Overview

Genetic, biochemical, and cytological studies of structural elements of chromosomes, with emphasis on telomeres, heterochromatin, and transposable elements. Studies of the coordination of nuclear and cytoplasmic activities. Analysis of the molecular mechanisms by which cells respond to stress, especially the molecular biology of the heat shock response.

Research Summary

Telomeres (the ends of chromosomes) have important roles in chromosome replication, in division, and in the cell-type-specific architecture of interphase nuclei. A wealth of data ties chromosome ends to cellular senescence, cell cycle checkpoints and organismal aging and tumorogenesis: telomere biology is relevant to many areas of biology. In addition, telomeres are located in heterochromatin, an intriguing but poorly understood form of chromatin. We study Drosophila telomeres, the original genetic and cytological model for telomeres. Despite their functional similarity to other telomeres, Drosophila telomeres have an unexpected molecular difference: Drosophila telomeres are maintained, not by telomerase, but by two transposable elements.

Transposable elements are abundant in the genomes of higher organisms but are usually thought to affect cells only incidentally, by transposing in or near a gene and influencing its expression. Surprisingly, we found that a non-LTR retrotransposon, HeT-A, maintains the telomeres of Drosophila chromosomes. A second Drosophila telomere-specific retrotransposon, TART, was found by R. Levis. (The relation between HeT-A and TART is intriguing. Why does Drosophila have two telomere-specific transposons?) HeT-A and TART are the first transposable elements with identified roles in chromosome structure. We have suggested that these telomere-specific elements may be evolutionarily related to telomerase; in both cases an enzyme extends the end of a chromosome by adding DNA copied from an RNA template. The evolutionary origin of the Drosophila telomere is an important question; however, whatever the origin, these variant telomeres offer new insights into telomere structure and function.

Phylogenetic studies of telomere-specific retrotransposons. The rapid sequence divergence of telomeric transposons makes it more difficult to study these elements but also increases the probability that features conserved in spite of this sequence change are of biological importance. We have recently found that HeT-A and TART form telomeres in D. virilis (65 My separation from D. melanogaster), and therefore this mechanism of telomere maintenance must predate the separation of the extant Drosophila species. HeT-A and TART appear to have different evolutionary origins yet our studies suggest that they cooperate, rather than compete, at telomeres. All D. melanogaster stocks and Drosophila species studied have both elements. In spite of significant sequence divergence of these elements, the basic features of the Drosophila telomere have been conserved in all species studied.

Potential roles for Gag proteins in retroelement targeting. Many Drosophila non-LTR retrotransposons actively transpose into internal, gene-rich, regions of chromosomes but do not transpose onto chromosome ends. HeT-A and TART are remarkable exceptions; they form telomeres of Drosophila by repeated transpositions onto the ends of chromosomes and never transpose to internal regions of chromosomes. Both telomeric and non-telomeric non-LTR elements transpose by target primed reverse transcription, and their targets are not determined simply by DNA sequence, so it is not clear why these two kinds of elements have non-overlapping transposition patterns. To explore roles of retrotransposon-encoded proteins in transposition, we analyzed intracellular targeting of Gag proteins from five non-LTR retrotransposons, HeT-A, TART, jockey, Doc, and I factor. All were expressed as GFP-tagged proteins in cultured Drosophila cells. These Gag proteins have high levels of sequence similarity, but they have dramatic differences in intracellular targeting. As expected, HeT-A and TART Gags are efficiently transported to nuclei where HeT-A Gag localizes at telomeres and directs TART Gag to telomeres. These patterns are cell cycle-dependent, disappearing during mitosis. In contrast, little, if any, of the other Gags moved into the nucleus; instead each formed characteristic clusters in the cytoplasm. These experiments demonstrate that closely related retrotransposon Gag proteins can have different intracellular localizations, presumably because they interact differently with cellular components. We suggest that these interactions reflect mechanisms by which the cell influences the level of transposition of an element, hindering transposition of “parasitic elements” at many steps, while enabling efficient transport of their telomere elements.

Novel retroelement promoters. Although HeT-A and TART have hallmarks of non-LTR retrotransposons, they also have novel features. We found that the HeT-A promoter resembles an evolutionary intermediate between promoters of LTR and non-LTR retrotransposons. It is at the 3´ end of the element and promotes transcription of the adjacent downstream element. Thus, the HeT-A promoter is identical to the 3´ end of the element it promotes and could be considered a pseudo-long terminal repeat, equivalent to the LTR promoter. TART also has a 3´ promoter but this promotes anti-sense transcription of the element that contains it. The TART sense strand promoter is in the 5´ untranslated region of TART.

Heterochromatin and promoters. Heterochromatin is a poorly-understood type of chromatin found, among other places, at telomeres and generally thought to be genetically inert. Our studies of the HeT-A promoter provide the first molecular dissection of a promoter that is active in heterochromatin. Our initial characterization of the HeT-A promoter was done by testing the ability of different fragments of sequence from HeT-A to act as promoters for a bacterial lac Z reporter gene in transiently transfected Drosophila cultured cells. The test system was chosen as the best way to approximate a heterochromatic environment. We have now used P-element-mediated transfection to insert the constructs in euchromatic regions of the chromosome to test the effect of chromosomal environment. We have characterized the stage- and tissue-modulation of HeT-A RNA expression to compare to expression of the reporter. In euchromatic locations these reporter constructs are expressed and show at least some aspects of appropriate stage- and tissue-specific regulation.

Selected Publications

Pardue, M.-L. and DeBaryshe, P.G. Retrotransposons provide an evolutionarily robust non-telomerase mechanism to maintain telomeres. Ann. Rev. Genetics 37:485-511 (2003).

Rashkova, S., Athanasiadis, A., and Pardue, M.-L. Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons. J. Virol. 77:6376-6384 (2003).

Casacuberta, E. and Pardue, M.-L. Transposon telomeres are widely distributed in the Drosophila genus: TART elements in the virilis group. Proc. Natl. Acad. Sci. USA 100:3363-3368 (2003).

George, J.A., and Pardue, M.-L. The promoter of the heterochromatic Drosophila telomeric retrotransposon, HeT-A, is active when moved into euchromatic locations. Genetics 163:625-635 (2003).

Danilevskaya,O.N., Arkhipova, I.R., Traverse, K.L., and Pardue, M.L. Promoting in tandem; the promoter for the telomere transposon HeT-A and implications for evolution of retroviral LTRs. Cell 88:647-655 (1997).

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