Abstract
Methylation of plant promoters is often associated with transcriptional gene silencing, while methylation of the transcribed region is associated with post-transcriptional gene silencing. Promoter complexes that include the first untranslated exon and intron, such as maize ubiquitin1 and rice actin1, have been widely used in monocot transformation because they support higher levels of transient transgene expression than the core promoter does. However, persistent problems with transgene silencing driven by these promoter complexes brought up a troubling question: were higher initial levels of transgene expression at the expense of long-term expression stability? Here we report that methylation of an untranslated exon and intron in the promoter complex is correlated with transcriptional transgene silencing in barley. Two sibling sublines, designated T330 and T331, were identified in a homozygous T3 population from a single transgenic parental line. In the T6 generation, all progeny of one subline, T330, expressed ubiquitin-driven bar and uidA, but both transgenes were transcriptionally silenced in the other subline, T331. Although the structure of the transgene locus is complex, no differences in the physical structure or location of the locus were detected between the two sublines. Transcriptional transgene silencing in T331 correlated with two molecular events: methylation of the first untranslated exon and 5′ end of the intron of the promoter complex, and condensation of the chromatin in regions containing transgenes. Passage of the non-silenced subline through in vitro culture recreated the silenced phenotype of T331 and the molecular events leading to its silencing.
Similar content being viewed by others
References
Assaad, F.F., Tucker, K.L. and Signer, E.R. 1993. Epigenetic repeatinduced gene silencing (RIGS) in Arabidopsis. Plant Mol. Biol. 22: 1067–1085.
Banks, J.A., Masson, P. and Fedoroff, N. 1988. Molecular mechanisms in the developmental regulation of the maize Suppressormutator transposable element. Genes Dev. 2: 1364–1380.
Bregitzer, P. and Tonks, D. 2003. Inheritance and expression of transgenes in barley, Hordeum vulgare L. Crop Sci. 43: 4–12.
Bregitzer, P., Zhang, S., Cho, M.-J. and Lemaux, P.G. 2002. Reduced somaclonal variation in barley is associated with culturing highly differentiated, meristematic tissues. Crop Sci. 42: 1303–1308.
Chandler, V.L., Eggleston, W.B. and Dorweiler, J.E. 2000. Paramutation in maize. Plant Mol. Biol. 43: 121–145.
Cho, M.J., Choi, H.W., Buchanan, B.B. and Lemaux, P.G. 1999. Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants. Theor. Appl. Genet. 98: 1253–1262.
Cho, M.J., Choi, H.W., Jiang, W., Ha, C.D. and Lemaux, P.G. 2002. Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants. Physiol. Plant. 115: 144–154.
Christensen, A.H. and Quail, P.H. 1996. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5: 213–218.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675–689.
Cogoni, C. and Macino, G. 1999. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399: 166–169.
Cone, K. 1989. Yet another rapid plant DNA prep. Maize Genet. Coop. Newsl. 63: 68.
Dalmay, T., Hamilton, A., Rudd, S., Angell, S. and Baulcombe, D.C. 2000. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101: 543–553.
Fagard, M. and Vaucheret, H. 2000. (Trans) gene silencing in plants: how many mechanisms? Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 167–194.
Finnegan, E.J. and Kovac, K.A. 2000. Plant DNA methyltransferases. Plant Mol. Biol. 43: 189–201.
Finnegan, E.J., Peacock, W.J. and Dennis, E.S. 1996. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc. Natl. Acad. Sci. USA 93: 8449–8454.
Fu, X., Kohli, A., Twyman, R.M. and Christou, P. 2000. Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice. Mol. Gen. Genet. 263: 106–118.
Iyer, L.M., Kumpatla, S.P., Chandrasekharan, M.B. and Hall, T.C. 2000. Transgene silencing in monocots. Plant Mol. Biol. 43: 323–346.
Jakowitsch, J., Papp, I., Moscone, E.A., Van Der Winden, J., Matzke, M. and Matzke, A.J. 1999. Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans. Plant J. 17: 131–140.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.
Jorgensen, R.A., Cluster, P.D., English, J., Que, Q. and Napoli, C.A. 1996. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and singlecopy vs. complex T-DNA sequences. Plant Mol. Biol. 31: 957–973.
Kilby, N.J., Leyster, H.M.O. and Furner, I.J. 1992. Promoter methylation and progressive transgene inactivation in Arabidopsis. Plant Mol. Biol. 20: 103–112.
Kohli, A., Gahakwa, D., Vain, P., Laurie, D.A. and Christou, P. 1999. Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing. Planta 208: 88–97.
