LINE-1 retrotransposons: Multifaceted functions and regulatory mechanisms in health and disease

Abstract

Long interspersed nuclear element-1 (LINE-1), a prominent class of retrotransposons, is abundantly distributed throughout mammalian genomes and plays critical roles in development, aging, and disease. In this review, we provide a comprehensive overview of the multifaceted functions of LINE-1, with a focus on its regulatory impact on gene transcription and cellular responses across the DNA, RNA, and protein levels. We highlight recent advances in understanding the transcriptional and epigenetic mechanisms controlling LINE-1 transcription. Furthermore, we explore the current challenges and future opportunities in LINE-1 research, emphasizing its potential as a novel therapeutic target. Continued investigation into LINE-1 biology holds promise for deepening our understanding of human health and disease.

Graphical abstract

This review summarizes the physiological and pathological duality of LINE-1, outlining its transcriptional and epigenetic regulation and how LINE-1 DNA, RNA, and proteins influence development and tissue repair while driving genomic instability and inflammation in cancer and aging, highlighting LINE-1 as a promising biomarker and therapeutic target.

Introduction

Transposable elements (TEs), commonly known as jumping genes, are highly repetitive and autonomously migrating DNA sequences first discovered by Barbara McClintock in the 1940s.¹ Studies have shown that their existence not only lays the foundation for the structural diversity of the genome but also has an important impact on the regulation of gene expression.²

Transposons are mainly divided into two types: type 1, also known as retrotransposons, uses a "copy-and-paste" mechanism. They are first transcribed into RNA, then reverse transcribed into DNA, which integrates into a new genomic location. This group includes long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). Type 2 adopts the mechanism of direct translocation of DNA by means of the "cut-and-paste" method.³

Approximately 17% of the human genome consists of LINE-1. LINE-1 is a type of transposon that can replicate itself and insert into the host genome. LINE-1 transposons exert a regulatory influence on the general functionality of the genome by affecting chromatin structure and gene connections.^4,5 They can act as regulatory elements, such as enhancers or silencers, to affect the expression patterns of distant genes. Moreover, the activity of LINE-1 transposons constitutes a crucial source of genomic diversity, since their insertions and deletions can induce genomic rearrangements, resulting in novel gene architectures and expression patterns that underpin species adaptability to environmental changes. Consequently, LINE-1 transposons are essential for genome evolution. While LINE-1 activity has shaped genome evolution over millions of years, its aberrant activation in somatic cells is a double-edged sword, leading to genomic instability and diseases such as cancer and neurodegenerative diseases.⁶

Full-length LINE-1 is approximately 6 kb and consists of four parts, including the 5′ untranslated region (UTR) as the promoter, open reading frame 1 (ORF1), open reading frame 2 (ORF2), and a 3′ UTR with a poly(A) tail. On both terminals of LINE-1, there are segments of approximately 6–20 bp identical host DNA sequence, known as target site duplications (TSDs). The 5′ UTR provides two internal promoters: a sense promoter (SP) and an antisense promoter (ASP). Driven by the SP, ORF1 encodes an RNA-binding protein (ORF1p), while ORF2 encodes an endonuclease and a reverse transcriptase that are crucial for reverse transcription.⁷ The LINE-1 mRNA transcribed from the SP is transported to the cytoplasm and binds to ORF1p and ORF2p to form the LINE-1 ribonucleoprotein (RNP) complex.⁸ Then this complex is transported back to the nucleus, and ORF2p cuts the genomic DNA and exposes a segment of oligonucleotide. Once transported into the nucleus, ORF2p uses the exposed 3′-hydroxyl (3′-OH) of the nicked genomic DNA as a primer to initiate reverse transcription of the LINE-1 mRNA. This process, known as target-primed reverse transcription (TPRT), is the predominant mechanism for inserting a new LINE-1 copy into the genome. In addition to the canonical ORF1 and ORF2, primate LINE-1 elements encode a previously unrecognized upstream open reading frame, ORF0, which is initiated from the ASP, translated in human cells, and localizes to the nucleus. ORF0 enhances LINE-1 retrotransposition and represents a primate-specific regulatory layer of LINE-1 activity, further expanding the functional complexity of LINE-1-encoded proteins (Figure 1).⁹

Figure 1.

Structure and life cycle of LINE-1 elements

(A) Autonomous transcription of LINE-1s can be promoted at the 5′ UTR and terminates at the polyadenylation signal in the 3' UTR. The SP drives the expression of ORF1 (encoding ORF1p) and ORF2 (encoding endonuclease and reverse transcriptase), while the ASP directs the synthesis of the ORF0 transcript. (B) The LINE-1 is transcribed in the nucleus, followed by mRNA export to the cytoplasm for translation. ORF1p and ORF2p assemble with the transcript to form a ribonucleoprotein (RNP) complex, which re-enters the nucleus to mediate integration through TPRT.

Physiological roles and pathological consequences of LINE-1

LINE-1 has been demonstrated to play crucial roles in normal development as well as in disease progression. A precise temporal expression of LINE-1 is critical for early embryonic development and cell fate commitment. During aging, altered LINE-1 activity exacerbates genomic instability and increases susceptibility to diseases. In cancer, aberrant activation of LINE-1 may result in genomic instability and contribute to carcinogenesis. In neurodegenerative diseases, dysregulated LINE-1 expression has been linked to disease progression and neuronal damage (Figure 2).

Figure 2.

LINE-1 influences the processes of development, aging, and disease

Development: during early embryogenesis, LINE-1 expression is essential for zygotic genome activation and increasing chromatin accessibility. Aging: the loss of heterochromatin during aging leads to the aberrant accumulation of cytoplasmic LINE-1 cDNA, which triggers IFN-I-driven sterile inflammation. Diseases: dysregulated LINE-1 activity can promote oncogenes or disrupt tumor suppressor genes, drive oxidative stress and neuronal injury, and activate chronic inflammation.

