L1 insertions. Somatic L1 retrotransposition can cause aberrant splicing of tumor suppressor genes by utilizing cryptic splice donor (SD) sites such as the nucleotide 97 SD in the L1 5’UTR or disrupting repressive regulatory regions of proto-oncogenes, inducing aberrant activation, leading to cancer. In contrast to these reported mechanisms, here we report the first evidence of a driver oncogene activation due to somatic acquisition of an L1 promoter in a pediatric high-grade glioma (HGG). While investigating activation of FOXR2 (forkhead box R2), a known oncogene in CNS embryonal tumors, in a recurrent pediatric diffuse HGG that showed a methylation profile of CNS FOXR2-activated tumors, we identified overexpression of a non-canonical FOXR2 isoform. Transcription of FOXR2 initiated from ~11kb upstream of exon 2 at a site harboring a somatic structural variation (SV) reminiscent of an L1 insertion. Targeted PCR amplification and subsequent PacBio sequencing revealed a 5’ inverted/deleted L1 insertion (~3kb sequence) containing an intact L1 5’UTR, presence of a 3’ transduction sequence, two poly-A tails, and target site duplications. The 3’ transduction sequence matches the sequence downstream an intact polymorphic full-length L1 element at 6p24.1, the likely source L1 element that initiated the FOXR2 somatic insertion. Strikingly, tumor RNA-seq data reveals FOXR2 transcription initiated from the intact sense pol-II promoter of the inserted L1 5’UTR, generating a fusion transcript joining the L1 5’UTR 97 SD to a canonical splice acceptor within exon 2 of FOXR2. Targeted bisulfite sequencing of the FOXR2 L1 5’UTR revealed a hypomethylated state, further supporting its role in FOXR2 transcription initiation. FOXR2 activation is likely a tumor initiating event as the following was observed in the primary tumor that occurred two years prior: a matching recurrent tumor methylation profile, the presence of L1/FOXR2 fusion transcripts, and overexpression of FOXR2. Additional driver mutations, clonal TP53 R175H mutation and PDGFRA amplification, identified in the recurrent tumor but absent in the primary tumor, support the somatic L1 insertion as a tumor initiating event occurring prior to later acquired alterations. Our data represent the first example of oncogenic activation by L1 “promoter donation” via somatic retrotransposition as a novel tumor initiating mechanism. Future large-scale research and development of new computational methods for detecting rearranged L1 insertions are required to determine the prevalence of L1 “promoter donation” in cancer or other diseases. Citation Format: Xiaolong Chen, Diane A. Flasch, Bensheng Ju, Heather L. Mulder, John Easton, Lu Wang, Suzanne J. Baker, Jason Chiang, Jinghui Zhang. Oncogenic activation of FOXR2 driven by somatic acquisition of a LINE-1 promoter in pediatric high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1462.

RNA sensor MDA5 suppresses LINE-1 retrotransposition by regulating the promoter activity of LINE-1 5\u2032-UTR

BackgroundType 1 long interspersed elements, or LINE-1, are the only retroelements that replicate autonomously in human cells. The retrotransposition process of LINE-1 can trigger the activation of the innate immune system and has been proposed to play a role in the development of several autoimmune diseases, including Aicardi-Goutières syndrome (AGS). In contrast, all known AGS-associated proteins, except MDA5, have been reported to affect LINE-1 activity. Thus, MDA5 is likely to also function as a LINE-1 suppressor.ResultsMDA5 was found to potently suppress LINE-1 activity in a reporter-based LINE-1 retrotransposition assay. Although MDA5 is an endogenous RNA sensor able to activate the innate immune system, increased interferon (IFN) expression only contributed in part to MDA5-mediated LINE-1 suppression. Instead, MDA5 potently regulated the promoter activity of LINE-1 5′-UTR, as confirmed by transiently expressed myc-tagged MDA5 or knockdown of endogenous MDA5 expression. Consequently, MDA5 effectively reduced the generation of LINE-1 RNA and the subsequent expression of LINE-1 ORF1p and ORF2p. Interestingly, despite MDA5 being a multi-domain protein, the N-terminal 2CARD domain alone is sufficient to interact with LINE-1 5′-UTR and inhibit LINE-1 promoter activity.ConclusionOur data reveal that MDA5 functions as a promoter regulator; it directly binds to the LINE-1 5′-UTR and suppresses its promoter activity. Consequently, MDA5 reduces LINE-1 RNA and protein levels, and ultimately inhibits LINE-1 retrotransposition. In contrast, MDA5-induced IFN expression only plays a mild role in MDA5-mediated LINE-1 suppression. In addition, the N-terminal 2CARD domain was found to be a functional region for MDA5 upon inhibition of LINE-1 replication. Thus, our data suggest that besides being an initiator of the innate immune system, MDA5 is also an effector against LINE-1 activity, potentially forming a feedback loop by suppressing LINE-1-induced innate immune activation.

