(Lines) and the Short (Sines) of It: Altered Methylation As a Precursor to Toxicity

TOXICOLOGICAL SCIENCES 75, 229–235 (2003) DOI: 10.1093/toxsci/kfg138

REVIEW The Long (LINEs) and the Short (SINEs) of It: Altered Methylation as a Precursor to Toxicity

Ammie N. Carnell and Jay I. Goodman1

Department of Pharmacology and Toxicology, Michigan State University, B-440 Life Science Building, East Lansing, Michigan 48824 Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021

Received March 28, 2003; accepted April 28, 2003

DNA methylation plays a key role in maintaining stability of Although once thought of as “junk” DNA, the importance of the genome by silencing the expression of mobile, retrotrans- interspersed elements in the genome has become increasingly posable elements. Altered DNA methylation, a secondary, epi- appreciated in recent years. In a broad sense these are collectively genetic change (Goodman and Watson, 2001), may facilitate referred to as transposable elements, which encompass both trans- expression of these elements leading to a cascade of detrimen- posons and retrotransposons. The latter include long interspersed tal events resulting in mutation and decreased stability of the nuclear elements (LINEs) and short interspersed nuclear elements genome. This review extends the general notion that the study (SINEs). Expression of these elements leads to genetic instability. of altered DNA methylation has come of age in toxicological Therefore, it is important that they remain transcriptionally si- sciences (Watson and Goodman, 2002) by focusing upon a lenced, and DNA methylation plays a key role in this regard. A specialized area. We put forth a framework for understanding framework for understanding the possible interplay between al- the interplay between epigenetic and mutational events and tered DNA methylation, an epigenetic change, and mutational events is presented. A case is made as to how retrotransposable indicate how retrotransposable elements are likely to emerge as elements, specifically LINEs and SINEs, are likely to emerge as an important player in furthering our understanding of mech- key players in furthering our understanding of mechanisms un- anisms underlying a variety of toxicities, including carcinogen- derlying a variety of toxicities, including carcinogenesis but not esis but not limited to this endpoint. limited to this endpoint. Key Words: DNA methylation; epigenetics; LINEs, retrotrans- Overview of Retroelements posons; transposable elements; SINEs. Although once thought of as “junk” DNA, the importance of interspersed elements in the genome has become increasingly Conventional thinking has supported the notion that muta- appreciated in recent years. It has been estimated that at least tion/altered genome stability, produced in response to treat- one third of the mammalian genome consists of these elements ment with a chemical or physical agent, has as its basis one or in various forms (Yates et al., 1999). In a broad sense they are a combination of the following: adduct formation, inhibition of collectively referred to as transposable elements, which en- apoptosis, strand breaks in DNA, altered DNA repair, and compass both transposons and retrotransposons (Fig. 1). Trans- increased cell proliferation resulting in an increased chance for posons have inverted terminal repeats, encode a transposase “errors” during DNA replication (Preston and Hoffmann, activity, and move from one site to another through a “cut and 2001). However, another key factor may be involved. This paste” mechanism (Smit and Riggs, 1996). Retrotransposons, review focuses on (1) mutagenesis and/or genome stability as which move by a “copy and paste” mechanism, proceed a consequence of transcriptional activation of mobile elements through an RNA intermediate largely dependent on their en- and (2) how altered DNA methylation, an indirect, secondary coded reverse transcriptase activity. However, they may utilize mechanism, may underlie the increased expression of these the host’s reverse transcriptase (Ostertag and Kazazian, 2001). elements. It is appropriate to reflect upon the toxicological In this manner a copy of the original can be integrated into a significance of these mobile elements. new genomic location. Therefore, stability of the genome de- pends upon keeping these movable and amplifiable elements 1 To whom correspondence should be addressed. Fax: 517-353-8915. E- transcriptionally repressed. DNA methylation plays a key role mail: [email protected]. in the regulation of gene expression overall, including keeping Toxicological Sciences 75(1), © Society of Toxicology 2003; all rights reserved. transposable elements transcriptionally silent (Robertson and

229 230 REVIEW

FIG. 1. Transposable elements encompass both transposons and retrotransposons. (1) Transposons have inverted terminal repeats (ITR), which act as cis elements in the integration process, and two (or more) open reading frames (ORF), one of which encodes a transposase activity. These elements move by a “cut and paste” mechanism. (2) Retro- transposons are divided into autonomous and nonautonomous elements. Autono- mous elements include long terminal repeat (LTR) and non-LTR subgroups. LTR-containing elements are structurally similar to retroviruses although they lack Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021 a functional env gene. Non-LTR elements contain an internal promoter for RNA polymerase II, a 5Ј untranslated region (UTR) and a 3Ј deoxyadenosine (A)-rich tract. Nonautonomous elements (SINEs) contain an internal promoter for RNA polymerase III and a 3Ј A-rich tract. Modiﬁed from Deininger and Roy- Engel (2002) and reprinted with permission from ASM Press, Washington, DC.