LINE-1 in development

In early embryonic development, LINE-1 functions to activate genes associated with zygotic genome activation (ZGA).^10,11 When the embryo enters the 2-cell (2C) stage, LINE-1 needs to be highly expressed to promote the activation of ZGA.^{12,13,14,15,16} Moreover, several studies have reported that LINE-1 and other transposable elements must be downregulated to enable the transition from the 2C stage to subsequent differentiation stages.¹⁷

LINE-1 plays as a regulator that influences gene expression throughout early development.¹⁸ In embryonic stem cells, LINE-1 activity supports pluripotency by interacting with core regulatory networks. L1Md_T (a LINE-1 subfamily) 5′ UTRs function as enhancers to regulate naive pluripotency in embryonic stem cells.¹⁹ Similarly, LINE-1 mediates cis-acting transcriptional control in human pluripotent stem cells and is essential for modulating early neural development.²⁰ In addition, LINE-1 expression changes dynamically as stem cells differentiate into endoderm, mesoderm, and ectoderm.²¹

During neural stem cell differentiation, LINE-1 mRNA under epigenetic regulation enables the PRC2 complex to bind and repress nearby gene activity through the H3K27me3 mark. Thus, LINE-1 expression is under strict regulation in neuronal development.^22,23

LINE-1 in aging

LINE-1 activity increases with age and contributes to genomic instability, mutation accumulation, and loss of cellular function.^24,25 Specifically, cytosolic accumulation of LINE-1-derived cDNAs triggers the cGAS-STING-mediated IFN-I response, driving sustained inflammation and the senescence-associated secretory phenotype (SASP), and accelerates systemic aging.^24,26,27 This mechanism also contributes to cardiac dysfunction by fostering an intracardiac inflammatory and senescent environment.²⁸ Dysregulated LINE-1 transcripts impair effector activity of CD4⁺ T cells, which is essential for the clearance of senescent cells.^29,30,31,32 Moreover, LINE-1 can disrupt circadian rhythm genes (e.g., BMAL1), further promoting aging.^{33,34,35,36,37} In premature aging syndromes, LINE-1 overactivation accelerates senescence and apoptosis.²⁶ Nucleoside reverse transcriptase inhibitors (NRTIs) treatment effectively antagonized not only the IFN-I response but also more broadly reduced age-associated chronic inflammation.³⁸ Thus, controlling LINE-1 activity may be a therapeutic avenue for delaying aging.

LINE-1 in disease

LINE-1 overexpression is also linked to cancer, neurodegeneration, and other diseases (Table 1).⁵⁵ In cancers, LINE-1 activation is increasingly recognized as an early event in neoplastic transformation, arising from loss of epigenetic repression that leads to elevated transcription and retrotransposition activity. Hypomethylation of the LINE-1 promoter is observed in precursor lesions of multiple cancers and correlates with increased genomic instability, insertional mutagenesis, and chromosomal rearrangements during early tumor evolution, as documented in esophageal squamous carcinoma^56,57,58 and ovarian cancer^59,60 precursor lesions. Loss of methylation at the LINE-1 promoter relieves transcriptional repression, resulting in the reactivation of LINE-1 retrotransposons. Reactivated LINE-1 elements promote insertional mutagenesis, induce DNA double-strand breaks, and trigger chromosomal instability, thereby compromising genomic integrity and facilitating the malignant transformation of normal epithelial cells into preneoplastic adenomas.^61,62 Somatic LINE-1 insertions are abundant across diverse cancer genomes and can induce large megabase-scale deletions, amplifications, and complex structural rearrangements that remove tumor suppressor genes or drive oncogene amplification.^59,63,64 LINE-1 insertions disrupt gene function or create chimeric transcripts, while hypomethylation enhances their mobilization.^65,66,67,68 For example, p53 mutations abolish suppression of LINE-1, elevating retrotransposition.⁶⁹ LINE-1 also alters chromatin states, recruits histone modifiers (e.g., KDM4B), and engages oncogenic pathways such as Wnt signaling and the PI3K/Akt pathway.^70,71 Beyond cancer, LINE-1 is elevated in Alzheimer’s and Parkinson’s disease, where it promotes oxidative stress and neuronal death.^72,73,74 Dysregulated LINE-1 is further linked to TDP-43 pathology, a hallmark of several neurodegenerative disorders.^75,76,77,78 In response to tissue injury, such as ischemia-reperfusion, LINE-1 upregulation appears to be a maladaptive stress response; inhibiting its expression has been shown to improve heart function.⁷⁹ In addition, the aberrant accumulation of LINE-1 DNA serves as a primary source of neuroinflammation and neurotoxicity by triggering IFN-I response.⁸⁰ The impact of LINE-1-driven inflammation is further exemplified in celiac disease, where LINE-1 hypomethylation correlates with the local inflammatory response, a condition that reverses upon adherence to a gluten-free diet.⁵⁴

Table 1.

