- Email: [email protected]
Transposons, DNA methylation and gene control
LETTER
Transposons, DNA methylation and gene control The issues raised by Adrian Bird and Tim Bestor concerning the role of DNA methylation in mammalian genomes1,2 might usefully be addressed by revisiting some old ideas in plants. Bestor has proposed that DNA methylation is restricted almost entirely to transposable elements, which constitute more than a third of the mouse genome. The primary role of DNA methylation might thus be to prevent transposition3 which would otherwise be expected to ‘lacerate the genome’. Bird points out that, if methylation were to regulate transposon spread, then it has been singularly unsuccessful, as large amplifications of transposons have occurred in recent vertebrate evolution, as well as in those insects, such as Drosophila, that do not have methylation. Bird proposes that the primary role of methylation is to limit ‘background’ transcriptional noise, including transcription from transposons themselves. These points of view might be reconciled by re-examining the role of DNA methylation and transposable elements in plant gene regulation as well as in mammals. In plants, the prevalence of methylated transposons has been known for some time4–6. Furthermore, methylation of transposase promoters does appear to repress transposon activity7–10. However, transposase (from cut-and-paste DNA transposons) interacts better with (hemi-) methylated than with unmethylated transposon substrates11. Furthermore, active transposons in Z. mays, have homologs in distantly related species (such as P. hybrida and Antirrhinum majus) suggesting an ancient origin. Thus transposons remain active over long evolutionary periods in heavily methylated genomes, despite the potential for high mutation rates, and the danger of turning their hosts into cabbages (Ref. 12, cited by Bestor). Transposons and viruses are parasites that have evolved numerous ways of doing as little damage as possible to their hosts13. One way is to transpose preferentially
into each other, resulting in large tracts of heterochromatin that contain no genes6,14. Another mechanism is to insert preferentially into regions of chromosomal genes where they will do least harm. Maize, yeast and Drosophila transposons carry signals for splicing and transcriptional control near the ends of the element that allow them to insert into introns and promoters without noticeable phenotypic effect13. In maize for example, when Robertson’s Mutator transposons are inserted into promoters, transcription is blocked. However, the resulting mutant phenotypes can be suppressed by restoration of transcription near the site of insertion15. Counter-intuitively, this transcription appears to require transposon methylation, although it is regulated normally in other ways15,16. Similarly, insertions of En/Spm transposons into chromosomal gene introns block transcriptional elongation when they are bound to transposase. When the transposase source is removed (whether by methylation
or deletion of the transposase gene), chromosomal gene expression is restored by read-through transcription and splicing17. As a result of these mechanisms, strains with multiple suppressible mutations are severely mutant when the transposons are unmethylated, but near normal when they are methylated10,18. A casual observer might well conclude that methylation played a key role in development, rather than simply interacting with transposons (Fig. 1). The suppression of transposon insertions in promoters occurs in maize, snapdragon and for that matter Neurospora, suggesting an ancient conserved mechanism for ‘hiding’ transposons in this way18–20. Furthermore, such epigenetic effects are not limited to organisms with DNA methylation: suppression of transposon mutations in yeast and Drosophila is mediated by basal transcription factors and chromatin proteins21,22. So it is likely that the mechanism of suppression involves the regulation of chromatin structure rather than transposon methylation per se23. How do these old observations relate to the role of vertebrate DNA methylation? Methylation-deficient mice die very early during
FIGURE 1. Cabbages or corn plants? Both these maize plants have a Robertson’s Mutator transposon mutation in bladeless2 causing threadlike leaves. The plant on the left has methylated elements, while the plant on the right has unmethylated elements. A casual observer might conclude that DNA methylation influenced development, but this influence is mediated by transposons. TIG JULY 1998 VOL. 14 NO. 7
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01518-2
263
LETTER embryogenesis, and fail to regulate imprinted and sex-linked genes24. So lethality might result from aberrant gene dosage. However, in organisms like mice, or maize, genome de-methylation would also reveal perhaps hundreds of mutations caused by the insertion of cryptic transposons, which might contribute to early lethality. In organisms with relatively few transposons, such as Arabidopsis thaliana25 these effects would be much less severe. Of course, Arabidopsis is nearly a cabbage already, but Arabidopsis DNA methylation mutants (ddm) are viable24,26 consistent with this idea. Interestingly, inbred ddm mutants in Arabidopsis accumulate epimutations at a small number of loci over time27. Perhaps some of these loci have transposon remnants that bring them under the control of methylation in the same way as Mutator transposons do in maize28. In fact, many epigenetic phenomena, including paramutation in plants29,30 and imprinting in plants and animals16 can be mimicked by genes with transposon insertions, perhaps suggesting a common mechanism in these and other cases31. Thus, DNA methylation masks the effects of transposon insertion by mechanisms that do not depend on regulating transposition, or on regulating (background) transcription directly. Rather, methylated transposons are ‘hidden’ from the genome by interactions with a wide variety of chromatin factors, some of which might be mediated by DNA methylation23. This property of transposons allows them to accumulate to high copy numbers with minimal effects on host gene regulation. The ancient relationship between chromatin and transposons might even suggest a common origin for some of these mechanisms. Upon the rediscovery of transposable elements in bacteria,
Barbara McClintock was reported to have become frustrated with the excitement surrounding mobile DNA. After discovering transposons in maize in the 1940s, she spent much of the next 30 years characterizing their effects on chromosomal gene expression, convinced that she had uncovered a novel aspect of gene control9,32. It can even be argued that McClintock discovered gene regulation in maize before Jacob and Monod did in bacteria. When interviewed at the height of the excitement, her comment was this: ‘The real secret to all of this is control, it is not transposition’. (N. Comfort 1997 PhD Thesis, S.U.N.Y. Stony Brook, USA).