Wolffe, 2000). Altering the methylation status of repetitive currently the object of a flurry of research concerning their elements may foster a surge of events leading to toxicity, and mechanism of transposition, their role in the activation and a consideration of this is the focal point of this review. movement of nonautonomous elements, and the health-related consequences of the movement of these elements within the Retrotransposable Elements: LINEs and SINEs genome. Retrotransposable elements are categorized as either auton- An additional, important category of non-LTR interspersed omous or nonautonomous elements, where autonomous refers elements is the nonautonomous retrotransposons. These are to the property of self-sufficiency for mobility. There are two short interspersed elements (SINEs), which are approximately classes of autonomous elements: long terminal repeat (LTR) 80–400 bp in length and require activities encoded by the and non-LTR retrotransposons. Similar in structure to retrovi- autonomous retrotransposons and/or the host for their mobility ruses, although lacking a functional env gene, LTR retrotrans- (Ostertag and Kazazian, 2001). Unlike LINEs, SINEs have an Ј posons encode proteins necessary for retrotransposition. Like- internal promoter for RNA polymerase III and a 3 A-rich tract wise, non-LTR retrotransposons also encode a reverse (Weiner, 2002). The most prominent SINEs are the human Alu transcriptase and endonuclease that play a role in their ability elements and the rodent B1 elements. to mobilize themselves and other nonautonomous elements Movement of Transposable Elements (Ostertag and Kazazian, 2001). A basic difference between the LTR and non-LTR retrotransposons is their method of recom- Human L1 elements have been shown in vitro to ultimately bination. LTR retrotransposons move by first being transcribed move through a target-primed reverse transcription (TPRT) into RNA, followed by reverse transcription leading to a DNA mechanism (Cost et al., 2002). Briefly, full-length L1 elements copy that recombines with genomic DNA. Non-LTR retro- are transcribed from their internal promoter to produce a bi- transposons move through a somewhat different RNA-medi- cistronic mRNA (Ostertag and Kazazian, 2001). Association of ated event, discussed below (Eickbush and Malik, 2002). Up to a 7.5kb, sense strand mouse L1 RNA with protein from ribo- several kb in length, the non-LTR retrotransposons are com- nucleoproteins (RNP) has been demonstrated (Martin, 1991). monly referred to as long interspersed elements (LINEs). ORF1 protein from both mouse and human embryonal carci- Structurally, they contain an internal promoter for RNA poly- noma cells will bind L1 RNA, and this has been suggested as merase II, a 5Ј untranslated region (UTR), two open reading a mechanism of “coating” the RNA in the process of forming frames (ORFs), and a 3Ј terminal polyadenylation site (Loeb et. a RNP (Kolosha and Martin, 1997). Similarly, the 40kDa al., 1986). The ORF1 protein is an RNA binding protein product of the first ORF of L1Hs (Homo sapiens) associates (Kolosha and Martin, 1997), while ORF2 encodes both a with L1Hs RNA to form a large multimeric complex (Hohjoh reverse transcriptase and a DNA endonuclease (Weiner, 2002). and Singer, 1996). Once entry into the nucleus is obtained, L1 Most notable among the lines are the L1 elements, which are RNA is reverse transcribed via its own encoded reverse tran- REVIEW 231 scriptase or an endogenous enzyme (Evans and Palmiter, 1991) pears to be the prime regulator in terms of inhibition of and integrated into the targeted site via TPRT (Ostertag and transcription and formation of heterochromatin (Fahrner et al., Kazazian, 2001). Truncations, producing inactive elements, 2002). The consequences of aberrant DNA methylation in frequently occur just prior to integration as a consequence of terms of LINEs and SINEs is potentially far reaching when RNase H digestion or the inability of the reverse transcriptase taking into account that methylation may regulate expression to efficiently copy the entire L1 RNA before dissociating and movement of not only the parent LINEs but also SINEs (Ostertag and Kazazian, 2001). These L1 element fragments (Kajikawa and Okada, 2002). are functionally inactive and hence “litter” the genome. Addi- The concept of methylation as a genome defense system tionally, LINEs may facilitate the movement of SINEs. Re- assumes that retrotransposable elements are inherently detri- cently, using a HeLa cell retrotransposition assay, an eel (An- mental to the genome. Protection and conservation of the guilla japonica) SINE with a similar 3Ј end to a partner eel integrity and fidelity of an organism’s DNA serves as the