LINE-1 and diseases

Diseases type	Relationship between LINE-1 and diseases	Specific performance and impact
Hereditary diseases
Hemophilia39,40	gene mutation caused by LINE-1 retrotransposition	a hereditary hemorrhagic disease
Familial hypercholesterolemia41	the insertion of LINE-1 in the exon of the LDLR gene results	a genetic disorder in which total cholesterol and LDL cholesterol are raised
Progeria26	LINE-1 RNA accumulates in the cells	including Werner syndrome and Hutchinson-Gilford progeria, which cause premature aging
Chronic noncommunicable diseases
Cancer42	reactivation of LINE-1 in cancer cells	promotes tumor development by altering gene expression and cell function, affecting apoptosis, DNA damage, and repair
Neurodegenerative disease43 (Alzheimer’s disease)	elevated LINE-1 expression levels	in Alzheimer’s disease, the ORF1 protein encoded by LINE-1 has elevated immune reactivity in patients’ microglia
Mental illness44 (autism and schizophrenia)	upregulated expression and transposition of LINE-1 potentially contribute to this process	the specific mechanism of its action is not clear, but it may be involved in the occurrence and development of mental diseases by affecting gene expression and neurodevelopment
Cardiovascular disease45	LINE-1 methylation levels are associated with cardiovascular diseases, such as coronary artery disease	the specific mechanism may involve the change of gene expression, abnormal cell function, and inflammatory response
Chronic obstructive pulmonary disease46,47	increased expression of LINE-1 was observed in the lung tissue of COPD patients	involved in the senescence and apoptosis of lung cells, affecting the immune response
Diabetes mellitus48,49	altered methylation levels of LINE-1 in diabetic patients	the decrease in LINE-1 DNA methylation levels may lead to the activation of transposons, thereby affecting the expression of metabolism-related genes and promoting the development of type 2 diabetes
Atherosclerosis50	in human carotid atherosclerotic plaques, DNA methylation levels of LINE-1 are significantly reduced	destroy the function of vascular endothelial cells and cardiomyocytes, and promote atherosclerosis
Infections and immune diseases
Human immunodeficiency virus (HIV)51,52	HIV-1 infection results in the accumulation of LINE-1 DNA	during HIV infection, LINE-1 may play a role in modulating the host cell’s immune response and interferon signaling pathways
Autoimmune disease42,53	abnormal expression of LINE-1	this leads to an abnormal activation of the immune system, which may trigger inflammatory responses and autoimmune attacks
Celiac disease54	LINE-1 methylation significantly decreased	the low methylation of LINE-1 is significantly present in CeD and CeD-related SBA and is associated with inflammatory immune activities

Molecular mechanisms underlying LINE-1 functions

LINE-1 has been shown to play important roles in various biological processes. At the molecular level, LINE-1 regulates gene expression and biological functions at three levels: DNA (as non-coding functional elements), RNA (as functional non-coding RNAs), and protein (as a transposase).

LINE-1 as a DNA-level regulator and evolutionary driver

As an autonomous retrotransposon, it plays a crucial role in the evolution of the genome.⁸¹ Its evolutionary driving effect is mainly reflected in three aspects: firstly, continuous retrotransposition introduces heritable genetic variation, providing substrates for natural selection. Second, over long evolutionary timescales, LINE-1 sequences can be exapted by the host genome to generate regulatory elements or novel genes with important biological functions.⁸² Third, LINE-1-mediated DNA rearrangements promote structural genome variation, thereby accelerating the evolution of genome architecture across species.⁴

At the mechanistic level, LINE-1 insertions can function as alternative promoters or enhancers.^19,83,84 Recent research has revealed that active LINE-1 elements not only function as transcriptional units but also activate the expression of distal genes through epigenetic modifications (e.g., H3K27ac) in their 5′ UTR, potentially via LINE-1-gene loops formation.⁸³ LINE-1 has enhancer activity, facilitating the transcriptional activation of remote gene expression, especially during early embryonic development, where it contributes to ZGA and affects chromatin accessibility (Figure 3A).⁸³

Figure 3.

Molecular mechanisms of the LINE-1 functions

(A) Evolutionary driver and cis-regulatory roles at the DNA level: LINE-1 evolution over millions of years, L1HS is the youngest LINE-1 subfamily. It acts as an evolutionary driver through retrotransposition and structural rearrangements. Besides transposition, LINE-1 can act as a promoter or enhancer to regulate the expression of adjacent or long-range genes. (B) Regulatory roles at the RNA level: LINE-1 mRNA can act as a noncoding RNA to modulate epigenetic state and influence gene expression. The cytoplasmic accumulation of LINE-1-derived dsRNA activates PKR-mediated physiological inflammation, whereas cDNA triggers cGAS-STING-mediated IFN response. (C) Function at the protein level: ORF1p and ORF2p of LINE-1 alter gene structure and expression, and generate novel chimeric transcripts and fusion proteins. The fusion proteins may promote tumor proliferation and activate the immune response, while serving as potential diagnostic and therapeutic targets.

Context-dependent outcomes of LINE-1 retrotransposition in somatic tissues

Despite its central role in genome evolution, the consequences of LINE-1 retrotransposition at the DNA level in somatic tissues are more complex. In somatic contexts, the most well-established physiological outcome of LINE-1 activity is its contribution to somatic mosaicism, especially within neuronal lineages, which may increase neuronal diversity.^85,86,87

Outside these restricted physiological contexts, LINE-1 retrotransposition in somatic cells is more commonly associated with pathological consequences, particularly in cancer. Certain LINE-1 insertions located upstream of oncogenes can generate novel promoters, thereby leading to aberrant overexpression of these oncogenes.⁶³ In addition, LINE-1 retrotransposition can also repress gene transcription by disrupting native promoter regions, leading to loss of normal gene expression. The FGGY gene exemplifies a tumor suppressor gene whose expression is meticulously regulated under normal settings, hence supporting the maintenance of standard cellular physiological processes. The retrotransposition of LINE-1 can suppress its expression, thereby facilitating the onset and advancement of lung squamous cell carcinoma (LUSC).⁶⁵ Moreover, LINE-1 can intensify genomic instability by causing chromosomal rearrangements or facilitating the transposition of additional transposons (Figure 3A).^88,89

