Co-expression of distinct L1 retrotransposon coiled coils can lead to their entanglement.

L1 (LINE1) non-LTR retrotransposons are ubiquitous genomic parasites and the dominant transposable element in humans having generated about 40% of their genomic DNA during their ~ 100 million years (Myr) of activity in primates. L1 replicates in germ line cells and early embryos, causing genetic diversity and defects, but can be active in some somatic stem cells, tumors and during aging. L1 encodes two proteins essential for retrotransposition: ORF2p, a reverse transcriptase that contains an endonuclease domain, and ORF1p, a coiled coil mediated homo trimer, which functions as a nucleic acid chaperone. Both proteins contain highly conserved domains and preferentially bind their encoding transcript to form an L1 ribonucleoprotein (RNP), which mediates retrotransposition. However, the coiled coil has periodically undergone episodes of substantial amino acid replacement to the extent that a given L1 family can concurrently express multiple ORF1s that differ in the sequence of their coiled coils. Here we show that such distinct ORF1p sequences can become entangled forming heterotrimers when co-expressed from separate vectors and speculate on how coiled coil entanglement could affect coiled coil evolution.

Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma.

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) results from a series of genetic alteration in squamous cells. This particular type of cancer considers one of the most aggressive malignancies to control because of its frequent local invasions to the regional lymph node. Although several biomarkers have been reported, the key marker used to predict the behavior of the disease is largely unknown. Here we report Long INterpersed Element-1 (LINE1 or L1) retrotransposon activity in post-operative oral cancer samples. L1 is the only active retrotransposon occupying around 17% of the human genome with an estimated 500,000 copies. An active L1 encodes two proteins (L1ORF1p and L1ORF2p); both of which are critical in the process of retrotransposition. Several studies report that the L1 retrotransposon is highly active in many cancers. L1 activity is generally determined by assaying L1ORF1p because of its high expression and availability of the antibody. However, due to its lower expression and unavailability of a robust antibody, detection of L1ORF2p has been limited. L1ORF2p is the crucial protein in the process of retrotransposition as it provides endonuclease and reverse transcriptase (RT) activity. METHODS: Immunohistochemistry and Western blotting were performed on the post-operative oral cancer samples and murine tissues. RESULTS: Using in house novel antibodies against both the L1 proteins (L1ORF1p and L1ORF2p), we found L1 retrotransposon is extremely active in post-operative oral cancer tissues. Here, we report a novel human L1ORF2p antibody generated using an 80-amino-acid stretch from the RT domain, which is highly conserved among different species. The antibody detects significant L1ORF2p expression in human oral squamous cell carcinoma (OSCC) samples and murine germ tissues. CONCLUSIONS: We report exceptionally high L1ORF1p and L1ORF2p expression in post-operative oral cancer samples. The novel L1ORF2p antibody reported in this study will serve as a useful tool to understand why L1 activity is deregulated in OSCC and how it contributes to the progression of this particular cancer. Cross-species reactivity of L1ORF2p antibody due to the conserved epitope will be useful to study the retrotransposon biology in mice and rat germ tissues.

Invasive and Noninvasive Nonfunctioning Gonadotroph Pituitary Tumors Differ in DNA Methylation Level of LINE-1 Repetitive Elements.

PURPOSE: Epigenetic dysregulation plays a role in pituitary tumor pathogenesis. Some differences in DNA methylation were observed between invasive and noninvasive nonfunctioning gonadotroph tumors. This study sought to determine the role of DNA methylation changes in repetitive LINE-1 elements in nonfunctioning gonadotroph pituitary tumors. METHODS: We investigated LINE-1 methylation levels in 80 tumors and normal pituitary glands with bisulfite-pyrosequencing. Expression of two LINE-1 open reading frames (L1-ORF1 and L1-ORF2) was analyzed with qRT-PCR in tumor samples and mouse gonadotroph pituitary cells treated with DNA methyltransferase inhibitor. Immunohistochemical staining against L1-ORF1p was also performed in normal pituitary glands and tumors. RESULTS: Hypomethylation of LINE-1 was observed in pituitary tumors. Tumors characterized by invasive growth revealed lower LINE-1 methylation level than noninvasive ones. LINE-1 methylation correlated with overall DNA methylation assessed with HM450K arrays and negatively correlated with L1-ORF1 and L1-ORF2 expression. Treatment of αT3-1 gonadotroph cells with 5-Azacytidine clearly increased the level of L1-ORF1 and L1-ORF2 mRNA; however, its effect on LßT2 cells was less pronounced. Immunoreactivity against L1-ORF1p was higher in tumors than normal tissue. No difference in L1-ORF1p expression was observed in invasive and noninvasive tumors. CONCLUSION: Hypomethylation of LINE-1 is related to invasive growth and influences transcriptional activity of transposable elements.