LINE was mobilized by the retrotransposition machinery of overriding goal. Therefore, an important aspect of DNA meth- Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021 that partner LINE (Kajikawa and Okada, 2002). This, then, ylation is its connection to the host-defense system, which acts supports the notion that SINEs may exploit LINEs as a mech- to offset the threats from these largely parasitic sequences by anism of retrotransposition. maintaining them in a methylated, transcriptionally silent state (Yoder et al., 1997). Genome instability is a common feature Distribution and Abundance of tumorigenesis (Loeb, 2001). Extensive hypomethylation results in genome instability reflected by an increase in muta- Overall, approximately 33% of the human genome consists tion frequency (Chen et al., 1998). Hypomethylation-induced of transposable elements (Yoder et al., 1997), and they are transcriptional activation of LINEs and SINEs contributes to distributed in a nonrandom fashion. According to the Human this instability (Takai et al., 2000). Furthermore, hypomethy- Genome Project Data, ϳ26% of the sequences on the X chro- lation of LINE-1 sequences has been observed in various mosome are L1 sequences, while the percentages in chromo- cancers, such as colon cancer and chronic lymphocytic leuke- somes 21 and 22 are approximately 13% and 10%, respectively mia (Dante et al., 1992). More recently, when nine human (Hattori et al., 2000). In 2001, Ovchinnikov et al. hypothesized hepatocellular carcinoma samples were tested, eight samples that distribution of L1 elements occurs as a result of selection showed evidence of L1 hypomethylation. Even more interest- either for the retention of L1s in regions of low GC content or ing is the finding that hypomethylated L1 sequences in the for the loss of L1s from regions of high GC content. The same surrounding noncancerous liver tissue were absent (Takai et reasoning may not apply to SINEs, in particular Alu elements. al., 2000). The story is much the same for urothelial carcino- Imprinted regions of the genome carry a very low density of mas, where hypomethylation of the L1 sequences is consis- SINE elements such as Alu elements (Greally, 2002). This tently seen as compared to the normal bladder mucosa (Ju¨r- raises questions concerning the accessibility of LINEs to im- gens, 1996). This led to the speculation that demethylation of printed regions versus a possible active selection process main- LINE-1 sequences may promote genomic instability and facil- taining the intolerance to SINEs (Greally, 2002). The impor- itate tumor progression (Ju¨rgens, 1996). tance of these elements has been stressed in the context of An early study which looked at the link between DNA mammalian genomes, but it should be noted that they, partic- methylation and gene expression found that infectious proviral ularly SINEs, are present in both mammalian and nonmamma- DNA could be generated following removal of methyl groups lian species (Borodulina and Kramerov, 1999). from an inactive viral genome (Harbers et al., 1981). This initial look at the transcriptional regulation of an endogenous DNA Methylation Silences Transposable Elements retroviral genome helped to spur a parallel interest in DNA It is instructive to consider the role of altered methylation as methylation influence over retrotransposons. L1 sequences in an epigenetic mechanism for the activation of retrotransposable the mouse, human, and rat have been characterized structurally elements leading to their expression and possible retrotranspo- as having a promoter region which controls the expression of sition (Fig. 2). Given the sheer number and distribution of these the two open reading frames, ORF1 and ORF2 (Nur et al., elements, both