Research Spotlight

Dr. Marjori Matzke and Dr. Antonius Matzke

Gregor Mendel Institute of Molecular Plant Biology
Austrian Academy of Sciences

Vienna, Austria

Research interests: Our lab has a long standing interest in epigenetic gene silencing in plants. In early experiments, we discovered a homology-dependent gene silencing phenomenon in which a trans-acting, sequence-specific signal triggered promoter methylation and transcriptional gene silencing (TGS) (Matzke et al., 1989; Neuhuber et al., 1994). We proposed that promoter-directed RNAs might provide the sequence-specific signal for methylation (Park et al., 1996) and went on to show that a transcribed inverted DNA repeat containing promoter sequences could induce promoter methylation and TGS of homologous promoters in trans (Mette et al., 1999, 2000). The double stranded RNA was processed to short RNAs 21-24 nt in length, thereby establishing a mechanistic link between RNA-mediated TGS and post-transcriptional gene silencing or RNA interference (RNAi) (Mette et al., 2000).

The early work that identified the molecular trigger for TGS was carried out using tobacco. This knowledge allowed us to establish well-defined transgene systems in Arabidopsis for conducting forward genetic screens to identify factors required for RNA-directed DNA methylation (RdDM) and TGS. Our basic approach uses a two-component system consisting of a silencer locus, which contains a transcribed inverted DNA repeat of target promoter sequences, and an unlinked target locus, which contains the target promoter driving expression of a reporter gene. Forward genetic screens using two different target promoters identified several conserved proteins required for RdDM/TGS, including the CG methyltransferase MET1 (Aufsatz et al., 2004) and a histone deacetylase (HDA6) (Aufsatz et al., 2002) as well as several novel, plant-specific factors. These include DRD1, a putative SNF2-like chromatin remodelling protein (Kanno et al., 2004) and subunits of a novel RNA polymerase called Pol IV (Kanno et al., 2005). Work from several laboratories including ours has established that there are two isoforms, Pol IVa and Pol IVb, which are distinguished by their unique largest subunit, NRPD1a and NRPD1b, respectively. Both Pol IV isoforms share the same second largest subunit, NRPD2a. Pol IVa is needed to produce or amplify the small RNA trigger, whereas Pol IVb together with DRD1 acts downstream of this step to somehow facilitate de novo methylation at the small RNA targeted site (Matzke et al., 2007).

Genetic screens from our lab and others as well as deep sequencing efforts to identify endogenous small RNAs have revealed that plants possess a complex machinery for making and using small RNAs to induce methylation of homologous DNA sequences. Methylation can either be maintained during DNA replication or lost through passive or active means (Matzke et al., 2007). RdDM thus appears to be important for facilitating epigenetic plasticity in plants. Various transcriptome approaches have demonstrated that intergenic transposons and repeats in euchromatin are frequent targets of RdDM mediated by Pol IVb and DRD1 (Huettel et al., 2006). Because of the potentially reversible nature of RdDM and the presence of potential targets of RdDM in the vicinity of many plant genes, we and others have proposed roles for this silencing pathway in stress responses and plant development (Matzke et al., 2007).

We are currently mapping mutants obtained from a recent screen based on a system in which a meristem-specific enhancer is targeted for RdDM/TGS and have already identified a previously uncharacterized protein related to structural maintenance of chromosomes (SMC) proteins. The continued discovery of novel factors in forward genetic screens demonstrates that there is still much to be learned about the molecular machinery required for RdDM as well as the mechanism of this epigenetic process.

References:
Aufsatz W., Mette, M.F., Matzke, A.J.M. and Matzke, M.A. (2004) Role of MET1 in RNA-directed de novo and maintenance methylation of CG dinucleotides. Plant Mol. Biol. 54: 793-804.

Aufsatz, W., Mette, M.F., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. (2002) HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double stranded RNA. EMBO J. 21, 6832-6841.

Huettel, B., Kanno, T., Daxinger, L., Aufsatz, W., Matzke, A.J.M. and Matzke, M. (2006) Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis. EMBO J. 25, 2828-2836.

Kanno, T., Huettel, B., Mette, M.F., Aufsatz, W., Jaligot, E., Daxinger, L., Kreil, D.P., Matzke, M.A. and Matzke, A.J.M. (2005) A typical RNA polymerase subunits required for RNA-directed DNA methylation. Nat. Genet. 37, 761-765.

Kanno, T., Mette, M.F., Kreil, D.P., Aufsatz, W., Matzke, M.A. and Matzke, A.J.M. (2004) Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation. Curr. Biol. 14, 801-805.

Matzke, M.A., Kanno, T., Huettel, B., Daxinger, L. and Matzke, A.J.M. (2007) Targets of RNA-directed DNA methylation. Curr. Opin. Plant Biol. 10, 512-519.

Matzke, M.A., Primig, M., Trnovsky, J. and Matzke, A.J.M. (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8, 643-649.

Mette, M.F., Aufsatz, W., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. (2000) Transcriptional gene silencing and promoter methylation triggered by double stranded RNA. EMBO J. 19, 5194-5201.

Mette, M.F., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. (1999) Production of aberrant promoter transcipts contributes to methylation and silencing of unlinked homologous promoters in trans. EMBO J. 18, 241-248.

Neuhuber, F., Park, Y.-D., Matzke, A.J.M. and Matzke, M.A. (1994) Susceptibility of transgene loci to homology-dependent gene silencing. Mol.Gen. Genet. 244, 230-241.

Park, Y.-D., Papp, I., Moscone, E.A., Iglesias, V.A., Vaucheret, H., Matzke, A.J.M. and Matzke, M.A. (1996) Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J. 9, 183-194.