References 1 Bird, A. (1997) Trends Genet. 13, 469–470 2 Bestor, T.H. and Yoder, J.A. (1997) Trends Genet. 13, 470–472 3 Yoder, J.A., Walsh, C.P., Bestor, T.H. (1997) Trends Genet. 13, 335–340 4 White, S.E., Habera, L.F. and Wessler, S.R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11792–11796 5 Timmermans, M.C., Das, O.P. and Messing, J. (1996) Genetics 1434, 1771–1783 6 SanMiquel, P. et al. (1996) Science 274, 765–768. 7 Chandler, V.L. and Walbot, V. (1986) Proc. Natl. Acad. Sci. U. S. A. 836, 1767–1771 8 Chomet, P.S., Wessler, S. and Dellaporta, S.L. (1987) EMBO J. 62, 295–302 9 Banks, J.A., Masson, P. and Fedoroff, N. (1988) Genes Dev. 211, 1364–1380 10 Martienssen, R. and Baron, A. (1994) Genetics 1363, 1157–1170 11 Wang, L., Heinlein, M. and Kunze, R. (1996) Plant Cell 84, 747–758 12 Fedoroff, N.V. (1992 in The Dynamic Genome (Botstein, D. and Fedoroff, N.V., eds), p. 414, Cold Spring Harbor Laboratory Press 13 Kidwell, M.G. and Lisch, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7704–7711
14 Heslop-Harrison, J.S. et al. (1997) Genetica 100, 197–204 15 Barkan, A. and Martienssen, R.A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3502–3506 16 Martienssen, R.A. and Richards, E.J. (1995) Curr. Opin. Genet. Dev. 5, 234–242 17 Menssen, A. et al. (1990) EMBO J. 910, 3051–3057 18 Martienssen, R. (1996) in Epigenetic Mechanisms of Gene Regulation (Russo, V.E.A., Martienssen, R.A. and A. Riggs, eds), pp. 593–608, Cold Spring Harbor Laboratory Press 19 Chatterjee, M. and Martin, C. (1997) Plant J. 114, 759–771 20 Cambareri, E.B. et al. (1996) Genetics 1431, 137–146 21 Hartzog, G.A., Wada, T., Handa, H. and Winston, F. (1998) Genes Dev. 123, 357–369 22 Gdula, D.A., Gerasimova, T.I. and Corces, V.G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9378–9383 23 Kass, S.U., Pruss, D. and Wolffe, A. (1997) Trends Genet. 13, 444 24 Li., E., Beard, C. and Jaenisch, R. (1993) Nature 366, 362–365 25 Bevan, M. et al. (1998) Nature 391, 485–488 26 Vongs, A., Kakutani, T., Martienssen, R.A. and Richards, E.J. (1993) Science 260, 1926–1928 27 Kakutani, T. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12406–12411 28 Martienssen, R.A. (1996) Trends Genet. 12, 508 29 Martienssen, R. (1996) Curr. Biol. 67, 810–813 30 Richards, E.J. (1997) Trends Genet. 13, 319–323 31 Matzke, A.J.M., Eggleston, W.B. and Matzke, M. (1996) Trends Plant Sci. 1, 382–388 32 McClintock, B. (1958) Carnegie Inst. Wash. Year Book 57, 415–429
Rob Martienssen [email protected] Delbruck-Page Department, Cold Spring Harbor Laboratory, Bungtown Road, Cold Spring Harbor, NY 11724-2203, USA.
