Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells

648–665 Nucleic Acids Research, 2008, Vol. 36, No. 2 doi:10.1093/nar/gkm1045 Published online 10 December 2007 Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells Alexander Kirilyuk1, Genrich V. Tolstonog1, Annette Damert2, Ulrike Held2, Silvia Hahn2, Roswitha Löwer2, Christian Buschmann2, Axel V. Horn2, Peter Traub1 and Gerald G. Schumann2,* 1 Max-Planck-Institut für Zellbiologie, Rosenhof, D-68526 Ladenburg and 2Paul-Ehrlich-Institut, Section PR2/Retroelements, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany Received March 24, 2007; Revised November 1, 2007; Accepted November 2, 2007 INTRODUCTION LINE-1 (L1) is a highly successful autonomous nonLTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia. LINE-1s (Long Interspersed Nuclear Element-1s or L1s) represent a family of abundant non-long-terminal repeat (non-LTR) retrotransposons in mammalian genomes. More than 99.8% of L1s are retrotransposition-defective because they are 50 truncated, contain internal rearrangements or harbor mutations within their open reading frames (ORFs) (1–3). However, the average human genome is estimated to contain 80–100 retrotransposition-competent (RC) L1s, and
10% of these elements are classified as highly active or ‘hot’ (4). By comparison, the mouse genome is estimated to contain at least 3000 active L1s (5,6). L1s have shaped mammalian genomes through a number of mechanisms (7,8). First, they have greatly expanded the genome both by their own retrotransposition and by providing the machinery necessary for the retrotransposition of other mobile elements, such as Alus and processed pseudogenes. Second, they have shuffled non-L1 sequences throughout the genome by a process termed transduction. Third, they have affected gene expression by several mechanisms. The best-studied L1s are those found in mice and men. There are at least 599 000 copies of mouse L1 (L1Md) that are interspersed in all chromosomes, and together comprise
19% of the genomic DNA (9). RC L1Mds are
6500 nt in length. Full-length RC-L1s have a 50 untranslated region (UTR) with internal promoter activity, two ORFs, a 30 UTR that ends in an AATAAA polyadenylation signal and a poly A tail. The role of the ORF1-encoded protein (ORF1p) is poorly understood. ORF1p represents a basic protein that co-fractionates as a *To whom correspondence should be addressed. Tel: +49 6103 773 105; Fax: +49 6103 771 265; Email: Correspondence may also be addressed to Peter Traub. Tel: +49 228 739 573; Fax: +49 228 739 004; Email: ; Genrich V. Tolstonog. Tel: +49 4048 051 235; Fax: +49 4048 051 117; Email: Present addresses: Alexander Kirilyuk, Georgetown University, Lombardi Cancer Center, Washington, DC 20007, USA. Genrich V. Tolstonog, Heinrich-Pette Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, D-20251 Hamburg, Germany. Peter Traub, Institut für Zelluläre und Molekulare Botanik, Universität Bonn, D-53115 Bonn, Germany. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Nucleic Acids Research, 2008, Vol. 36, No. 2 649 cardiac tissue after ischemia-reperfusion and the presence of L1-encoded proteins could be demonstrated (34). Here we describe isolation and characterization of the first functional rat LINE-1 retrotransposon from a pool of 10 different cDNAs generated from actively transcribed endogenous full-length L1Rn elements which are all coding for intact ORF1ps in rat chloroleukemia (RCL) cells. We determined the retrotransposition activity of this active L1Rn element in HeLa cells as well as in the RCL cell line from which the cDNAs originated. This is the first report on a functional endogenous L1 element that is mobilized in the tumor cell line in which it was shown to be endogenously expressed. In order to investigate L1 translation and subcellular localization of L1-encoded proteins in rat cells, we generated monoclonal antibodies directed against recombinant ORF1p and ORF2p. Our experiments revealed that both L1-encoded proteins are expressed from endogenous elements in RCL cells. While endogenous ORF1 proteins appeared almost exclusively in the nucleus, transiently expressed ORF1p localized predominantly to the cytoplasm. Localization studies with ORF1p deletion mutants indicate that the N-terminal third of ORF1p has a function in cytoplasmic retention and targeting to cytoplasmic intermediate filaments. Our data demonstrating expression and retrotransposition of functional endogenous L1 elements in RCL cells support the idea that L1 activity might be at least one cause for the accumulation of molecular and chromosomal abnormalities leading to genomic instability which is characteristic for the progression of leukemia. MATERIALS AND METHODS Cell culture and UV irradiation RCL cells growing in suspension culture (30) and HeLa S3 cells were kindly provided by K. Servomaa and by Dr A. Shatkin, respectively, and maintained in DMEM containing 10% FCS. Rat embryo fibroblasts (REFs) and mouse embryo fibroblasts (MEFs) from whole embryos were cultured in DMEM/10% FCS. Cells were transfected using FUGENE 6 lipid reagent (Roche). RCL cells were UV irradiated under following conditions: 312 nm (VL-215M; Bioblock Scientific), 4.65 mW/cm2, as measured with a VLH-3W radiometer equipped with (...truncated)