Alternative polymerase genome regulation in Zea mays

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