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation.

DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR)genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1Gy (low) and 2.0Gy (high) doses of γ-radiation and proliferated for 48h and 72h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM)method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72h. At 2.0Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48h and 72h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.

Line-1 methylation in different vessels, vascular and blood cells in human atherosclerosis.

Background and Aims: Decreased LINE-1 methylation was associated with cardiovascular diseases. However, LINE-1 methylation in vascular cells is poorly investigated in atherosclerosis. Here we studied DNA methylation of LINE-1 promoter in human carotid atherosclerotic plaque, intact artery, and vein, as well as in vascular smooth muscle cells (VSMC) and macrophages (MPH) isolated from plaques, and in blood cells.

In utero exposure to chlordecone affects histone modifications and activates LINE-1 in cord blood.

Environmental factors can induce detrimental consequences into adulthood life. In this study, we examined the epigenetic effects induced by in utero chlordecone (CD) exposure on human male cord blood as well as in blood-derived Ke-37 cell line. Genome-wide analysis of histone H3K4me3 distribution revealed that genes related to chromosome segregation, chromatin organization, and cell cycle have altered occupancy in their promoters. The affected regions were enriched in ESR1, SP family, and IKZF1 binding motifs. We also observed a global reduction in H3K9me3, markedly in repeated sequences of the genome. Decrease in H3K9me3 after CD exposure correlates with decreased methylation in LINE-1 promoters and telomere length extension. These observations on human cord blood were assessed in the Ke-37 human cell line. H3K4me3 and the expression of genes related to immune response, DNA repair, and chromatin organization, which were affected in human cord blood were also altered in CD-exposed Ke-37 cells. Our data suggest that developmental exposure to CD leads to profound changes in histone modification patterns and affects the processes controlled by them in human cord blood.

Early maternal care restores LINE-1 methylation and enhances neurodevelopment in preterm infants

BackgroundPreterm birth affects almost 9–11% of newborns and is one of the leading causes of childhood neurodevelopmental disabilities; the underlying molecular networks are poorly defined. In neurons, retrotransposons LINE-1 (L1) are an active source of genomic mosaicism that is deregulated in several neurological disorders; early life experience has been shown to regulate L1 activity in mice.MethodsVery preterm infants were randomized to receive standard care or early intervention. L1 methylation was measured at birth and at hospital discharge. At 12 and 36 months, infants’ neurodevelopment was evaluated with the Griffiths Scales. L1 methylation and CNVs were measured in mouse brain areas at embryonic and postnatal stages.ResultsHere we report that L1 promoter is hypomethylated in preterm infants at birth and that an early intervention program, based on enhanced maternal care and positive multisensory stimulation, restores L1 methylation levels comparable to healthy newborns and ameliorates neurodevelopment in childhood. We further show that L1 activity is fine-tuned in the perinatal mouse brain, suggesting a sensitive and vulnerable window for the L1 epigenetic setting.ConclusionsOur results open the field on the inspection of L1 activity as a novel molecular and predictive approach to infants’ prematurity-related neurodevelopmental outcomes.Trial registrationClinicalTrial.gov (NCT02983513). Registered on 6 December 2016, retrospectively registered.

