Workshop 5 : Epigenetics
Chair: Eric Richards, Washington University, USA.
email: richards@wustlb.wustl.edu
The workshop highlighted the recent development of several experimental tools and systems in Arabidopsis to study epigenetic regulation. Epigenetic regulation may be viewed as propagation of alternate information states of the genome without alteration of primary nucleotide sequence. The study of plant epigenetic regulation is most highly developed in maize where a number of sophisticated and complex epigenetic phenomena have been dissected for several decades. Arabidopsis, by comparison, has suffered from the lack of sufficiently well-described epigenetic phenomena but this situation is changing as evidenced by this workshop.The first two presentations dealt with the most conspicuous candidate mechanisms for epigenetic regulation: DNA modification and chromatin structure. The first speaker in the workshop, Eric Richards (Washington University), described work on Arabidopsis ddm mutants which contain reduced levels of genomic cytosine methylation. The current status of investigation of ddm1 mutations was summarized and a description of more recently isolated alleles of a second locus (DDM2) was presented. The DDM2 locus appears to correspond to the cytosine methyltransferase gene, MET1, originally isolated by Jean Finnegan and colleagues (CSIRO). The second talk, presented by Rob Oosterling (Utrecht), focused on chromatin structure. A picture of higher-order chromatin organization was sought from the definition of matrix attachment regions (MARs) in a 16 kb genomic region around the plastocyanin gene. Three MARs were found in intergenic regions by a heterologous (rodent) nuclear matrix binding assay. While these MARs correspond to the general design of previously identified MARs in plants and other organisms (e.g., AT-rich) a novel degenerate 21-bp motif was found in all three plastocyanin locus MARs.
The balance of the workshop was devoted to description of epigenetic systems and the role of cytosine methylation in regulating these systems. Ian Furner (Cambridge University) began with a description of his group's work on epigenetic lesions of the TT4 locus which encodes an anthocyanin biosynthesis enzyme chalcone synthetase. The lesions, which cause reduction in CHS gene expression and anthocyanin pigmentation and correlate with increased cytosine methylation of the CHS gene, were apparently established by sense-suppression (or "co-suppression") after introduction of a TT4 transgene. The lesions persisted even after the removal of the transgene. While 5azadeoxycytidine did not reactivate the silenced endogenous CHS gene, introduction of the tt4 epiallele into a background homozygous for the hypomethylation mutation, ddm1, led to loss of CHS gene methylation and activation of the gene. This work provides one of the first examples of the formation and inheritance of epigenetic lesions of an endogenous gene in Arabidopsis. Further, the sensitivity of the tt4 epialleles to ddm1 suggests that at least the maintenance, if not the establishment, of the silenced state involves DNA methylation.
Craig Pikaard (Washington University) then presented results on nucleolar dominance, a classical epigenetic phenomenon involving the silencing of the entire set of rRNA genes from one parent of a hybrid or allotetraploid. The Pikaard lab investigated nucleolar dominance in Brassica allotetraploids at the molecular level and showed that the rRNA genes of one parent in each of the three allotetraploid Brassica species are transcriptionally silenced. Similar results demonstrating nucleolar dominance in the Arabidopsis allotetraploid, A. suecica, were presented. 5azadeoxycytidine treatment of the Brassica allotetraploids leads to activation of the silenced rRNA gene complement indicating that DNA methylation is involved in maintenance of the transcriptionally inactive state.
The importance of DNA methylation for proper Arabidopsis development is clearly demonstrated by the cytosine methyltransferase antisense gene expression studies presented by Jean Finnegan (CSIRO). A severe reduction in cytosine methylation occurs in some of the group's MET1 methyltransferase antisense lines (down to 10-15% of wild-type levels). This reduction in methylation leads to a number of abnormal morphological phenotypes, including homeotic transformations in the flowers. MET1 antisense expression also is associated with the ectopic expression in the leaves of the normally flower-specific floral identity genes, AG and AP3. The similarities between the morphological and gene expression phenotypes of the MET1 antisense plants and mutants in the curly leaf (clf) polycomb-like gene led to the suggestion that DNA methylation and chromatin factors, such as polycomb-class proteins, may collaborate to ensure proper gene expression.
The final speaker of the workshop, Tetsuji Kakutani (National Institute of Agrobiological Resources, Tsukba), presented the results of genetic and phenotypic analysis of ddm1 mutants which argue further for the importance of cytosine methylation in plant development. ddm1 homozygotes exhibit dramatic morphological phenotypes, including alterations in apical dominance, leaf shape and flowering time, after propagation for a number of generations by self-pollination. Genetic analysis indicates that the morphological abnormalities are caused by the accumulation of heritable lesions at genomic sites unlinked to the potentiating ddm1 mutations. The lesions which have been analyzed are stable in DDM1 backgrounds and may correspond to traditional genetic mutations or meiotically transmissible epimutations. In any case, the results draw a potential connection between alteration of DNA methylation and the maintenance of the genomic information, either genetic or epigenetic.