In maize, mouse, and other eukaryotes, paramutation refers to a process by which heritable changes in gene regulation are facilitated by interactions between alleles on homologous chromosomes. As typically described, alleles conferring relatively high gene action invariably change to a repressed expression state when heterozygous with specific alleles or allelic states. Operationally, paramutation violates the first law of Mendelian inheritance that alleles segregate unchanged from a heterozygote and thus has important implications for normal genome function and evolution.

By mutation selection and positional cloning, I show in this thesis that the largest subunit of the presumed maize Pol IV is involved in paramutation and normal maize development as well as in the biogenesis of small interfering RNAs (siRNAs). Nuclear run-on transcription assays indicate that Pol IV does not engage in efficient RNA synthesis typical of the three major eukaryotic DNA-dependent RNA polymerases. Additionally, I show that specific haplotypes of the purple plant1 (pl1) locus, encoding an anthocyanin pigment regulator, acquire and retain a spatially expanded expression domain in the kernel aleurone following transmission from siRNA biogenesis mutants. This conditioned expression pattern is progressively enhanced over generations in Pol IV mutants and remains heritable after restoration of Pol IV function. pl1 expression in kernels is associated with promoter-proximal transposable element (TE) fragments, but is independent of sequences required for paramutation. These results indicate that trans-generational Pol IV action defines the expression patterns of haplotypes using co-opted transposon-derived sequences as regulatory controlling elements. Finally, I present nascent RNA libraries of Pol IV mutants and wild-type siblings that represent the first global, transcription-based analyses in plants. The transcription profiles of both endogenous genes and TEs in the maize genome are altered in Pol IV mutants in both the sense and antisense orientation. The results presented in this thesis indicate that Pol IV employs abnormal RNA polymerase activities to achieve genome-wide silencing and that disruption of its function by mutation affects both heritable epigenetic changes to gene regulation and maize development.

Paramutation describes an interaction between specific alleles which results in heritable epigenetic changes in gene expression. To study the mechanisms underlying paramutation, genetic screens were performed to identify factors required to maintain repression (rmr) of paramutant states. For my thesis research I mapped and identified rmr1 as encoding a novel, plant-specific Rad54-like Snf2 protein. Additionally, I have participated in the identification of rmr6 as encoding RPD1, the largest subunit of the plant specific RNA polymerase IV (Pol IV), and rmr7 as encoding RPD2a, the second largest subunit of either Pol IV and / or an additional plant-specific RNA polymerase, Pol V. Pol IV and Pol V, along with RMR1 and related Snf2 proteins, have evolved exclusively in plants to mediate RNA-based epigenetic repression. While the epigenetic changes mediated by rmr1, rmr6, and rmr7 appear to indirectly affect the paramutation mechanism, the identification of these factors has facilitated cross-species comparisons of the RNA-based repression mechanisms and provided new evidence for the role of these types of regulatory mechanisms in plant development and evolution.

Plants have acquired and maintained an expanded suite of DNA-dependent RNA polymerases (RNAPs) compared to other eukaryotes. Although their exact roles remain unclear, plant-specific RNAPs (Pol IV and Pol V) are involved in epigenetic silencing of transposable elements (TEs). Zea mays (maize) Pol IV is required for proper plant development as well as the establishment and maintenance of paramutations, which are trans-homolog interactions that facilitate heritable gene silencing. Maize has duplications of Pol IV catalytic subunits, which define multiple Pol IV subtypes, and accessory proteins associated with these subtypes define distinct Pol IV complexes. Understanding the roles of Pol IV will require identifying the composition and function of these Pol IV subtypes and complexes. In exploring interactions between the genes encoding a Pol IV catalytic subunit and a putative accessory protein, I identified two new alleles of the Pol IV subunit and a family of potential Pol IV accessory proteins conserved in multicellular plants. Together, these Pol IV complexes are proposed to function through two, potentially overlapping, general mechanisms to control of gene function: direct competition with Pol II and/or generation of 24-nucleotide RNAs that guide de novo cytosine methylation. I analyzed nascent transcriptome data of seedlings lacking Pol IV and their wild-type siblings, which identified a global effect of Pol IV on gene boundary transcription. Pol IV-affected loci serve as molecular and phenotypic models for dissecting the involvement of various Pol IV subtype and complex components. Such analysis implicated a Pol IV-affected allele as being controlled by Pol IV competition with

Pol II. Through expanded sequencing of a paramutation participating allele (Pl1-Rhoades), I identified a new Pol IV target of five tandem repeats that may serve as an enhancer element. I used the Pl1-Rhoades allele in particular to compare and contrast the effects of Pol IV loss with that of Pol IV accessory proteins. Together, the work presented here explores the involvement of an uncharacterized protein family of putative Pol IV accessory proteins in

Pol IV complexes, expands the roles of Pol IV complexes in controlling maize gene function, and identifies new Pol IV-affected loci for future study.