Transposition-independent regulatory functions of LINE-1 RNA

In addition to the classic transposition function, LINE-1 RNA also exhibits significant non-transposition-dependent molecular regulatory activity. LINE-1 mRNA can act as a noncoding RNA to modulate gene expression and affect cell identity.^22,90 LINE-1 RNA can recruit specific TFs or regulatory proteins to designated genomic regions, therefore modulating epigenetic states and gene expression levels.^26,91 For example, FTO mediates the m⁶A demethylation of LINE-1 RNA, regulating the abundance of LINE-1 RNA and the local chromatin state, thus having an impact on H3K4 methylation.⁹² LINE-1 RNA can act as a long non-coding RNA (ncRNA) to participate in the developmental regulation of the cerebral cortex. It maintains the homeostasis of proliferation and differentiation of neural progenitor cells precisely and interacts with chromatin remodeling complexes such as PRC2, mediating the migration and maturation processes of neurons.²² Ultimately, precise and specific regulation was implemented on the key gene expression programs of neural development.²²

In addition, LINE-1s also affect the three-dimensional (3D) genome as ncRNAs. Elevated LINE-1 expression enhances the co-localization of LINE-1 RNA with corresponding DNA in regions enriched for HP1α and H3K9me3, promoting heterochromatin formation and compaction.⁹³ This expansion of dense heterochromatin strengthens the anchoring of chromosomal fragments to the nucleolus and nuclear periphery, bringing relevant domains into closer spatial proximity and altering their 3D organization.⁹⁴

The ncRNA function of LINE-1 is also evident in its role as a constituent of stress granules. Studies indicate that the MxB host restriction factor can associate with LINE-1 RNP within stress granules, obstructing LINE-1 RNP from accessing the nucleus and thereby impeding the retrotransposition of LINE-1.⁹⁵ In addition, aberrant cytoplasmic accumulation of LINE-1-derived nucleic acids can activate innate immune pathways. During the process of tissue repair, LINE-1-formed dsRNA can activate a strong and transient immune response through the PKR-mediated inflammatory response pathway in the early stage, thereby positively regulating the matrix formation of osteocytes.⁹⁶ The cytoplasmic double-stranded DNA produced by LINE-1 mRNA reverse transcription is recognized by cGAS, which synthesizes cyclic GMP-AMP (cGAMP) to activate STING, and ultimately triggers IFN-I response and induces expression of pro-inflammatory cytokines (Figure 3B).^24,28

Protein-level functions of LINE-1

At the protein level, LINE-1-encoded ORF1p and ORF2p mediate both retrotransposition and broader cellular effects. ORF1p has been considered as a potential cancer biomarker.^97,98,99 ORF1p may engage with other cellular proteins to modulate intracellular signal transmission, gene expression, or cell cycle mechanisms.^100,101 ORF2p constitutes the central component of the LINE-1 transposition machinery, and this establishes LINE-1 as a significant model for investigating the replication and infection mechanisms of retroviruses like HIV,⁵¹ while also elucidating the possible involvement of LINE-1 in genome evolution, gene expression regulation, and disease manifestation. The retrotransposition of LINE-1 may enhance the proliferation, invasion, and metastasis of tumor cells by suppressing tumor suppressor gene expression, inducing chromosomal rearrangements, or producing fusion proteins with carcinogenic potential.⁶⁵

Beyond only replicating LINE-1, the retrotranscription mechanism of LINE-1 may recombine with the mRNAs of other genes to create chimeric transcripts and fusion proteins. These novel chimera proteins may modify cellular signal transduction pathways and influence critical processes such as cell proliferation, death, and differentiation.¹⁰² In cancer, the fusion proteins generated by LINE-1 may serve as characteristic molecules of tumor cells, offering novel targets for disease diagnostics and therapy (Figure 3C).^103,104

Regulatory factors of LINE-1 transcription

LINE-1 transcription under TFs regulation

Given its indispensable functions in multiple biological processes, the expression of LINE-1 is subject to strict and finely tuned regulation. The 5′ UTR of LINE-1 contains an internal bidirectional promoter and includes multiple TF-binding motifs, including those for general TFs and cell-type-specific regulators. More recently, genome-wide CRISPR-Cas9 screening in combination with LINE-1 reporter systems has identified both activators and suppressors of LINE-1 expression.⁸³ Furthermore, proximity labeling using dCas9-APEX2 and guide RNAs targeting the LINE-1 promoter has revealed additional candidate proteins that bind to the promoter and modulate LINE-1 transcription (Figure 4; Table 2).^118,119

Figure 4.

LINE-1 expression is regulated by TFs and epigenetic factors

Spatial partitioning within the 3D genome regulates LINE-1 activity, where sequestration in B compartments maintains silencing while localization to A compartments facilitates activation. Concurrently, LINE-1 is tightly regulated by the coordination of transcription factors (e.g., YY1) and epigenetic modifiers (e.g., DNMTs). Its silencing is maintained by DNA methylation (5 mC), repressive histone marks (e.g., H3K9me3 and H3K27me3), and chromatin remodeling complexes (e.g., NuRD and ATRX/DAXX), whereas activation is driven by active histone modifications (e.g., H3K27ac and H4K16ac) and specific TFs (e.g., RUNX3 and SOX2).

Table 2.

