Prior to publication of the Arabidopsis thaliana genome sequence in the year 2000, there were three known eukaryotic, multisubunit RNA Polymerases, abbreviated as Pol I, II and III. The Arabidopsis genome sequence revealed that plants have two additional RNA Polymerases, named Pol IV and V. Affinity purification and protein identification by mass spectrometry revealed that Pol IV and Pol V evolved as specialized forms of RNA Polymerase II, with six of the twelve subunits of Pol II, IV and V encoded by the same genes. Pol IV and Pol V have independent and essential roles in a pathway that ultimately contributes to proper plant morphological development, large scale heterochromatin organization and the silencing of transposable elements and other repeats. Pol IV, in collaboration with the RNA Dependent RNA Polymerase RDR2 and the Dicer-Like protein DCL3, is required to produce small interfering RNAs that interact with non-coding transcripts generated by Pol V to recruit DNA methylating and histone modifying enzymes to the adjacent chromatin. This epigenetic remodeling inhibits localized transcription by RNA Polymerases I, II and III. Three subunits differ between Pol IV and Pol V and presumably account for their unique functions. There are approximately ~6000 sites in the Arabidopsis genome that are subject to Pol IV and Pol V cytosine methylation;however, we know of very few DNA sequences bound by either Pol IV or Pol V. It is not clear whether specific DNA sequences are primarily responsible for recruiting Pol IV and Pol V to DNA or whether chromatin structure plays a primary role. It is also unclear how Pol IV and Pol V activities are coordinated with other activities within the RNA-directed DNA methylation pathway. During this fellowship period, my goal is to address many of these questions by defining the genome-wide targets of Pol V and the transcripts it produces. Furthermore, I will seek to understand the mechanism by which the Pol V polymerase selects its targets. I will also attempt to define the proteins that interact with Pol V so that we have a clearer picture of how Pol V remodels chromatin and silences genes.
In diverse eukaryotes, including humans, flies, worms and fission yeast, non-coding RNAs direct essential processes through chromatin modifications. Alterations in DNA methylation and other chromatin modifications are implicated in Rett, Prader-Willi, Angelman and Fragile X syndromes, as well as numerous forms of cancer. The experiments and analyses detailed in this proposal should contribute to the long-term goal of understanding the role of non-coding RNA and chromatin modifications in gene regulation, thereby contributing to the understanding of human diseases.