The lab is currently engaged in two main projects. The goal of the first project, which is funded by the National Institutes of Health, is to understand the mechanisms responsible for nucleolar dominance. The goal of the second project, also funded by the National Institutes of Health, with additional support from Monsanto Company, is to understand the role of RNA polymerases IV and V and small RNA pathways in RNA-directed DNA methylation and gene silencing in Arabidopsis. A recently completed project was a collaborative, service-oriented effort, funded by the National Science Foundation, to generate community resources and tools to enable the study of chromatin proteins in a variety of epigenetic phenomena. These tools include Gateway-compatible plant transformation vectors we developed for high-throughput construction of epitope-tagged proteins or for generating fusions of target proteins to one of several fluorescent proteins (GFP, YFP, CFP) in Arabidopsis or maize. Other resources include full-length cDNA sequences and generating transgenic maize and Arabidopsis lines for transgene-induced RNAi-mediated knockdown of chromatin modifying activities.

These projects are discussed briefly here, with additional detail on nucleolar dominance and Pol IV/Pol V available by linking to the web pages devoted to those subjects.

Nucleolar Dominance. In all offspring there are genes that are expressed from the chromosomes inherited from only one parent. Often a maternal or paternal imprint dictates which parental allele will be active. However, this is not the case for the uniparental expression of ribosomal RNA (rRNA) genes in genetic hybrids, which occurs independent of maternal or paternal effects. This epigenetic phenomenon, known as nucleolar dominance, occurs both in plants and animals but is best studied in plants because non-sterile hybrids can be generated and exist in nature. We've shown that rRNA gene silencing involves concerted changes in both DNA methylation and histone modification and we have proposed a model whereby DNA and histone modifications are each upstream of one another in a self-reinforcing, circular pathway. Recent evidence indicates that siRNA-directed de novo cytosine methylation is involved in nucleolar dominance and may help explain how one set of rRNA genes is singled out for silencing. Members of the lab are identifying the chromatin modifying activities required for nucleolar dominance and how their actions are intertwined to comprise an epigenetic on-off switch.

RNA polymerases IV and V (Pol IV and Pol V) are plant-specific polymerases required for small interfering RNA (siRNA)-directed DNA methylation and silencing of transgenes, transposable elements, pericentromeric repeats and essential rRNA genes. These structurally and functionally distinct polymerases act in a pathway that also includes DICER-LIKE3 (DCL3), ARGONAUTE4 (AGO4) and RNA-DEPENDENT RNA POLYMERASE2 (RDR2). To better understand Pol IV and Pol V functions, our current priorities include purifying and determining Pol IV subunit composition, template requirements (e.g. DNA vs. RNA) and enzymatic products.

Chromatin control of gene expression and gene silencing. Silencing of rRNA genes in nucleolar dominance, like most epigenetic phenomena, involves chromatin modifications. This realization inspired the formation of a consortium of plant epigeneticists, funded by the National Science Foundation (grants DBI-9975930 and DBI-0421619), to work together to advance epigenetic studies by annotating, cloning and sequencing (as necessary) and disrupting the expression of more than 200 chromatin genes in Arabidopsis and maize. The projects have included Richard Jorgensen, Vicki Chandler and Karen McGinnis at the University of Arizona, Judith Bender (Johns Hopkins U.), Karen Cone (U. Missouri), Stanton Gelvin (Purdue University), Shawn and Heidi Kaeppler (U. Wisconsin), David Mount (U. Arizona), Eric Richards (Washington U.) and Craig Pikaard. Project information can be found, or linked to, at the following website: http://www.chromdb.org/

Our consortium used transgene-induced RNA interference (RNAi) technology to degrade mRNAs encoding chromatin proteins, many of which modify either DNA or the histones that assemble DNA into nucleosomes (see figure above; histone proteins are on the inside, DNA on the outside). Protein families targeted in our projects included:

DNA methyltransferases (DMTs).
Methylcytosine Binding Domain Proteins (MBDs).
Histone acetyltransferases (HACs).
Histone deacetylases (HDAs).
Chromatin remodeling activities (CHA, CHB, CHC etc).
SET Domain-containing proteins (e.g. histone methyltransferases).
Chromodomain containing proteins (e.g. histone-binding repressor proteins).
Bromodomain containing proteins (e.g. acetyl-lysine binding proteins).
High Mobility Group (HMG) Proteins (DNA bending "architectural" proteins).
Proteins involved in siRNA-mediated chromatin modifying pathways