CHD Chromatin Remodelers Promote Epigenetic Control of Development in Arabidopsis thaliana
Chromatin remodeling is a vital biological process that facilitates activation and repression of gene expression as well as compaction of large eukaryotic genomes into compact nuclei. The CHD3 family of chromatin remodelers play key roles as epigenetic modifiers and transcriptional regulators in both plants and animals. PKL is a CHD3 remodeler that represses expression of seed genes during germination in Arabidopsis. Unlike animal CHD3 remodelers, PKL does not function as a member of a multi-subunit Mi-2 NuRD complex. Instead, PKL acts as a monomer in vivo and promotes the epigenetic modification H3K27me3, which is associated with transcriptional repression of tissue-specific genes by the PRC2. Thus, PKL plays an important role in promoting tissue identity by facilitating deposition and/or maintenance of H3K27me3. Many questions remain about how PKL facilitates expression of developmentally regulated genes. For example, PKL is necessary for repression of seed genes after germination, but its contributions to development in the seed and to other aspects of reproduction are comparatively unknown. To address these questions, we investigated the role of PKL and its seed-specific paralog PKR2 in reproductive development in Arabidopsis. We identified new roles for PKL in multiple reproductive processes in the maternal plant including pollen tube growth, development of the female gametophyte, and in specification of seed size. Interestingly, PKR2 appears to act antagonistically with respect to PKL in regards to seed size, revealing a novel interaction between these genes in the developing seed. Another aspect of PKL activity that is poorly understood is its role in transcriptional activation: recent studies have indicated that PKL is involved in activation of H3K27me3-enriched loci through an unknown mechanism. We are undertaking a novel forward genetic screen using the inducible PKL-GR construct to identify additional pathways by which PKL activates or represses gene expression. From a similar screen, we have determined that PKL acts in the same pathway as does the SWR1 ortholog PIE1, which is necessary for deposition of the histone variant H2A.Z in Arabidopsis. H2A.Z promotes gene inducibility in response to internal and external stimuli. RNA-seq analysis of pkl seedlings reveals that PKL is necessary for expression of stress response genes. Further, ChIP analyses indicate that PKL, like PIE1, can promote H2A.Z levels in chromatin. Taken together, these results reveal a new epigenetic pathway associated with PKL and implicate PKL in the regulatory mechanisms that govern expression of stress response genes. We have undertaken a variety of additional analyses to investigate the contributions of PKL and H3K27me3 to development in Arabidopsis. We have identified a potential link between the PKL pathway and H3K56ac, an epigenetic modification that is known to interact with the H2A.Z pathway in S. cerevisiae. Further, we are undertaking a domain deletion analysis of PKL to identify domains of sequence conservation that are important for targeting and/or activity of the PKL chromatin remodeler. Finally, we have collaborated with Dr. Stan Gelvin to identify a role for H3K27me3-related processes in determining susceptibility to Agrobacterium-mediated transformation of Arabidopsis. This provides a potential new avenue of investigation into methods of increasing transformation efficiency in plants. As a whole, the research detailed in this thesis advances our understanding of the role of PKL in plant reproduction and of the epigenetic pathways by which PKL affects expression of developmentally important genes. Further, our mutant screen has identified enhancer of pkl lines that will serve as the basis for future investigations into the developmental and epigenetic pathways in which PKL acts.
Ogas, Purdue University.
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