Cell Culture-Induced Gradual and Frequent Epigenetic Reprogramming of Invertedly Repeated Tobacco Transgene Epialleles1[W]

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.

[1]  M. Fraga,et al.  Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells , 2008, PloS one.

[2]  G. Wenzel,et al.  Variation in DNA methylation patterns of grapevine somaclones (Vitis vinifera L.) , 2008, BMC Plant Biology.

[3]  G. Raidl,et al.  CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing. , 2007, The Plant journal : for cell and molecular biology.

[4]  R. Hellens,et al.  The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro. , 2007, Plant biology.

[5]  R. Hellens,et al.  Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype. , 2007, Plant & cell physiology.

[6]  R. Arroyo-García,et al.  Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli. , 2006, Journal of plant physiology.

[7]  P. Lemaux,et al.  Nature of stress and transgene locus influences transgene expression stability in barley , 2006, Plant Molecular Biology.

[8]  K. Křížová,et al.  The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco , 2006, Nucleic acids research.

[9]  E. Richards.  Inherited epigenetic variation — revisiting soft inheritance , 2006, Nature Reviews Genetics.

[10]  J. Bender,et al.  H3 Lysine 9 Methylation Is Maintained on a Transcribed Inverted Repeat by Combined Action of SUVH6 and SUVH4 Methyltransferases , 2005, Molecular and Cellular Biology.

[11]  J. Fulnecek,et al.  Dedifferentiation of Tobacco Cells Is Associated with Ribosomal RNA Gene Hypomethylation, Increased Transcription, and Chromatin Alterations1[w] , 2005, Plant Physiology.

[12]  V. Hemleben,et al.  Organization, differential expression and methylation of rDNA in artificial Solanum allopolyploids , 2004, Plant Molecular Biology.

[13]  B. Arnholdt‐Schmitt.  Stress-Induced Cell Reprogramming. A Role for Global Genome Regulation? , 2004, Plant Physiology.

[14]  R. Margis,et al.  Suppression of post-transcriptional gene silencing by callus induction and virus infection reveals the existence of aberrant RNAs , 2004 .

[15]  M. Labra,et al.  Specific hypomethylation of DNA is induced by heavy metals in white clover and industrial hemp , 2004 .

[16]  F. Meins,et al.  Meiotic transmission of epigenetic changes in the cell-division factor requirement of plant cells , 2003, Development.

[17]  Vincent Colot,et al.  Changes in 5S rDNA Chromatin Organization and Transcription during Heterochromatin Establishment in Arabidopsis Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017467. , 2003, The Plant Cell Online.

[18]  A. Depicker,et al.  Epigenetic Switch from Posttranscriptional to Transcriptional Silencing Is Correlated with Promoter Hypermethylation1 , 2003, Plant Physiology.

[19]  N. Morozova,et al.  Chromatin reorganization accompanying cellular dedifferentiation is associated with modifications of histone H3, redistribution of HP1, and activation of E2F‐target genes , 2003, Developmental dynamics : an official publication of the American Association of Anatomists.

[20]  J. S. Heslop-Harrison,et al.  Retroelements, transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation , 2003, Plant Molecular Biology.

[21]  C. Vannini,et al.  Analysis of cytosine methylation pattern in response to water deficit in pea root tips , 2002 .

[22]  T. Kakutani.  Epi-alleles in plants: inheritance of epigenetic information over generations. , 2002, Plant & cell physiology.

[23]  E. Finnegan.  Epialleles - a source of random variation in times of stress. , 2002, Current opinion in plant biology.

[24]  H. Vaucheret,et al.  Post-transcriptional gene silencing in plants. , 2001, Journal of cell science.

[25]  M. Matzke,et al.  Transcriptional silencing and promoter methylation triggered by double‐stranded RNA , 2000, The EMBO journal.

[26]  M. Van Montagu,et al.  Both sense and antisense RNAs are targets for the sense transgene-induced posttranscriptional silencing mechanism , 2000, Molecular and General Genetics MGG.

[27]  S. Kaeppler,et al.  Epigenetic aspects of somaclonal variation in plants , 2000, Plant Molecular Biology.

[28]  J. Verdeil,et al.  Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis , 2000, Plant Cell Reports.

[29]  R. Matyášek,et al.  Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units , 2000, Chromosoma.

[30]  Tamas Dalmay,et al.  An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus , 2000, Cell.

[31]  M. Van Montagu,et al.  Cross‐talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes , 2000, FEBS letters.

[32]  A. Depicker,et al.  Drug‐induced hypomethylation of a posttranscriptionally silenced transgene locus of tobacco leads to partial release of silencing , 2000, FEBS letters.

[33]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[34]  S. Melquist,et al.  Arabidopsis PAI gene arrangements, cytosine methylation and expression. , 1999, Genetics.

[35]  M. Grandbastien.  Activation of plant retrotransposons under stress conditions , 1998 .

[36]  P. Meyer,et al.  Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing , 1997, Molecular and General Genetics MGG.

[37]  M. Montagu,et al.  Post-transcriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3' flanking regions , 1997 .

[38]  B. Koukalová,et al.  Hypermethylation of tobacco heterochromatic loci in response to osmotic stress , 1997, Theoretical and Applied Genetics.

[39]  M. Smulders,et al.  Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants , 1995, Theoretical and Applied Genetics.

[40]  M. Van Montagu,et al.  Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[41]  S. Kaeppler,et al.  Genetic instability of plant tissue cultures: breakdown of normal controls. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[42]  S. Kaeppler,et al.  Tissue culture-induced DNA methylation variation in maize. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[43]  H. Hirochika.  Activation of tobacco retrotransposons during tissue culture. , 1993, The EMBO journal.

[44]  G. Giuliano,et al.  DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs , 1989, Theoretical and Applied Genetics.

[45]  M. Mette,et al.  Local DNA features affect RNA-directed transcriptional gene silencing and DNA methylation. , 2008, The Plant journal : for cell and molecular biology.

[46]  I. Mitsuhara,et al.  Release from post-transcriptional gene silencing by cell proliferation in transgenic tobacco plants: possible mechanism for noninheritance of the silencing. , 2002, Genetics.

[47]  H. Vaucheret,et al.  Transcriptional gene silencing in plants: targets, inducers and regulators. , 2001, Trends in genetics : TIG.

[48]  M. Van Montagu,et al.  Gene silencing results in instability of antibody production in transgenic plants , 1999, Molecular and General Genetics MGG.

[49]  A. Karp.  On the current understanding of somaclonal variation , 1991 .

[50]  M. Matzke,et al.  Gene interactions and epigenetic variation in transgenic plants. , 1990 .