their movement and expression can lead to 1988; Severynse et al., 1992; Swergold, 1990). Expression of unstable conditions within the genome. The adverse conse- these regions, encoding an RNA binding protein, a reverse quences associated with the movement of these elements are transcriptase, and an endonuclease, is required for integration diverse and will be discussed below. In order for the genome to of a new copy of the original element into the genome. For this maintain its integrity, retrotransposable elements need to be reason, a closer look at the methylation status of the promoter regulated. Methylation of cytosines to produce 5-methyl cyto- region is a focus of investigation. A foundation was laid by sine plays an important role as a transcriptional regulator Thayer in 1993 with the finding that hypomethylation of the 5Ј (Jones and Baylin, 2002; Roberston and Wolffe, 2000). In region of L1Hs correlated with increased amounts of ORF1 general, increased methylation is associated with decreased protein, and specific nucleotides were alluded to as critical gene expression. Indeed, in mammalian cells, methylation ap- regulatory sites for methylation. Subsequent to these findings, 232 REVIEW Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021

FIG. 2. Schematic representation of an epigenetic change as a precursor to expression and movement of retrotransposable elements. (1) An epigenetic change, e.g., hypomethylation of the retrotransposable element allows for (2) enhanced transcriptional activity. (3) RNA processing and (4) mRNA export ensue. (5) Translation and (6) posttranslational modification precede the formation of a ribonucleoprotein particle in which ORF1 and ORF2 encoded proteins are associated with the original mRNA. (7) Once entry into the nucleus has occurred, (8 and 9) reverse transcription and integration are achieved via the encoded reverse transcriptase and endonuclease through a mechanism termed target primed reverse transcription (TPRT). Modified from Ostertag and Kazazian, Jr. (2001), and reprinted with permission from the Annual Review of Genetics, vol. 35 © 2001, by Annual Reviews, www.annualreviews.org. the effect of methylation on transcription was examined in vivo the role of methylation in silencing SINEs (Kochanek et al., by transfecting a plasmid containing a L1 promoter linked to 1993). In addition, cell-free transcription experiments sug- chloramphenicol acetyl transferase reporter (CAT) gene into gested that alu element transcription in vitro can be inhibited HeLa cells and in vitro by performing transcription studies by 5ЈCG 3Ј methylation (Kochanek et al., 1993). Alu se- with mutagenized templates (Hata and Sakaki, 1997). Here it quences are structurally set up as a right and left monomer was found that methylation of the L1 promoter completely linked by an oligo-d(A) tract. The left monomer, only, contains suppressed the transcription of the CAT gene, and methylation the functional two box RNA polymerase III (pol III) promoter, of four particular CpG sites was necessary and sufficient to but curiously no terminator sequence is found within the alu inhibit L1 transcription in vitro (Hata and Sakaki, 1997). element; downstream RNA polymerase III terminators are SINEs, being much a counterpart to LINEs, also fall under utilized (Fuhrman et al., 1981). Based on artificial methylation the influence of methylation status. Certain human alu ele- of CpG sites, methylation anywhere within or near the internal ments associated with the genes for ␣1 globin, tissue plasmin- pol III promoter can result in transcriptional repression (Liu ogen activator, adrenocorticotropic hormone, and angiogenin, and Schmid, 1993). However, the context of the methylation were found to display a high degree of methylation supporting patterning may also positively affect the transcriptional re- REVIEW 233 sponse (Vorce et al., 1994). It was postulated that transcrip- refer to Table 1 of Ostertag and Kazazian (2001). Additionally, tional inhibition could be additionally dependent on a methyl- altered regulation of gene expression by insertion of L1 ele- ated DNA binding protein. The loss of inhibition and, in fact, ments as a direct mutation has been documented numerous activation