RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo.

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.

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.

LINE-1 retrotransposon encoded ORF1p expression and promoter methylation in oral squamous cell carcinoma: a pilot study.

Oral squamous cell carcinoma (OSCC) is highly predominant in India due to excessive use of tobacco. Here we investigated Long INterpersed Element 1 (LINE or L1) retrotransposon activity in OSCC samples in the same population. There are almost 500,000 copies of L1 occupied around 30%  of the human genome. Although most of them are inactive, around 150-200 copies are actively jumping in a human genome. L1 encodes two proteins designated as ORF1p and ORF2p and expression of both proteins are critical for the process of retrotransposition. Here we have analyzed L1 ORF1p expression in a small cohort (n = 15) of paired cancer-normal tissues obtained from operated oral cancer patients. Immunohistochemistry (IHC) with the human ORF1 antibody showed the presence of ORF1p in almost 60%  cancer samples, and few of them also showed aberrant p53 expression.  Investigating L1 promoter methylation status, showed certain trends towards hypomethylation of the L1 promoter in cancer tissues compared to its normal counterpart. Our data raise the possibility that L1ORF1p expression might have some role in the onset and progression of this particular type of cancer.

Expression of L1 retrotransposon open reading frame protein 1 in gynecologic cancers.

LINE-1 (L1) retrotransposons are mobile genetic elements capable of "copy-and-pasting" their own sequences into random genomic loci, and one of the proteins it uses to achieve mobility is LINE-1 open reading frame 1 protein (L1ORF1p). L1ORF1p expression is found across many epithelial cancers, including small cohorts of ovarian and endometrial cancers, and is highly expressed in cancers with mutant p53 expressions. Here we aimed to gain insights into L1ORF1p expression levels within specific histotypes of ovarian cancers: high-grade serous (n = 585), low-grade serous (n = 26), clear cell (n = 132), endometrioid (n = 148), and mucinous (n = 32) ovarian cancers, as well as endometrial cancers (n = 607) using tissue microarray (TMA's). We demonstrated that L1ORF1p expression is associated with advanced stage and serous histotype in gynecological cancers. Like previous studies, we found a higher proportion of L1ORF1p expression in cases with aberrant p53 expression. We evaluated the expression of L1ORF1p in serous tubal intraepithelial carcinomas (STICs) (n = 6) and p53 signature lesions (n = 2) in fallopian tubes. Three STIC cases displayed aberrant p53 overexpression with corresponding L1ORF1p expression in the same tissues, but such correlation was not seen in the two p53 signature lesions, suggesting that L1 protein may be expressed after dysplastic transformation. The remaining three STIC cases have TP53 nonsense mutations with absent p53 expression but a strong and clear L1ORF1p expression within the STIC lesions. While L1ORF1p may not be prognostic in gynecological cancers, it may be useful clinically as a diagnostic IHC marker for p53 null STIC lesions and this warrants further investigation.

LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated ovarian cancers.

OBJECTIVE: Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic elements activated in many cancers capable of moving neighboring DNA through 3' transductions. Here we investigated the involvement of specific L1-mediated transductions in EAOCs. METHODS: Through whole genome sequencing, we identified active L1-mediated transductions originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations detected using multiplex PCR and next generation sequencing. RESULTS: TTC28-L1 mediated transductions were identified in at least three tumor samplings in all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites while other selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. CONCLUSION: The TTC28-L1 transductions along with classical driver mutations were near ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal relationships and to track ovarian cancer progression.

A LINE-1-encoded reverse transcriptase-dependent regulatory mechanism is active in embryogenesis and tumorigenesis.

LINE-1 (long interspersed nuclear elements) retrotransposons constitute a large family of retrotransposable elements, accounting for 17% of the human genome. They encode proteins required for their own mobilization, including a reverse transcriptase (RT) enzyme highly expressed in mouse embryos and mouse and human cancer cells and repressed in somatic differentiated healthy cells. We have found that reverse transcription takes place in early murine embryos, yielding an increase in LINE-1 copy number during preimplantation development, which also occurs in tumor progression. RT inhibition irreversibly arrests embryo development, reduces cancer cell proliferation, promotes differentiation, antagonizes tumor growth, and causes a global reprogramming of transcription profiles. These results strongly suggest that a previously unrecognized RT-dependent regulatory mechanism operates during preimplantation development, is repressed during differentiation to normal tissues, and, when erroneously reactivated in adult life, promotes cell transformation and cancer progression by "resurrecting" embryonic transcriptional pathways. The RT-dependent mechanism emerges as a major source of genetic and epigenetic changes with physiological, pathological, and evolutionary implications.