Cell Type-specific Expression of LINE-1 Open Reading Frames 1 and 2 in Fetal and Adult Human Tissues

Abstract

The LINE-1 (L1) family of non-long terminal repeat retrotransposons is a major force shaping mammalian genomes, and its members can alter the genome in many ways. Mutational analyses have shown that coexpression of functional proteins encoded by the two L1-specific open reading frames, ORF1 and ORF2, is an essential prerequisite for the propagation of L1 elements in the genome. However, all efforts to identify ORF2-encoded proteins have failed so far. Here, applying a novel antibody we report the presence of proteins encoded by ORF2 in a subset of cellular components of human male gonads. Immunohistochemical analyses revealed coexpression of ORF1 and ORF2 in prespermatogonia of fetal testis, in germ cells of adult testis, and in distinct somatic cell types, such as Leydig, Sertoli, and vascular endothelial cells. Coexpression of both proteins in male germ cells is necessary for the observed genomic expansion of the number of L1 elements. Peptide mass fingerprinting analysis of a ϳ130-kDa polypeptide isolated from cultured human dermal microvascular endothelial cells led to the identification of ORF2-encoded peptides. An isolated ϳ45-kDa polypeptide was shown to derive from nonfunctional copies of ORF2 coding regions. The presence of both ORF1-and ORF2-encoded proteins in vascular endothelial cells and its apparent association with certain stages of differentiation and maturation of blood vessels may have functional relevance for vasculogenesis and/or angiogenesis. LINE-1 (long interspersed nuclear element) retrotransposons (L1)1 cover about 17% of the human genome (1) and play a significant role in shaping the mammalian genome, not only through their own expansion but also through the mobilization of non-L1 sequences. Although the average haploid human genome harbors ϳ516,000 L1 copies, the subgroup of active L1s is fairly small, encompassing 80 -100 elements (2, 3). So far, 82 retrotransposition-competent, full-length L1 elements were isolated and characterized (2-6). Remarkably, 84% of the retrotransposition capability of those elements was shown to be present in six highly active L1s (3). L1s affected the genome by (i) insertion of truncated L1s into new sites, (ii) intrachromosomal homologous recombination between L1s, (iii) transduction of 3Ј-flanking sequences during retrotransposition, (iv) aiding trans generation of processed pseudogenes and retrotransposition of Alu elements (7), and (v) by causing genome instability through substantial deletions (8, 9). A retrotransposition-competent, functional L1 element (RC-L1) covers ϳ6.1 kb and contains a 5Ј-untranslated region with an internal, CpG-rich promoter, a 1-kb ORF1 encoding a protein (p40) of approximately 40 kDa with RNA binding capability, followed by a 3.8-kb ORF2 coding for a protein (p150) with a predicted molecular mass of about 150 kDa with endonuclease (EN) and reverse transcription (RT) activities and a cysteine-histidine-rich domain. The 3Ј-end of L1 is terminated by a short 3Ј-untranslated region and a poly(A) tail (10) (Fig. 1A). L1 mRNAs ar...

Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression

“…In control (pS-neo) cells, the L1-encoded protein exhibits a discrete localization, close or adjacent to the nuclear envelope (panels a and b); in contrast, no signal is detected in the pS-L1 interfered cells (panels c and d). Western blot analysis ( Figure 2B) revealed the full-length protein (150 kDa) encoded by L1 ORF2, as well as lower molecular weight protein species, as expected (Ergun et al, 2004). Roles of LINE-1 and HERV-K in tumorigenesis E Oricchio et al relative to tubulin signals confirmed a drastic reduction (about 80%) of L1-encoded proteins in interfered (pS-L1i) compared to control (pS-neo) cell extracts ( Figure 2C).…”

“…The effectiveness of interference against L1 expression compared to control cells infected with empty vector (pS-neo) was assessed by immunofluorescence (IF), RT-PCR and immunoblotting analyses. Figure 2A shows an IF assay using an antibody (anti-L1EN), raised against polypeptides encoded by the endonuclease (EN)-domain of L1 ORF2 (Ergun et al, 2004), in A-375 cells infected with either vector alone (pS-neo), or with L1-interfering construct (pS-L1i). In control (pS-neo) cells, the L1-encoded protein exhibits a discrete localization, close or adjacent to the nuclear envelope (panels a and b); in contrast, no signal is detected in the pS-L1 interfered cells (panels c and d).…”

“…Fixed cells were preincubated in 20% goat serum for 30 min at 371C in a humidified chamber and incubated for 1 h with chicken polyclonal anti L1-EN antibody (Ergun et al, 2004;1:40 dilution) or with rabbit anti-env HERV-K antibody (1:70 dilution; Austral Biologicals, San Ramon, CA, USA). After three washes in phosphate-buffered saline (PBS) containing 0.05% Tween-20, cells were incubated with rhodamineconjugated anti-chicken or anti-rabbit antibody (1:600 dilution; Jackson Immunoresearch Laboratories, Westgrove, PA, USA, JIR).…”

“…Protein concentrations were determined using the Coomassie colorimetric assay (Pierce, Pero, Italy). Samples (20 mg) were fractionated on NuPAGE Novex 10% Bis-Tris gel (Invitrogen), transferred onto nitrocellulose membranes and processed for immunoblotting using chicken polyclonal anti L1-EN antibody (1:40 dilution) (Ergun et al, 2004) or mouse anti-atubulin (1:1000) (Sigma, T5168). HRP-conjugated anti-chicken IgY (Jackson Immunoresearch Laboratories, 703-035-155) or HRP-conjugated anti-mouse (Biorad, Segrate, Italy, 170-6516) secondary antibodies were used and signals were visualized using the enhanced chemiluminescence system (ECL Plus; Amersham Biosciences, Piscataway, NJ, USA).…”

Inhibition of endogenous reverse transcriptase antagonizes human tumor growth

“…At 72 h after L1-i transfection, expression of both ORF1 and ORF2 was reduced by almost 80% compared to cells transfected with noninterfering oligonucleotide (Figure 5c). To ascertain that the RT protein product was consistently downregulated in L1-i interfered cells, we made use of a recently developed antibody against LINE-1 ORF2-encoded RT (termed EN-L1, Ergun et al, 2004). Immunoprecipitation and Western blot assays indicate that RT protein levels 72 h after transfection are significantly reduced in L1-i cells (Figure 5d).…”