Letters to the Editor We welcome letters on any topic of interest to geneticists and developmental biologists. Write to: Mark Patterson [email protected] • Fax 01223 464430 Trends in Genetics, Elsevier Trends Journals, 68 Hills Road, Cambridge, UK CB2 1LA. TIG JULY 1998 VOL. 14 NO. 7
264
Transposons, DNA methylation and gene control The issues raised by Adrian Bird and Tim Bestor concerning the role of DNA methylation in mammalian genomes1,2 might usefully be addressed by revisiting some old ideas in plants. Bestor has proposed that DNA methylation is restricted almost entirely to transposable elements, which constitute more than a third of the mouse genome. The primary role of DNA methylation might thus be to prevent transposition3 which would otherwise be expected to ‘lacerate the genome’. Bird points out that, if methylation were to regulate transposon spread, then it has been singularly unsuccessful, as large amplifications of transposons have occurred in recent vertebrate evolution, as well as in those insects, such as Drosophila, that do not have methylation. Bird proposes that the primary role of methylation is to limit ‘background’ transcriptional noise, including transcription from transposons themselves. These points of view might be reconciled by re-examining the role of DNA methylation and transposable elements in plant gene regulation as well as in mammals. In plants, the prevalence of methylated transposons has been known for some time4–6. Furthermore, methylation of transposase promoters does appear to repress transposon activity7–10. However, transposase (from cut-and-paste DNA transposons) interacts better with (hemi-) methylated than with unmethylated transposon substrates11. Furthermore, active transposons in Z. mays, have homologs in distantly related species (such as P. hybrida and Antirrhinum majus) suggesting an ancient origin. Thus transposons remain active over long evolutionary periods in heavily methylated genomes, despite the potential for high mutation rates, and the danger of turning their hosts into cabbages (Ref. 12, cited by Bestor). Transposons and viruses are parasites that have evolved numerous ways of doing as little damage as possible to their hosts13. One way is to transpose preferentially
into each other, resulting in large tracts of heterochromatin that contain no genes6,14. Another mechanism is to insert preferentially into regions of chromosomal genes where they will do least harm. Maize, yeast and Drosophila transposons carry signals for splicing and transcriptional control near the ends of the element that allow them to insert into introns and promoters without noticeable phenotypic effect13. In maize for example, when Robertson’s Mutator transposons are inserted into promoters, transcription is blocked. However, the resulting mutant phenotypes can be suppressed by restoration of transcription near the site of insertion15. Counter-intuitively, this transcription appears to require transposon methylation, although it is regulated normally in other ways15,16. Similarly, insertions of En/Spm transposons into chromosomal gene introns block transcriptional elongation when they are bound to transposase. When the transposase source is removed (whether by methylation
or deletion of the transposase gene), chromosomal gene expression is restored by read-through transcription and splicing17. As a result of these mechanisms, strains with multiple suppressible mutations are severely mutant when the transposons are unmethylated, but near normal when they are methylated10,18. A casual observer might well conclude that methylation played a key role in development, rather than simply interacting with transposons (Fig. 1). The suppression of transposon insertions in promoters occurs in maize, snapdragon and for that matter Neurospora, suggesting an ancient conserved mechanism for ‘hiding’ transposons in this way18–20. Furthermore, such epigenetic effects are not limited to organisms with DNA methylation: suppression of transposon mutations in yeast and Drosophila is mediated by basal transcription factors and chromatin proteins21,22. So it is likely that the mechanism of suppression involves the regulation of chromatin structure rather than transposon methylation per se23. How do these old observations relate to the role of vertebrate DNA methylation? Methylation-deficient mice die very early during
FIGURE 1. Cabbages or corn plants? Both these maize plants have a Robertson’s Mutator transposon mutation in bladeless2 causing threadlike leaves. The plant on the left has methylated elements, while the plant on the right has unmethylated elements. A casual observer might conclude that DNA methylation influenced development, but this influence is mediated by transposons. TIG JULY 1998 VOL. 14 NO. 7
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01518-2
263
LETTER embryogenesis, and fail to regulate imprinted and sex-linked genes24. So lethality might result from aberrant gene dosage. However, in organisms like mice, or maize, genome de-methylation would also reveal perhaps hundreds of mutations caused by the insertion of cryptic transposons, which might contribute to early lethality. In organisms with relatively few transposons, such as Arabidopsis thaliana25 these effects would be much less severe. Of course, Arabidopsis is nearly a cabbage already, but Arabidopsis DNA methylation mutants (ddm) are viable24,26 consistent with this idea. Interestingly, inbred ddm mutants in Arabidopsis accumulate epimutations at a small number of loci over time27. Perhaps some of these loci have transposon remnants that bring them under the control of methylation in the same way as Mutator transposons do in maize28. In fact, many epigenetic phenomena, including paramutation in plants29,30 and imprinting in plants and animals16 can be mimicked by genes with transposon insertions, perhaps suggesting a common mechanism in these and other cases31. Thus, DNA methylation masks the effects of transposon insertion by mechanisms that do not depend on regulating transposition, or on regulating (background) transcription directly. Rather, methylated transposons are ‘hidden’ from the genome by interactions with a wide variety of chromatin factors, some of which might be mediated by DNA methylation23. This property of transposons allows them to accumulate to high copy numbers with minimal effects on host gene regulation. The ancient relationship between chromatin and transposons might even suggest a common origin for some of these mechanisms. Upon the rediscovery of transposable elements in bacteria,
Barbara McClintock was reported to have become frustrated with the excitement surrounding mobile DNA. After discovering transposons in maize in the 1940s, she spent much of the next 30 years characterizing their effects on chromosomal gene expression, convinced that she had uncovered a novel aspect of gene control9,32. It can even be argued that McClintock discovered gene regulation in maize before Jacob and Monod did in bacteria. When interviewed at the height of the excitement, her comment was this: ‘The real secret to all of this is control, it is not transposition’. (N. Comfort 1997 PhD Thesis, S.U.N.Y. Stony Brook, USA).
