Following an exciting first day at the plant biology worldwide summit, over 800 attendees participated  at the plenary session II which answered questions on the mechanisms of epigenetic inheritance and reprogramming in plants.  Rob Martienssen while introducing the session, noted that although there has been a long history of studies about epigenetic inheritance in plants, reprogramming changes from one generation to the next. New sequencing tools have brought about a better understanding of the pathways involved epigenetic inheritance including DNA and histone methylation, chromatin remodeling and small RNA biogenesis. These mechanism were further discussed by the four plenary speakers.

Craig S. Pikaard- Pol IV-RDR2 complex generates two siRNAs that guide transcription

RNA- mediated DNA methylation (RdDM) is an essential process in silencing transposable elements involved in maintaining genomic stability in plants.  Craig S. Pikaard described his work in identifying the pathways for this process. Two enzymes: RNA Polymerase IV (Pol IV) and RNA-dependent RNA polymerase (RDR2) associate with one another to service the siRNA precursor which guides the DNA methylation pathway. Despite the amount of work done in understanding this pathway, there are still some unanswered questions as to the features, size and synthesis of these siRNA precursors in Arabidopsis.

Through sequencing the precursors from the Pol IV and RDR2 strand in vitro, Craig and his team reported that a Pol IV-RDR2 complex is involved in the generation of the 24- and 23-nt siRNAs. Pol IV generates precursor RNAs that are cleaved by the Dicer endonuclease (DCL3) to produce 24-nt siRNAs and this process is fully dependent on RDR2. Some of the interesting facts from this mechanism include: Pol IV products seem to be noisier sizewise than RDR2 products; dsRNA generated by RDR2 is very important at the dicing step of the DNA methylation process, and the termination of Pol IV is essential for the initiation of RDR2. Craig also touched upon other areas of his work including the mechanism involved in coupling the siRNA to Argonaute protein families, primarily AGO4 or AGO6, to guide the transcription of DNA sequences by Pol V

Ian Henderson – Telomeric recombination is dominant in asy1 mutants

Meiotic recombination events vary between species and some regions of the chromosomes experience more crossovers than others. This process shapes the patterns of genetic variation in plants. Ian Henderson and his group study the mechanisms involved in controlling variations in recombination along different arms of the chromosomes using the model plant Arabidopsis. He noted that these genetic variations are not only due to crossovers but also genetic diversity, recombination and DNA methylation events. In addition, the presence of heterochromatin and other protein structures that form along the chromosomes bring about some of the recombination landscapes. One of those proteins is called ASY1 (ASYNAPTIC 1), a HORMA domain protein.

Ian further described their work to understand how the protein structures shape the chromosome axis and enrich the telomere-centromere gradients. Through mapping genome wide crossovers in the absence of ASY1, they observed that telomeric crossovers predominate in asy1 mutants. This was demonstrated by the homologous chromosomes that pull apart indicating recombination at the terminal locations, while in asy1/+ heterozygotes the subtelomeric regions were dominant. Ian also showed that the crossover events are ASY1 gene dose sensitive and in wild type, most of the crossover events show interference. An implication of this result is that changing ASY1 gene dosage or expression could be a good way to change crossover landscapes in crops and might push recombination into regions where breeders need that to happen.

Daniel Grimanelli- Dynamics of DNA Methylation During Maize Reproductive Development

DNA methylation occurs in contexts of genes and transposable elements in angiosperms. For example, the maize genome has a fewer number of genes involved in DNA methylation and demethylation processes when compared to Arabidopsis. Daniel Grimanelli’s work focuses on identifying these functionally important genes involved in reproduction in maize using epigenomic mapping and functional analysis of meiotic cell division. The genes involved in this methylation process are the Argonaut genes (AGO 104, 105 and 119) which form very important architectures for the small RNA nucleotides; and some alleles for the transferase genes (ZMET 2, 3, 5 and 7).

Daniel and his group developed a great set of reporters to track DNA methylation in real time - with the exception of CHG methylation. Similarly, they used RNA-seq data to explain the relationship between expression and methylation levels in meiocytes. They came up with the following points: the methylation levels are significantly reduced in those ago104 mutants but not in ago105 and ago119; the level of DNA methylation in gene promoters is inversely proportional with expression levels; and ZMET5 is a major regulator of CHG methylation in maize. Finally, during meiosis, methylation is dynamic and atypical and is a tissue and stage-specific process in maize. Daniel’s high-resolution microscopy representation informed that male sterility is the consequence of perturbing non-CG methylation in maize.

Julie Law- A CLASSY Way to control the epigenome

DNA and histone methylation are heritable processes that play important roles in many processes such as genomic stability, disease, agriculture, adaptation, etc. In Arabidopsis, DNA methylation occurs in transposons and repetitive DNA elements, associated with transcriptional silencing and occurs in all sequence contexts. Julie Law’s research is to understand how chromatin modulations are controlled under normal circumstances and how their varying patterns can lead to developmental processes. The questions they seek to answer include: what controls where DNA methylation is deposited, how is DNA methylation connected to gene regulation, and how do chromatin modifications influence gene stability.

As mentioned by other speakers, the RdDM pathway involves different kinds of RNA polymerases (RNA Pol IV and Pol V). To understand what controls the patterns of DNA methylation, Julie and her group used a genetic and genomic approach to identify a family of four putative chromatin remodeling factors, CLASSY (CLSY) 1–4, required for the regulation of the methylation process.  These CLSY’s are highly locus specific and work together with the Pol IV enzyme to regulate the production of the 24nt-sRNAs. In addition, these CLSY’s regulate the methylation process in a tissue-specific manner. This information will assist researchers to understanding the different cellular machinery involved in generating epigenetic diversity in plants.

Each of the talk was followed by a live Q & A session and through the chat window, the speakers individually responded to questions they didn’t attempt during the live session. The vibrant discussion session provided a great networking opportunity for participants as well.