may then be explained by high template concentra- times. Britten (1997) cited twenty-one examples of sequence tions, which effectively titrate out the methylated DNA binding element inclusion from Drosophila, sea urchin, human, and protein (Vorce et al., 1994). One more possibility for selective mouse genomes that serve a function in terms of transcriptional control over these elements lies with the context of the sur- competency. rounding regions. The methylation and chromatin patterns of Counterpart to insertions are deletions and duplications, regions or genes surrounding alu elements could also mediate which can arise from unequal crossing-over and mispairing of the transcriptional activity of alu elements rendering transcrip- homologous L1 sequences (Kazazian and Goodier, 2002). As tionally competent elements incompetent (Schmid, 1991) or much as a 3% frequency of DNA deletions due to L1 retro-

vice versa. transposition has been proposed (Kazazian and Goodier, 2002). Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021 The mouse genome has proved useful for SINE research in Gilbert et al. (2002) observed a large deletion of the genomic utilizing the B1 and B2 elements, counterparts to human alu DNA following the retrotranspositional event. A common elements. On par with topics discussed for alu elements is that mechanism preceding this deletion, among other alterations, of the function and genomic target sites of de novo and main- was shown to involve cleavage of the genomic top strand. tenance methylation enzymes. When asking whether B1 ele- Variations of this model also suggest that chimaeric L1s, large ments provide a target for de novo methylation, an 838-base deletions, and long duplications are also possible (Gilbert et pair methylation center located upstream of the mouse adenine al., 2002). Clinically, inactivating mutations arising from L1- phosphoribosyltransferase gene was targeted. This region was mediated recombination can lead to the accumulation of mu- previously described as serving as a de novo methylation signal tations in specific target genes during cancer and development upon transfection into mouse embryonal carcinoma (EC) cells (Viel et al., 2002). This highlights the fact that L1 elements are (Yates et al., 1999). Two tandem B1 elements located at the 3Ј capable of reshaping the genome through direct mutation. end of the methylation center are methylated at relatively high Mutation as a result of movement of L1 elements is not the levels in embryonic cells with severe maintenance methylation only method for creating instability within the genome. Aber- deficiency but intact de novo activity (Yates et al., 1999). Upon rant activity in terms of expression of both LINEs and SINEs transfection, the B1 elements became methylated de novo, can result in altered gene expression and/or production of indicating a significant role in the methylation center (Yates et mRNAs bearing some sequence combination of the transpos- al., 1999). able element, the original gene, or both. Antisense promoters provide a recent example of the impact of altered gene expres- Genomic Consequences sion (Speek, 2001). The power and capability of antisense At the forefront of genomic consequences due to retrotrans- promoters is not limited by distance from protein coding se- poson expression and movement is insertional mutagenesis. quences, and they are scattered over the chromosomes, which Insertion of these elements, whether random or targeted, rep- only increases their importance in terms of transcriptional resents a mutation, and therefore, retrotransposition poses a control and command (Speek, 2001). In addition to antisense clear risk to the stability of the genome. Not only is movement promoters, transcriptional interference and, hence, altered gene of these elements critical but also their capability to transduce expression, can arise when a retrotransposon interrupts a gene. surrounding DNA sequences. At times this may promote This creates an environment for internal initiation via the genomic diversification (Moran et al., 1999), but more appar- transposable element promoter or disrupted transcriptional ini- ent is the possible contribution to mutagenicity. On a larger tiation via the