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.

L1-ORF1p and Ago2 are involved in a siRNA-mediated regulation for promoter activity of L1-5'UTR.

INTRODUCTION: Long interspersed nuclear elements-1 (L1), as the only one self-active retrotransposon of the mobile element, was found to be generally activated in tumor cells. The 5'UTR of L1 (L1-5'UTR) contains both sense and antisense bidirectional promoters, transcription products of which can generate double-strand RNA (dsRNA). In addition, L1-ORF1p, a dsRNA binding protein encoded by L1, is considered to engage in some RNA-protein (RNP) formation. Ago2, one of the RISC components, can bind to dsRNA to form RISC complex, but its role in L1 regulation still remains unclear. Due that the 5'UTR of L1 (L1-5'UTR) contains both sense and antisense bidirectional promoters, so the activities in both string were identified. A dsRNA-mediated regulation of L1-5'UTR, with the feedback regulation of L1-ORF1p as well as other key molecules engaged (Ago1-4) in this process, was also investigated. MATERIAL AND METHODS: Genomic DNA was extracted from HEK293 cells and subjected to L1-5'UTR prepa-ration by PCR. Report gene system pIRESneo with SV40 promoter was employed. The promoter activities of different regions in L1-5'UTR were identified by constructing these regions into pIRESneo, which SV40 region was removed prior, to generate different recombinant plasmids. The promoter activities in recombinant plasmids were detected by the luciferase expression assay. Western blot and co-immunoprecipitation were employed to identify proteins expression and protein-protein interaction respectively. RESULTS: Ago2 is a member of Agos family, which usually forms a RISC complex with si/miRNA and is involved in post- transcriptional regulation of many genes. Here L1-ORF1p and Ago2 conducts a regulation as a negative feedback for L1-5'UTR sense promoter. L1-ORF1p could form the immune complexes with Ago1, Ago2 and Ago4, respectively. CONCLUSIONS: L1-5'UTR harbors both sense and antisense promoter activity and a dsRNA-mediated regulation is responsible for L1-5'UTR regulation. Agos proteins and L1-ORF1p were engaged in this process.

Purification of L1-Ribonucleoprotein Particles (L1-RNPs) from Cultured Human Cells.

Almost two-thirds of the human genome is repetitive DNA, mostly derived from different kinds of transposon and retrotransposon sequences. Although most of these sequences are stable in the genome, one class called long interspersed element (LINE1 or L1) is actively jumping in the human genome, particularly in brain, germ cells, and certain types of cancer. Recent estimates predict that L1 activity combined with L1-mediated activity is responsible for a new insertion in 1 out of 25 newborns. In humans, more than 100 single-gene disease cases have been reported due to L1 activity. An active L1 encodes two proteins designated as ORF1p and ORF2p. L1 jumps by a target primed reverse transcription (TPRT) mechanism where L1 RNA forms L1-RNPs after binding with L1 proteins. L1-RNPs then enter into the nucleus where L1 RNA is converted to cDNA at the site of integration which subsequently integrates into the genome with the help of the L1 proteins (ORF1p and ORF2p) and other cellular factors. Although L1 is continuously jumping in the human genome the basic mechanism and requirement of other cellular factors in L1 retrotransposition are relatively unknown due to the difficulty in purifying intact L1-RNPs. Here we describe a detailed protocol for purification of L1-RNPs by an immunoaffinity method.