LINE-1 transcription under TFs regulation

Transcription factor	Mechanism of regulation	Notes/References
YY1	active TF family LINE-1 expression by binding to the 5′ UTR	Athanikar et al.105; Sakamoto et al.106; Meier et al.107; Saha et al.108
RUNX3	enhance sense and antisense transcription/transposition by binding distinct regions of the 5′ UTR	Yang et al.109
SOX2/6/11	enhancing expression through the binding site of the LINE-1 promoter, regulating chromatin accessibility, and neuronal diversity	Muotri et al.86; Tchénio et al.110; Bodea et al.111; Orqueda et al.112
IRF4	in resting CD4+ T cells, the LINE-1 transcripts are retained in the chromatin to maintain the resting state; after activation, the transcripts are degraded	Marasca et al.29
p53	directly combine the LINE-1 sequence, or indirectly by using SETDB1 and G9A to deposit the H3K9me3 mark to silence LINE-1	Li et al.113; Paul et al.114; Neri et al.115
MYC	the recruitment of the PRC2 complex increases the level of H3K27me3, and through the H3K9me3/H3K27me3 modifications in heterochromatin regions, it silences LINE-1	Neri et al.115; Sun et al.116
SAFB	inhibits the activity of the LINE-1 promoter, while maintaining the chromatin contact domain boundaries, and restricts chimeric transcription and abnormal genomic interactions	Hong et al.117

YY1 is one of the best-characterized LINE-1-binding factors, essential for initiating LINE-1 transcription during embryonic stem cell stages and early development by facilitating RNA polymerase II recruitment.^105,106,107 Notably, YY1 selectively activates the TF but not the Gf LINE-1 subfamily via the GCCAT motif.¹⁰⁸ RUNX3 directly binds the human LINE-1 5′ UTR to enhance both sense and antisense transcription, thereby promoting retrotransposition and influencing nearby gene regulation.¹⁰⁹ Members of the SOX family (SOX2, SOX6, and SOX11) activate LINE-1 and influence chromatin accessibility, transcriptional regulation, and neuronal diversity.^{86,110,111,112} In contrast, p53^113,114,115 and MYC^115,116 act as key repressors, enforcing epigenetic silencing through H3K9me3 and H3K27me3 deposition. Additionally, IRF4²⁹ and SAFB¹¹⁷ regulate LINE-1 through chromatin retention and higher order genome organization, highlighting multilayered transcriptional and epigenetic control of LINE-1 activity.

LINE-1 expression under epigenetic regulation

Beyond TFs, LINE-1 transcription is subject to tight epigenetic factor regulation (Table 3). DNA methylation and repressive histone marks (e.g., H3K9me3) together with chromatin-remodeling complexes (e.g., human silencing hub [HUSH] and NuRD) maintain LINE-1 silencing, while recruitment of activating histone modifiers (e.g., H3K4me3 and H3K27ac) promotes locus-specific LINE-1 activation. In addition, RNA-based modifications and RNA-binding protein (RBP) modulate LINE-1 transcript stability, processing, and nuclear localization, providing context-dependent regulation during development, aging, and diseases (Figure 4).

Table 3.

Epigenetic factors regulating LINE-1 expression

Molecules	Epigenetic mark	Active/repress	Reference
DNMT1	DNA methylation 5mC	repress	Maugeri et al.120
TET	DNA methylation 5mC	active	de la Rica et al.121
KAP1/TRIM28	DNA methylation 5mC	active	Percharde et al.91
RB1	DNA methylation 5mC	active	Tan et al.122
ALKBH1	DNA methylation 6mA	active	Wu et al.123
N4 Cytosine Methylation	DNA methylation 4mC	active	Walker et al.124
METTL3	RNA methylation m6A	active	Li et al.125
YTHDF2	RNA methylation m6A	active	Li et al.125
FTO	RNA methylation m6A	repress	Wei et al.92
SUV39H1/SUV39H2	histone modification (H3K9me3)	repress	Healton et al.126
SETDB1	histone modification (H3K9me3)	repress	Healton et al.126
EHMT2 (G9a)	histone modification (H3K9me2)	repress	Di Giacomo et al.127
KAP1 (TRIM28)	histone modification (H3K9me3)	repress	Healton et al.126
KDM4A/JMJD2A	histone modification (H3K9me3)	repress	Cheng et al.128
KDM5B	histone modification (H3K4me3)	active	Chen et al.129; Zhang et al.130
LSD1	histone modification (H3K4me3)	repress	Ancelin et al.131; Sheng et al.132
EZH2/EZH1	histone modification(H3K27me3)	repress	Mangoni et al.22
piRNA	non-coding RNA	repress	Kalmykova et al.133
miR-128	non-coding RNA	repress	Idica et al.134; Hamdorf et al.135
NuRD complex	chromatin remodeling	repress	Bojang et al.136; Montoya-Durango et al.137
ATRX/DAXX complex	chromatin remodeling	repress	Elsässer et al.138; Solovyov et al.139

DNA methylation

DNA methylation plays a vital role in silencing LINE-1 expression. Under normal physiological conditions, the CpG-rich regions within LINE-1 sequences are generally substantially methylated;¹⁴⁰ therefore, reducing the LINE-1’s transcriptional activity. Nevertheless, changes in DNA methylation patterns may occur under various environmental stresses or pathogenic conditions, such as cancer or genetic illnesses, which would demethylate CpG islands and hence activate LINE-1 transcription.¹⁴¹ In addition to 5-methylcytosine (5mC), N6-methyl-2′-deoxyadenosine (6mA) has now been identified in both prokaryotic and eukaryotic genomes, drawing growing interest due to its potential regulatory functions in development and diseases.^{123,142,143,144} In transposable element regions, particularly in young LINE-1 elements, 6mA is enriched contributing to their epigenetic silencing. Deletion of the 6mA demethylase ALKBH1 reinforces this repression, leading to sustained LINE-1 silence.¹²³ Recent studies have shown that another form of DNA methylation, N⁴-cytosine methylation (4mC), precisely regulates transposable element activity, thereby playing a critical role in normal plant germ cell development and maintaining genome stability.¹²⁴

RNA methylation

LINE-1 transcripts are major m⁶A-modified RNAs in human cells due to their high-density RRACH sequences.¹⁴⁵ Notably, the expression regulatory mechanisms mediated by m⁶A-modifying enzymes can only be effectively exerted in LINE-1 elements with intact 5′ UTRs.¹⁴⁵ Functional studies show that the m⁶A methyltransferase METTL3 significantly promotes LINE-1 retrotransposition activity by increasing the m⁶A modification level of LINE-1 RNA, while the m⁶A demethylase ALKBH5 inhibits LINE-1 retrotransposition by reducing the abundance of m⁶A modification on LINE-1.¹⁴⁶ In mouse embryonic stem cells (mESCs), the m⁶A reader protein YTHDC1 recognizes modified LINE-1 transcripts and recruits histone methyltransferases SETDB1, TRIM28, and nucleolin to form a transcriptional silencing complex, which ultimately achieves epigenetic silencing of retrotransposons by promoting the enrichment of H3K9me3 in chromatin regions.^147,148 This mechanism is of significant biological importance for maintaining genomic stability and regulating cellular pluripotency.