endogenous promoter. Resulting from this, chi- scale, L1 integrants and/or their transduced sequences can maeric mRNA molecules may be generated (Robertson and result in chromosomal rearrangements (Symer et al., 2002). Wolffe, 2000). In simpler terms, retrotransposons can increase Medically, muscular dystrophy, characterized by a progressive the level of nonfunctional or error-prone mRNAs. B2 SINEs loss of muscle strength which develops around age 10 in were implicated in providing a pol II promoter at sites through- humans, has been associated with a L1 insertion within exon out the genome, likely a method for creating ectopic start sites 48 of the dystrophin gene (Holmes et al., 1994). These findings leading to erroneous mRNA production (Ferrigno et al., 2001). supported recent L1 retrotransposition activity (Holmes et al., SINEs scattered throughout introns allow for pol III-derived 1994) and directly demonstrated the consequential toxicity transcription of genes. In addition to this, pol II transcription associated with the aberrant regulation of these elements. L1 initiated from neighboring regions may also alter the nature of insertions account for 13 isolated cases of human disease, mRNA products. Decreased mRNA levels and subsequent while alu insertions account for 19 cases (Ostertag and Kaza- protein levels, along with truncated proteins, may also be a zian, 2001). For a comprehensive and detailed list of human result of aberrant activity of LINEs and SINEs. Antisense disease resulting from movement of transposable elements, mechanisms initiating degradation of the message have been 234 REVIEW suggested as a possible cause of this (Robertson and Wolffe, patterns of long interspersed repeated DNA and alphoid repetitive DNA 2000). from human cell lines and tumors. Anticancer Res. 12, 559–564. Deininger, P. L., and Roy-Engel, A. M. (2002). Mobile elements in animal and plant genomes. In Mobile DNA II (N. L. Craig, R. Craigie, M. Gellert, and Toxicological Implications A. M. Lambowitz, Eds.), pp. 1074–1092. ASM Press, Washington, DC. Deviant transposable element activity has been presented as Eickbush, T. H., and Malik, H. S. (2002). Origins and evolution of retrotrans- a possible mechanism underlying toxicity. The mutagenicity posons. In Mobile DNA II (N. L. Craig, R. Craigie, M. Gellert, and A. M. Lambowitz, Eds.), pp. 1111–1144. ASM Press, Washington, DC. and/or altered genome stability resulting from expression and Evans, J. P., and Palmiter, R. D. (1991). Retrotransposition of mouse L1 movement of transposable elements, in particular, LINEs and element. Proc. Natl. Acad. Sci. U.S.A. 88, 8792–8795. SINEs, may occur subsequent to epigenetic changes, e.g., Fahrner, J. A., Eguchi, S., Herman, J. G., and Baylin, S. B. (2002). Dependence altered DNA methylation. Interestingly, there are multiple of histone modifications and gene expression on DNA hypermethylation in ways that DNA methylation may be modified (Goodman and cancer. Cancer Res. 62, 7213–7218. Watson, 2002). This can occur, for example, as a consequence Ferrigno, O., Virolle, T., Djabari, Z., Ortonne, J. P., White, R. J., and Aberdam, Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021 of inhibition of DNA methyltransferases (Trinh et al., 2002), D. (2001). Transposable B2 SINE elements can provide mobile RNA altered levels of methyl donors, and disregulation of one- polymerase II promoters. Nat. Genet. 28, 77–81. carbon metabolism (Sibani et al., 2002). Furthermore, DNA Fuhrman, S. A., Deininger, P. L., LaPorte, P., Friedmann, T., and Geiduschek, adduct formation can also lead to altered methylation, which E. P. (1981). Analysis of transcription of the human Alu family ubiquitous repeating element by eukaryotic RNA polymerase III. Nucleic Acids Res. 9, might persist after the adduct is repaired (Turk et al., 1995). In 6439–6456. this manner, there exists a possible interplay between epige- Gilbert, N., Lutz-Prigge, S., and Moran, J. V. (2002). Genomic deletions netic changes, mutation, and altered stability of the genome, created upon LINE-1 retrotransposition. Cell 110, 315–325. bearing in mind that the altered DNA methylation leading to Goodman, J. I., and Watson, R. E. (2001). Altered DNA methylation: A transcriptional activation of LINEs and SINEs may occur by a secondary mechanism involved in carcinogenesis. Annu. Rev. Pharmacol. secondary, threshold-exhibiting mechanism (Goodman and Toxicol. 