Koprek, T., Rangel, S., McElroy, D., Louwerse, J.D., Williams-Carrier, R.E. and Lemaux, P.G. 2001. Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley. Plant Physiol. 125: 1354–1362.
Kumpatla, S.P. and Hall, T.C. 1998. Recurrent onset of epigenetic silencing in rice harboring a multi-copy transgene. Plant J. 14: 129–135.
Kumpatla, S.P., Teng, W., Buchholz, W.G. and Hall, T.C. 1997. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice. Plant Physiol. 115: 361–373.
Matzke, A.J.M., Neuhuber, F., Park, Y.D., Ambros, P.F. and Matzke, M.A. 1994. Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol. Gen. Genet. 244: 219–229.
Matzke, M.A., Mette, M.F. and Matzke, A.J.M. 2000. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol. Biol. 43: 401–415.
Matzke, M., Mette, M.F., Jakowitsch, J., Kanno, T., Moscone, E.A., van der Winden, J. and Matzke, A.J.M. 2001. A test for transvection in plants: DNA pairing may lead to trans-activation or silencing of complex heteroalleles in tobacco. Genetics 158: 451–461.
McElroy, D., Zhang, W., Cao, J. and Wu, R. 1990. Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2: 163–171.
Meng, L. and Lemaux, P.G. 2003. A simple, rapid method for nuclear run-on transcription assays in plants. Plant Mol. Biol. Rep. 21: 65–71.
Muskens, M.W.M., Vissers, A.P.A., Mol, J.N.M. and Kooter, J.M. 2000. Role of inverted DNA repeats in transcriptional and posttranscriptional gene silencing. Plant Mol. Biol. 43: 243–260.
Paszkowski, J. and Whitham, S.A. 2001. Gene silencing and DNA methylation processes. Curr. Opin. Plant Biol. 4: 123–129.
Pawlowski, W.P., Torbert, K.A., Rines, H.W. and Somers, D.A. 1998. Irregular patterns of transgene silencing in allohexaploid oat. Plant Mol. Biol. 38: 597–607.
Ros, F. and Kunze, R. 2001. Regulation of activator/dissociation transposition by replication and DNA methylation. Genetics 157: 1723–1733.
Rossi, V., Motto, M. and Pellegrini, L. 1997. Analysis of the methylation pattern of the maize opaque-2 (O2) promoter and in vitro binding studies indicate that the O2 b-Zip protein and other endosperm factors can bind to methylated target sequences. J. Biol. Chem. 272: 13758–13765.
Stark, K., Kirk, D.L. and Schmitt, R. 2001. Two enhancers and one silencer located in the introns of regA control somatic cell differentiation in Volvox carteri. Genes Dev. 15: 1449–1460.
Vaucheret, H. 1993. Identification of a general silencer for 19S and 35S promoters in a transgenic tobacco plant: 90 bp of homology in the promoter sequence are sufficient for trans-inactivation. C.R. Acad. Sci. Sér. III Sci. Vie 316: 1471–1483.
Voinnet, O. 2001. RNA silencing as a plant immune system against viruses. Trends Genet. 17: 449–459.
Walker, E.L. 1998. Paramutation of rl locus of maize is associated with increased cytosine methylation. Genetics 148: 1973–1981.
Wan, Y. and Lemaux, P.G. 1994. Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104: 37–48.
Wan, Y., Widholm, J.M. and Lemaux, P.G. 1995. Type I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.). Planta 196: 7–14.
Wassenegger, M. and Pelissier, T. 1998. A model for RNA-mediated gene silencing in higher plants. Plant Mol. Biol. 37: 349–362.
Waterhouse, P.M., Wang, M.-B. and Lough, T. 2001. Gene silencing as an adaptive defence against viruses. Nature 411: 834–842.
Zhang, S., Warkentin, D., Sun, B., Zhong, H. and Sticklen, M. 1996. Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.). Theor. Appl. Genet. 92: 752–761.
Zhang, S., Cho, M.J., Koprek, T., Yun, R., Bregitzer, P. and Lemaux, P.G. 1999. Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings. Plant Cell Rep. 18: 959–966.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Meng, L., Bregitzer, P., Zhang, S. et al. Methylation of the exon/intron region in the Ubi1 promoter complex correlates with transgene silencing in barley. Plant Mol Biol 53, 327–340 (2003). https://doi.org/10.1023/B:PLAN.0000006942.00464.e3
Issue Date:
DOI: https://doi.org/10.1023/B:PLAN.0000006942.00464.e3