Noncoding RNA

LINE-1 expression is also regulated by ncRNA. piRNA serves a vital function in the regulation of LINE-1 transcription.^149,150 It suppresses LINE-1 transcription primarily via three mechanisms: first, by promoting histone H3K9 methylation¹⁵¹ and H3K4 demethylation, resulting in chromatin condensation¹⁵²; second, by inducing LINE-1 DNA methylation, establishing a stable epigenetic repression^153,154; and third, by depleting LINE-1 mRNA through the ping-pong cycle mechanism.¹⁴⁹ These together prevent mutations caused by LINE-1 transposons and preserve genomic stability.

Histone modifications

The H3K9me3 modification of the LINE-1 region in chromatin can maintain a compact heterochromatin state.¹⁵⁵ This compact configuration obstructs TFs and RNA polymerase from reaching the DNA sequence of LINE-1, thereby limiting LINE-1 transcription.^156,157 HP1, the quintessential reader protein for H3K9me3, can identify and bind to this change, subsequently enlisting additional protein complexes to execute certain biological actions.¹⁵⁸ Recent studies indicate that the HUSH complex selectively targets intronic youthful and full-length LINE-1s for transcriptional suppression by promoting the deposition of H3K9me3 on these LINE-1 elements.^5,159 In early human development, especially during the critical phase before fetal implantation, the dynamic reprogramming of H3K9me3 is essential.¹⁶⁰ The unequal distribution of H3K9me3 between parental genomes significantly affects the silencing status of LINE-1 elements throughout the cell cycle, with this dynamic control being especially vital during the S phase (DNA synthesis phase).¹⁶¹ Research has demonstrated an inverse relationship between H3K9me3 levels and the transcriptional activity of LINE-1 elements. Thus, an elevation in H3K9me3 levels typically correlates with a reduction in LINE-1 element expression, which is essential for preserving cellular genomic integrity. The epigenetic regulator TNRC18 can specifically recognize the histone modification H3K9me3, thereby mediating the silencing of transposable elements associated with endogenous retroviruses.¹⁶² This process underscores the pivotal role of epigenetic regulation in transposable element silencing and, through this mechanism, influences gene expression as well as other biological processes. In addition, KDM5B specifically recruits the H3K9 methyltransferase SETDB1 to catalyze and promote the methylation modification of the histone H3K9 site, thereby effectively inhibiting the transcriptional expression of transposable elements.¹³⁰

Other histone modifications have also been demonstrated to regulate LINE-1 expression. In LINE-1 transcriptional regulation, H3K27me3 functions by engaging with the PRC complex.¹⁶³ The PRC can identify and attach to the LINE-1 region to facilitate the creation of H3K27me3 modifications.⁹⁴ The dynamic alterations in H3K27me3 are intricately linked to the possible activation of LINE-1 and the determination of cell fate, offering significant insights for the comprehensive investigation of disease causes and the advancement of innovative therapeutic strategies.⁹⁴ For activation, male-specific lethal (MSL) complex-mediated H4K16ac specifically enriches in LINE-1/long terminal repeat (LTR) regions of stem cells and cancer cells, forming enhancer-like structures that regulate distal gene expression via chromatin looping.¹⁶⁴ In early human embryos, H3K56ac reaches its peak at the two-cell phase and directly activates the transcription of young LINE-1. The LINE-1 transposition element participates in nucleosome remodeling by binding to chromatin remodeling complexes, adjusts chromatin accessibility during embryonic genomic activation, and promotes the expression of key genes.¹⁶⁵ For repression, H4K20me3 is recognized by DNMT1 to induce DNA methylation, synergistically repressing LINE-1 activity.¹⁶⁶

Chromatin remodeling

In addition to DNA methylation, noncoding RNA, and histone modifications, ATP-dependent chromatin remodeling plays a pivotal role in regulating LINE-1 expression. Chromatin remodelers are essential for altering nucleosome positioning, accessibility, and histone variant incorporation, thereby enabling or restricting the transcriptional activity of retrotransposons. These enzymes act in complex with histone modifiers and TFs to establish a chromatin environment that either silences or permits LINE-1 activity.

Specifically, the NuRD complex (composed of CHD4, MBD3, and HDAC1/2) integrates histone deacetylation with nucleosome sliding to repress LINE-1 by establishing compact chromatin at its loci.^136,137 Components of the BAF complex, such as SMARCA4/BRG1, have context-dependent roles; while BAF can promote chromatin opening, it also aids in retrotransposon repression during early development by stabilizing repressive complexes.¹⁶⁷ Furthermore, the ATRX/DAXX complex deposits the H3.3 histone variant at repetitive regions,¹³⁸ where it binds to H3K9me3 and may facilitate heterochromatin formation and LINE-1 silencing. The importance of this mechanism is underscored by the fact that loss of ATRX leads to aberrant LINE-1 expression and genome instability.¹³⁹ Chromatin remodeling does not act in isolation but coordinates with histone methyltransferases (SETDB1 and SUV39H1), DNA methylation (DNMTs), and 3D genome folding to enforce silencing. Remodelers often guide or scaffold these epigenetic modifiers to specific LINE-1 loci, creating a multilayered repression system. LINE-1 RNA can recruit chromatin modifiers in feedback loops that reinforce its own silencing.^22,26