42, 501–525. Watson, 2001). Within this context, it is important to note that Greally, J. M. (2002). Short interspersed transposable elements (SINEs) are DNA methylation is more stable in human cells as compared to excluded form imprinted region sin the human genome. Proc. Natl. Acad. Sci. U.S.A. 99, 327–332. rodent cells (reviewed in Goodman and Watson, 2002). There- Harbers, K., Schnieke, A., Stuhlmann, H., Jahner, D., and Jaenisch, R. (1981). fore, compounds that produce toxicity in rodents, through a DNA methylation and gene expression: Endogenous retroviral genome mechanism involving hypomethylation and consequent in- becomes infectious after molecular cloning. Proc. Natl. Acad. Sci. U.S.A. 78, creased transcriptional activity of transposable elements, may 7609–7613. have a relatively low potential to cause toxicity in humans. Hata, K., and Sakaki, Y. (1997). Identification of critical CpG sties for However, even though humans may possess a “built in repression of L1 transcription by DNA methylation. Gene 189, 227–234. safety factor” when compared to rodents, exploration of Hattori, M., Fujiyama, A., Taylor, T. D., Watanabe, H., Yada, T., Park, H.-S., epigenetic mechanisms initializing transposable element activ- Toyoda, A., Ishii, K., Totoki, Y., Choi, D.-K., et al. (2000). The DNA sequence of human chromosome 21. Nature 405, 311–318. ity should not simply be discounted as a basis for potential Hohjoh, H., and Singer, M. F. (1996). Cytoplasmic ribonucleoprotien com- toxicity. plexes containing human LINE-1 protein and RNA. EMBO J. 15, 630–639. Holmes, S. E., Dombroski, B. A., Krebs, C. M., Boehm, C. D., and Kazazian, H. H., Jr. (1994). A new retrotransposable human L1 element from the ACKNOWLEDGMENT LRE2 locus on chromosome 1q produces a chimaeric insertion. Nat. Genet. 7, 143–148. A.N.C. is a predoctoral fellow, supported by NIH-NIEHS Training Grant Jones, P. A., and Baylin, S. B. (2002). The fundamental role of epigenetic ES-04911. events in cancer. Nature Rev. 3, 415–428. Ju¨rgens, B., Schmitz-Dra¨ger, B. J., and Schulz, W. A. (1996). Hypomethyla- tion of L1 LINE Sequences prevailing in human urothelial carcinoma. REFERENCES Cancer Res. 56, 5698–5703. Kajikawa, M., and Okada, N. (2002). LINEs mobilize SINEs in the eel through Borodulina, O. R., and Kramerov, D. A. (1999). Wide distribution of short a shared 3Ј sequence. Cell 111, 433–444. interspersed elements among eukaryotic genomes. FEBS Lett. 457, 409– Kazazian, H. H., Jr., and Goodier, J. L. (2002). LINE Drive: Retrotransposition 413. and genome instability. Cell 110, 277–280. Britten, R. J. (1997). Mobile elements inserted in the distant past have taken on Kochanek, S., Renz, D., and Doerfler, W. (1993). DNA methylation in the Alu important functions. Gene 205, 177–182. sequences of diploid and haploid primary human cells. EMBO J. 12, Chen, R. Z., Pettersson, U., Beard, C., Jackson-Grusby, L., and Jaenisch, R. 1141–1151. (1998). DNA hypomethylation leads to elevated mutation rates. Nature 395, Kolosha, V. O., and Martin, S. L. (1997). In vitro properties of the first ORF 89–93. protein from mouse LINE-1 support its role in ribonucleoprotein particle Cost, G. J., Feng, Q., Jacquier, A., and Boeke, J. D. (2002). Human L1 element formation during retrotransposition. Proc. Natl. Acad. Sci. U.S.A. 94, target-primed reverse transcription in vitro. EMBO J. 21, 5899–5910. 10155–10160. Dante, R., Dante-Paire, J., Dominique, R., and Roizes, G. (1992). Methylation Liu, W. M., and Schmid, C. W. (1993). Proposed roles for DNA methylation REVIEW 235

