Characterization of the contribution of the CHD chromatin remodeler PKL to chromatin modification and gene expression in Arabidopsis thaliana

H3K27me3 is a transcriptional repressive epigenetic mark that plays vital roles in many biological processes in Arabidopsis thaliana. A number of biochemical and functional characterizations of PKL, an ATP-dependent CHD chromatin remodeler, suggest that PKL contributes to maintain the homeostasis of H3K27me3. To identify other factors that act with PKL together to contribute to the homeostasis of H3K27me3, we undertook an EMS-mutagenesis screen for pkl-associated phenotypes. This genetic screen suggests that PKL may contribute to maintaining the homeostasis of H3K27me3 in an H2A.Z associated or a Mediator associated pathway.

Here, we took advantage of a combined genetic and bioinformatic method to characterize the contribution of PKL in these two pathways as described above. Our analysis revealed a robust genetic interaction between HTA9, HTA11, and PKL in maintaining proper H2A.Z distribution and enrichment of H3K27me3. In addition, the characterization also sheds light on unexpected roles of PKL in promoting the homeostasis of H3K4me3 and acting with histone demethylases to promote removal of H3K27me3 in an H2A.Z dependent manner. Furthermore, our result also raised the possibility that the tail module of the Mediator complex also plays a critical role in the homeostasis of H3K27me3. While we were examining PKL-dependent chromatin features, we largely optimized the protocol for preparation ChIP-seq samples and libraries and implemented a gene-centric ChIP-seq bioinformatics pipeline for providing robust analysis.

Ultimately, the work presented in this thesis highlights several divergent pathways that PKL contributes to maintain chromatin homeostasis. By and large, the combined observation from this thesis advances our knowledge of how PKL interacts with other chromatin-associated machineries together to maintain proper epigenetic states and promote other more emergent DNA-templated processes, including replication and transcription.