Method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter

The methylation index of the LINE-1 promoter is one of the most commonly used markers for assessing the global level of genome methylation in various human cells and tissues. We developed an NGS-based protocol for DNA methylation analysis of the LINE-1 retrotransposon promoter. This approach allows assessment of the DNA methylation index of 19 CpG sites in the LINE-1 promoter that have the highest tissue- or tumor-specific variability. The method provides a DNA methylation profile for analyzing either the methylation index of each CpG site independently or the mean DNA methylation index across the LINE-1 promoter. The results obtained using the developed method corresponded well to the level of methylation assessed using a commercially available kit for DNA pyrosequencing. In addition, our method provides much more information: 1) the DNA methylation profile of a significant part of the LINE-1 promoter and 2) the level of DNA methylation at individual LINE-1 loci in the genome. The method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter can be used in large-scale studies of the global level of genome methylation in normal human cells or tumors.To accomplish this, we modified the targeted massive parallel sequencing method based on 16S Metagenomic Sequencing Library Preparation protocol (Illumina, USA) by:•Introduction of the stage of bisulfite conversion of DNA.•Development of specific primers for the LINE-1 sequence.

P53 directly represses human LINE1 transposons

p53 is a potent tumor suppressor and commonly mutated in human cancers. Recently, we demonstrated that p53 genes act to restrict retrotransposons in germline tissues of flies and fish but whether this activity is conserved in somatic human cells is not known. Here we show that p53 constitutively restrains human LINE1s by cooperatively engaging sites in the 5'UTR and stimulating local deposition of repressive histone marks at these transposons. Consistent with this, the elimination of p53 or the removal of corresponding binding sites in LINE1s, prompted these retroelements to become hyperactive. Concurrently, p53 loss instigated chromosomal rearrangements linked to LINE sequences and also provoked inflammatory programs that were dependent on reverse transcriptase produced from LINE1s. Taken together, our observations establish that p53 continuously operates at the LINE1 promoter to restrict autonomous copies of these mobile elements in human cells. Our results further suggest that constitutive restriction of these retroelements may help to explain tumor suppression encoded by p53, since erupting LINE1s produced acute oncogenic threats when p53 was absent.

Promoter methylation, transcription, and retrotransposition of LINE-1 in colorectal adenomas and adenocarcinomas

BackgroundThe methylation of the CpG islands of the LINE-1 promoter is a tight control mechanism on the function of mobile elements. However, simultaneous quantification of promoter methylation and transcription of LINE-1 has not been performed in progressive stages of colorectal cancer. In addition, the insertion of mobile elements in the genome of advanced adenoma stage, a precancerous stage before colorectal carcinoma has not been emphasized. In this study, we quantify promoter methylation and transcripts of LINE-1 in three stages of colorectal non-advanced adenoma, advanced adenoma, and adenocarcinoma. In addition, we analyze the insertion of LINE-1, Alu, and SVA elements in the genome of patient tumors with colorectal advanced adenomas.MethodsLINE-1 hypomethylation status was evaluated by absolute quantitative analysis of methylated alleles (AQAMA) assay. To quantify the level of transcripts for LINE-1, quantitative RT-PCR was performed. To find mobile element insertions, the advanced adenoma tissue samples were subjected to whole genome sequencing and MELT analysis.ResultsWe found that the LINE-1 promoter methylation in advanced adenoma and adenocarcinoma was significantly lower than that in non-advanced adenomas. Accordingly, the copy number of LINE-1 transcripts in advanced adenoma was significantly higher than that in non-advanced adenomas, and in adenocarcinomas was significantly higher than that in the advanced adenomas. Whole-genome sequencing analysis of colorectal advanced adenomas revealed that at this stage polymorphic insertions of LINE-1, Alu, and SVA comprise approximately 16%, 51%, and 74% of total insertions, respectively.ConclusionsOur correlative analysis showing a decreased methylation of LINE-1 promoter accompanied by the higher level of LINE-1 transcription, and polymorphic genomic insertions in advanced adenoma, suggests that the early and advanced polyp stages may host very important pathogenic processes concluding to cancer.