in alu transcriptional repression and mutational inactivation. Nucleic Acids Speek, M. (2001). Antisense promoter of human L1 retrotransposon drives Res. 21, 1351–1359. transcription of adjacent cellular genes. Mol. Cell. Biol. 21, 1973–1985. Loeb, D. D., Padgett, R. W., Hardies, S. C., Shehee, W. R., Comer, M. B., Swergold, G. D. (1990). Identification, characterization, and cell specificity of Edgell, M. H., and Hutchison, C. A., III. (1986). The sequence of a large a human LINE-1 promoter. Mol. Cell. Biol. 10, 6718–6729. Ј L1Md element reveals a tandemly repeated 5 end and several features Symer, D. E., Connelly, C., Szak, S. T., Caputo, E. M., Cost, G. J., Parmigiani, found in retrotransposons. Mol. Cell. Biol. 6, 168–182. G., and Boeke, J. D. (2002). Human L1 retrotransposition is associated with Loeb, L. A. (2001). A mutator phenotype in cancer. Cancer Res. 61, 3230– genetic instability in vivo. Cell 110, 327–338. 3239. Takai, D., Yagi, Y., Habib, N., Sugimura, T., and Ushijima, T. (2000). Martin, S. L. (1991). Ribonucleoprotein particles with LINE-1 RNA in mouse Hypomethylation of LINE1 retrotransposon in human hepatocellular carci- embryonal carcinoma cells. Mol. Cell. Biol. 11, 4804–4807. nomas, but not in surrounding liver cirrhosis. Jpn. J. Clin. Oncol. 30, 306–309. Moran, J. V., DeBerardinis, R. J., and Kazazian, H. H., Jr. (1999). Exon Thayer, R. E., Singer, M. F., and Fanning, T. G. (1993). Undermethylation of shuffling by L1 retrotransposition. Science 283, 1530–1534. specific LINE-1 sequences in human cells producing a LINE-1 encoded Nur, I., Pascale, E., and Furano, A. V. (1988). The left end of rat L1 (L1Rn, protein. Gene 133, 273–277. long interspersed repeated) DNA which is a CpG island can function as a Downloaded from https://academic.oup.com/toxsci/article/75/2/229/1655841 by guest on 28 September 2021 Trinh, B. N., Long, T. I., Nickel, A. E., Shibata, D., and Laird, P. W. (2002). promoter. Nucleic Acids Res. 16, 9233–9249. DNA methyltransferase deficiency modifies cancer susceptibility in mice Ostertag, E. M., and Kazazian, H. H., Jr. (2001). Biology of mammalian L1 lacking DNA mismatch repair. Mol. Cell. Biol. 22, 2906–2917. retrotransposons. Annu. Rev. Genet. 35, 501–538. Turk, P. W., Laayoun, A., Smith, S. S., and Weitzman, S. A. (1995). DNA Ovchinnikov, I., Troxel, A. B., and Swergold, G. D. (2001). Genomic char- adduct 8-hydroxyl-2Ј-deoxyguanosine (8-hydroxyguanine) affects function acterization of recent human LINE-1 insertions: Evidence supporting ran- of human DNA methyltransferase. Carcinogenesis 16, 1253–1255. dom insertion. Genome Res. 11, 2050–2058. Viel, A., Petronzelli, F., Della Puppa, L., Lucci-Cordisco, E., Fornasarig, M., Preston, R. J., and Hoffman, G. R. (2001). Genetic toxicology. In Cassarett Pucciarelli, S., Rovella, V., Quaia, M., Ponz de Leon, M., and Boiocchi, M., and Doull’s Toxicology The Basic Science of Poisons (C. D. Klaasen, Ed.), Genuardi, M. (2002). Different molecular mechanisms underlie genomic 6th ed., pp. 321–350. McGraw-Hill Medical Publishing Divison, New York. deletions in the MLH1 gene. Hum. Mutat. 20, 368–374. Robertson, K. D., and Wolffe, A. P. (2000). DNA methylation in health and disease. Nature Rev. 1, 11–19. Vorce, R. L., Lee, B., and Howard, B. H. (1994). Methylation- and mutation- dependent stimulation of alu transcription in vitro. Biochem. Biophys. Res. Schmid, C. W. (1991). Human alu subfamilies and their methylation revealed Comm. 203, 845–851. by blot hybridization. Nucleic Acids Res. 19, 5613–5617. Watson, R. E., and Goodman, J. I. (2002). Epigenetics and DNA methylation Severynse, D. M., Hutchinson, C. A., III, and Edgell, M. H. (1992). Identifi- come of age in toxicology. Toxicol. Sci. 67, 11–16. cation of transcriptional regulatory activity within the 5Ј A-type monomer sequence of the mouse LINE-1 retroposon. Mamm. Genome 2, 41–50. Weiner, A. M. (2002). SINEs and LINEs: The art of biting the hand that feeds Sibani, S., Melnyk, S., Pogribny, I. P., Wang, W., Hiou-Tim, F., Deng, L., you. Curr. Opin. Cell Biol. 14, 343–350. Trasler, J., James, S. J., and Rozen, R. (2002). Studies of methionine cycle Yates, P. A., Burman, R. W., Mummaneni, P., Krussel, S., and Turker, M. S. intermediates (SAM, SAH), DNA methylation and the impact of folate (1999). Tandem B1 elements located in a mouse methylation center provide deficiency on tumor numbers in Min mice. Carcinogenesis 23, 61–65. a target for de Novo DNA methyation. J. Biol. Chem. 274, 36357–36361. Smit, A. F. A., and Riggs, A. D. (1996). Tiggers and other DNA transposons Yoder, J. A., Walsh, C. P., and Bestor, T. H. (1997). Cytosine methylation and fossils in the human genome. Proc. Natl. Acad. Sci. U.S.A. 93, 1443–1448. the ecology of intragenomic parasites. Trends Genet. 13, 335–340.