Control of Chromatin by RNA-mediated Transcriptional Silencing

In multicellular eukaryotes, transposable elements (TE) make up a large part of the genomic content. These “selfish” genetic elements can propagate and expand the genome through their ability to replicate. However, this poses a significant risk to the stability of gene structure and overall genomic integrity which can lead to aberrant gene expression or products. To combat this threat, highly specific gene silencing mechanisms are utilized by the host to keep TEs in a constantly repressed state. In plants, transcriptional gene silencing is conducted through the RNA-directed DNA methylation (RdDM) pathway. The combined action of short interfering RNA (siRNA) and long non-coding RNA (lncRNA) direct deposition of DNA methylation at target TE regions. Subsequently, DNA methylation together with other repressive chromatin modifications turns the TE regions into a silenced state and prevent them from becoming active again. Although our understanding of the processes that lead up to the deposition of DNA methylation has been well-studied, the specific role and function of DNA methylation in gene silencing remains poorly understood. It is unclear how the presence of DNA methylation can affect the ability of transcription machinery from working at silenced regions. In contrast, DNA methylation does not hinder the transcriptional gene silencing machinery which suggests that DNA methylation plays a central role for distinguishing between different types of transcriptional activity in the DNA. In the first story, we determined how DNA methylation interacts with nucleosomes in the context of transcriptional silencing. DNA compaction and packaging in the nucleus entirely revolves around its interaction with nucleosomes. This interaction has numerous implications for regulation of gene expression through changes in accessibility of DNA to factors involved in transcription. Here we show that RdDM can direct both DNA methylation and nucleosome positioning. Nucleosomes established by RdDM have no detectable impact on DNA methylation. Instead, DNA methylation affects nucleosome positioning. This applies not only to CHH methylation established by RdDM but also to DNA methylation in CG and CHG contexts, which is maintained by MET1 and CMT3. We propose a model where DNA methylation serves as one of the determinants of nucleosome positioning. In the second story, we wanted to investigate the relationship between Pol V transcription and DNA methylation as a potential feedback mechanism where DNA methylation reinforces recruitment of Pol V transcription at silenced regions. Pol V transcribes in a pervasive manner throughout the genome implying that it does not require pre-existing chromatin marks such as DNA methylation to initiate transcription. However, previous studies have claimed that factors upstream of Pol V transcription that are able to bind to DNA methylation are required for the recruitment and initiation of Pol V transcription. Hence, the impact of DNA methylation on Pol V transcription remained unresolved. We found that loss of DNA methylation leads to a strong reduction of Pol V transcription. This occurs when DNA methylation is lost in all sequence contexts, which may happen not only in mutants defective in RdDM but also in mutants lacking maintenance DNA methyltransferases. Our results support a model where RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription with a strong crosstalk with other silencing pathways.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169773/1/mrmhafiz_1.pd