References 1 Bird, A. (1997) Trends Genet. 13, 469–470 2 Bestor, T.H. and Yoder, J.A. (1997) Trends Genet. 13, 470–472 3 Yoder, J.A., Walsh, C.P., Bestor, T.H. (1997) Trends Genet. 13, 335–340 4 White, S.E., Habera, L.F. and Wessler, S.R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11792–11796 5 Timmermans, M.C., Das, O.P. and Messing, J. (1996) Genetics 1434, 1771–1783 6 SanMiquel, P. et al. (1996) Science 274, 765–768. 7 Chandler, V.L. and Walbot, V. (1986) Proc. Natl. Acad. Sci. U. S. A. 836, 1767–1771 8 Chomet, P.S., Wessler, S. and Dellaporta, S.L. (1987) EMBO J. 62, 295–302 9 Banks, J.A., Masson, P. and Fedoroff, N. (1988) Genes Dev. 211, 1364–1380 10 Martienssen, R. and Baron, A. (1994) Genetics 1363, 1157–1170 11 Wang, L., Heinlein, M. and Kunze, R. (1996) Plant Cell 84, 747–758 12 Fedoroff, N.V. (1992 in The Dynamic Genome (Botstein, D. and Fedoroff, N.V., eds), p. 414, Cold Spring Harbor Laboratory Press 13 Kidwell, M.G. and Lisch, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7704–7711
14 Heslop-Harrison, J.S. et al. (1997) Genetica 100, 197–204 15 Barkan, A. and Martienssen, R.A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3502–3506 16 Martienssen, R.A. and Richards, E.J. (1995) Curr. Opin. Genet. Dev. 5, 234–242 17 Menssen, A. et al. (1990) EMBO J. 910, 3051–3057 18 Martienssen, R. (1996) in Epigenetic Mechanisms of Gene Regulation (Russo, V.E.A., Martienssen, R.A. and A. Riggs, eds), pp. 593–608, Cold Spring Harbor Laboratory Press 19 Chatterjee, M. and Martin, C. (1997) Plant J. 114, 759–771 20 Cambareri, E.B. et al. (1996) Genetics 1431, 137–146 21 Hartzog, G.A., Wada, T., Handa, H. and Winston, F. (1998) Genes Dev. 123, 357–369 22 Gdula, D.A., Gerasimova, T.I. and Corces, V.G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9378–9383 23 Kass, S.U., Pruss, D. and Wolffe, A. (1997) Trends Genet. 13, 444 24 Li., E., Beard, C. and Jaenisch, R. (1993) Nature 366, 362–365 25 Bevan, M. et al. (1998) Nature 391, 485–488 26 Vongs, A., Kakutani, T., Martienssen, R.A. and Richards, E.J. (1993) Science 260, 1926–1928 27 Kakutani, T. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12406–12411 28 Martienssen, R.A. (1996) Trends Genet. 12, 508 29 Martienssen, R. (1996) Curr. Biol. 67, 810–813 30 Richards, E.J. (1997) Trends Genet. 13, 319–323 31 Matzke, A.J.M., Eggleston, W.B. and Matzke, M. (1996) Trends Plant Sci. 1, 382–388 32 McClintock, B. (1958) Carnegie Inst. Wash. Year Book 57, 415–429
Rob Martienssen [email protected] Delbruck-Page Department, Cold Spring Harbor Laboratory, Bungtown Road, Cold Spring Harbor, NY 11724-2203, USA.
Letters to the Editor We welcome letters on any topic of interest to geneticists and developmental biologists. Write to: Mark Patterson [email protected] • Fax 01223 464430 Trends in Genetics, Elsevier Trends Journals, 68 Hills Road, Cambridge, UK CB2 1LA. TIG JULY 1998 VOL. 14 NO. 7
264