3 LTR retrotransposons and the evolution of eukaryotic enhancers John F. McDonald, Lilya V. Matyunina, Susanne Wilson, I. King Jordan, Nathan J. Bowen & Wolfgang J. Miller Department of Genetics, University of Georgia, Athens, GA 30602, USA Accepted 22 April 1997 Key words: long terminal repeat retrotransposon, transposable element, enhancer, gene expression, copia/Drosophila Abstract Since LTR retrotransposons and retroviruses are especially prone to regional duplications and recombination events, these viral-like systems may be especially conducive to the evolution of closely spaced combinatorial regulatory motifs. Using the Drosophila co pia LTR retrotransposon as a model, we show that a regulatory region contained within the element's untranslated leader region (ULR) consists of multiple copies of an 8 bp motif (TTGTGAAA) with similarity to the core sequence of the SV40 enhancer. Naturally occurring variation in the number of these motifs is correlated with the enhancer strength of the ULR. Our results indicate that inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and perhaps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to playa role in regulatory evolution. Introduction Retrotransposons are the most abundant and widely distributed class of eukaryotic transposable elements (Berg & Howe, 1989). These elements are distin- guished from other transposable elements by the fact that their replication involves an RNA intermediate and is reverse transcriptasedependent. Retrotranspo- son insertions adjacent to chromosomal genes fre- quently result in altered regulatory phenotypes. The molecular mechanisms that underlie these retrotrans- poson mediated regulatory mutations are varied and diverse (McDonald, 1995). For example, retrotrans- poson insertions into a gene's 5' flanking region may affect transcriptional initiation in a temporal-specific or tissue-specific manner. Such regulatory changes can be due to the read-through of transcripts initiated in the retrotransposon promoter or to the presence of positive or negative regulatory sequences within the element. Another way in which retrotransposons may influence gene expression is through insertion induced changes in chromatin structure which may, for example, insu- late a gene's promoter from enhancer sequences locat- ed distal to the site of insertion (Gerasimova et aI., 1995). Although the hypothesis that transposable elements may have a dramatic effect on regulatory evolution was first proposed by McClintock over 40 years ago (e.g., McClintock, 1951, 1956), it is only recently that experimental evidence has begun to accumulate which directly supports the hypothesis. Instances of retrotransposons contributing to the evolution of chro- mosome gene regulation have recently been described in vertebrates (e.g., Robins & Samuelson, 1992), Drosophila (e.g., Miller et aI., 1995) and plants (e.g., White, Habera & Wessler, 1994). Because it now seems likely that the regulatory evolution of at least some chromosomal genes has been influenced by retro- transposon insertions, the question arises as to what factors may be influencing the evolution of retrotrans- poson regulatory sequences in the first place. For the past several years, our laboratory has been studying the evolution of enhancer-like regions within the Drosophila copia LTR retrotransposon. LTR retro- transposons are closely related to infectious retrovirus- es and encode genes homologous to the retroviral gag P. Capy (ed.), Evolution and Impact of Transposable Elements © Kluwer Academic Publishers 1997