3D genome architecture

The 3D organization directly affects LINE-1 activity and regulatory potential by spatial chromatin arrangement. Genome-wide CRISPR-Cas9 screens have identified host factors such as SAFB that preserve chromatin contact-domain boundaries and thereby limit LINE-1-driven chimeric transcription and disruptive genomic interactions.^5,117 High-throughput chromosome conformation capture (Hi-C) shows that LINE-1s are preferentially enriched in B compartments, which sequester LINE-1 away from transcriptional activators.⁹³ By contrast, LINE-1 insertions that escape silencing tend to reside in A compartments, adjacent to active enhancers or euchromatic gene clusters, and are more susceptible to activation.

The topologically associating domains (TADs) constrain LINE-1 activity by restricting enhancer-promoter interactions. For instance, disruption of TAD boundaries through genetic, epigenetic, or pathological perturbations can reposition LINE-1 into transcriptionally permissive domains, provoking aberrant expression.¹⁶⁸ Architectural proteins such as CTCF bind near LINE-1 loci and influence their insulation and autonomous transcription.^119,169 Conversely, active LINE-1s can remodel 3D genome architecture themselves: LINE-1 5′ UTRs acquire activating marks (e.g., H3K4me3) that facilitate long-range chromatin loop or R-loop formation to activate distal genes,²⁶ and RNA modifications like m⁶A alter LINE-1 RNA secondary structure, modulating interactions with nuclear proteins and compartmental localization.¹⁴⁶ This bidirectional interplay highlights how LINE-1s both respond to and shape higher order genome folding, balancing regulatory with genome stability.

LINE-1 as a diagnostic and therapeutic target

The clinical relevance of LINE-1 stems from its role as both an indicator and a driver of cellular dysfunction. Mechanistically, aberrant LINE-1 activation reflects and amplifies epigenetic dysregulation. The erosion of heterochromatin and loss of repressive histone modifications or DNA methylation lead to LINE-1 derepression, while accumulated LINE-1 RNA can disrupt chromatin-regulatory complexes, further destabilizing epigenetic homeostasis.^{7,26,29,93,170} Within the nucleus, reactivated LINE-1 transcripts may reshape transcriptional programs through alternative promoter activity or cis-regulatory interactions with neighboring genes.^{83,155,171,172} Beyond transcriptional perturbation, active LINE-1 elements can retrotranspose into new genomic loci, introducing insertional mutations, triggering DNA double-strand breaks, and promoting chromosomal rearrangements, which collectively drive genomic instability and accelerate the mutational landscape of tumor cells.^{63,65,88,89,173} In the cytoplasm, accumulation of LINE-1-derived RNA and DNA/RNA hybrids activates innate immune sensing pathways, generating chronic inflammatory signaling that promotes tumor evolution and microenvironment remodeling.^24,28,96 In addition to these nucleic acid-mediated effects, LINE-1-encoded proteins and LINE-1-derived fusion proteins may modulate cellular signal transduction pathways and influence cell proliferation and differentiation.^{97,98,103,104} Collectively, these mechanistic insights have positioned LINE-1 as a versatile biomarker across multiple malignancies.

LINE-1 hypomethylation has been widely observed across multiple malignancies, including lung,¹⁷⁴ breast,¹⁷⁵ colorectal,¹⁷⁶ gastric,¹⁷⁷ ovarian,¹⁷⁸ bladder,¹⁷⁹ and hepatocellular carcinomas,¹⁸⁰ and is frequently associated with poor prognosis. Notably, elevated LINE-1 RNA and ORF1p levels disrupt key cellular pathways such as apoptosis, DNA damage responses, and stress signaling, thereby promoting tumor progression.^181,182,183 Beyond tumor tissues, LINE-1 methylation patterns and ORF1p in peripheral blood and plasma DNA have emerged as powerful non-invasive biomarkers for cancer risk stratification and early detection,^{184,185,186,187,188,189,190} as demonstrated by recent liquid biopsy approaches detecting LINE-1 hypomethylation across multiple cancer types (Table 4).¹⁹¹

Table 4.

Diagnostic and prognostic significance of the LINE-1 in cancer

Type	Source	Biomarker	Clinical	Reference
Lung cancer	tissue	methylation	prognosis	Saito et al.174
Breast cancer	tissue	methylation	prognosis	van Hoesel et al.175
Colorectal cancer	tissue	methylation	prognosis	Mima et al.176
Gastric cancer	tissue	methylation	diagnose/prognosis	Shigaki et al.177
Ovarian cancer	tissue	methylation	prognosis	Pattamadilok et al.178
Bladder cancer	tissue/plasma	methylation	diagnose	van Bemmel et al.179
Hepatocellular carcinomas	tissue	methylation	prognosis	Anwar et al.180
Esophageal carcinoma	tissue	ORF1p	diagnose	Doucet-O'Hare et al.181
Gastric cancer	plasma	methylation	prognosis	Ko et al.184
HNSCC	PBMC	methylation	diagnose	Arayataweegool et al.185
Esophageal adenocarcinoma	plasma	methylation	diagnose	Boldrin et al.186
Breast cancer	plasma	methylation	diagnose	Lee et al.187
Lung cancer	plasma	methylation	diagnose	Gainetdinov et al.188
Ovarian carcinoma	plasma	ORF1p	diagnose	de Santiago et al.189
Multicancer	plasma	methylation	diagnose	Michel et al.191

Importantly, LINE-1 is emerging as a promising therapeutic target, with its inhibition offering a strategy to restore a normal cellular phenotype. Strategies targeting LINE-1 across DNA, RNA, and protein levels have been developed. These include restoration of epigenetic repression,¹⁹² inhibition of transcription or enzymatic activity,¹⁰² and pharmacological suppression using reverse transcriptase or endonuclease inhibitors,¹⁹³ highlighting LINE-1 as both a biomarker and a mechanistically actionable vulnerability in cancer.^191,194,195 Future therapeutic development will likely require approaches that distinguish disease-driving LINE-1 loci from global epigenetic activation, enabling selective modulation of pathogenic activity while preserving physiological genomic regulation.

Challenges and prospects

LINE-1 in cancer and other diseases: From genome-wide burden to locus-specific effects

A central conceptual challenge in LINE-1 research is distinguishing locus-specific regulatory function from global epigenetic noise. It is still unclear whether LINE-1 contributes to tumorigenesis or other disease contexts (including neurodegeneration, autoimmunity, and aging-related disorders) primarily through a cumulative genome-wide burden or via the activation of specific high-impact loci that function as regulatory hubs or mutational drivers. While LINE-1 activity has traditionally been interpreted as a genome-wide indicator of epigenetic derepression, increasing evidence supports a model in which a subset of loci acts as cis-regulatory elements that modulate neighboring gene expression. The key question is therefore not whether LINE-1 is active, but which specific loci exert functional regulatory influence and through what molecular mechanisms.

Technical and conceptual challenges in detecting locus-specific LINE-1

LINE-1 elements are highly repetitive, polymorphic, and often overlapping with genes or generating chimeric transcripts, making locus-specific analysis difficult with hybridization assays, short-read RNA sequencing, or chromatin immunoprecipitation (ChIP) sequencing.^196,197 Young LINE-1 subfamilies show high sequence similarity, which hampers unambiguous read assignment, especially at the single-cell level resolution.¹⁹⁸

Recent advances in sequencing technologies and computational methods have markedly improved the resolution of LINE-1 analyses. In bulk datasets, Telescope¹⁹⁹ and TEtranscripts²⁰⁰ enhance quantitative accuracy by probabilistically assigning multi-mapping reads. For single-cell applications, SoloTE²⁰¹ and scTE²⁰² partially overcome repetitive sequence challenges by leveraging alignment scores to allocate reads to individual loci. MATES (multi-mapping alignment for TE loci quantification in single-cell) integrates deep learning with unique flanking sequences to achieve more accurate locus-level quantification,²⁰³ representing an important conceptual advance.

The advent of third-generation long-read sequencing technologies (PacBio and Oxford Nanopore) enables direct identification of chimeric LINE-1-host transcripts and allows precise attribution of expression to specific LINE-1 loci.^204,205 NanoATAC-seq2 uses long-read sequencing to accurately depict the chromatin state of repetitive elements.²⁰⁶ These approaches are increasingly central to elucidating the functional consequences of LINE-1 activation in development and disease.

Technical and conceptual challenges in studying locus-specific LINE-1

A major technical barrier to testing the function of locus-specific LINE-1 is achieving locus-specific manipulation without perturbing entire LINE-1 subfamilies. Because LINE-1 copies share extensive sequence homology, conventional RNA interference, reverse transcriptase inhibition, or epigenetic modulation typically produces global effects. Mechanistically precise targeting will require strategies that exploit locus-unique features, including flanking genomic sequences, local chromatin architecture, and 3D genome contacts. CRISPR interference or activation systems guided by flanking-sequence-specific single-guide RNAs (sgRNAs), epigenome editing tools that deposit or erase histone marks at defined coordinates, and allele-resolved long-read transcriptomics provide feasible frameworks for selective regulation of individual LINE-1 loci while preserving global LINE-1 homeostasis.

Equally important is establishing whether locus-specific LINE-1 activation is a causal regulator or a downstream consequence of chromatin remodeling. Resolving this requires causal inference frameworks combining temporally controlled perturbation, locus-resolved transcriptional profiling, and functional rescue experiments. Demonstrating the necessity, sufficiency, and reversibility of a specific LINE-1 locus for gene expression changes will be essential to distinguish regulatory drivers from passive epigenetic bystanders.

Translational potential and therapeutic opportunities toward precision LINE-1 biology

Despite unresolved challenges, LINE-1 biology offers substantial translational promise. Aberrant LINE-1 expression and methylation patterns are emerging as non-invasive biomarkers detectable in liquid biopsies, with potential applications in early cancer detection and disease monitoring.^207,208

Therapeutically, LINE-1 inhibition may mitigate genome instability or inflammatory signaling, while engineered LINE-1 elements could be repurposed as gene delivery vehicles in precision medicine.^193,209 However, these approaches raise critical concerns regarding specificity, off-target effects, and long-term safety, necessitating careful mechanistic and preclinical evaluation.

Together, these directions outline a transition from global descriptive profiling toward a mechanistically grounded and therapeutically actionable understanding of LINE-1 biology. With continued integration of long-read detection and functional perturbation systems, locus-specific LINE-1 is poised to emerge as a defined regulatory component of disease-relevant gene networks and a potential target for precision medicine.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC 32170596) to Z.L., and the Major Project of Guangzhou National Laboratory (GZNL2023A02010 and GZNL2023A02003) to Z.L. We apologize to the excellent colleagues whose publications were not cited due to space limitations and the specific focus of this review, and sincerely thank all the authors of the papers mentioned and reviewed in this manuscript. We also extend our gratitude to all the members of the laboratory for their valuable suggestions and feedback on this work.

Author contributions

X.L. and C.Z. wrote the manuscript and created the figures with the assistance of S.W. and Q.H.; Z.L. supervised the writing and reviewed and revised the manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT for grammatical error correction to improve language. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.