RNA Polymerases as Genetic Requirements for the Establishment and Maintenance of Epigenetic Silencing

The maize genome is highly complex and repetitive, with almost 85% of the genome encoding transposable elements (TE). The movement of TE can have deleterious effects on a genome. In maize, one model for transcriptional gene silencing (TGS) relies heavily on a pathway termed RNA dependent DNA Methylation (RdDM). The current model dictates that RdDM establishes a silent state at TE and other repetitive regions of the genome. It employs two plant specific DNA dependent RNA polymerases, (Pol IV and V). Transcription by Pol IV is modeled to result in the generation of 24 nucleotide (24nt) small interfering RNA (siRNA). While Pol V transcription is modeled to generate a scaffold transcript, required for the targeting of chromatin remodelers and de novo methyl transferases to regions of the genome for regulation and silencing. Current maize data supports this model; however, large gaps in our understanding of this pathway exist. Three transgenic constructs were used to test the necessity of three RNA polymerases in RNA mediated TGS. BTG-s, is regulated through a MOP1, RMR1, and RMR2 dependent pathway. A forward genetic screen was used to identify additional proteins required for silencing of BTG-s. Through this screen Tgr1 was identified as a regulator of BTG silencing. Mapping of tgr1-1 indicated a 12 Mbp window on chromosome 1. Complementation experiments confirmed tgr1-1 is an allele of GRMZM2G007681, which encodes the largest subunit of Pol IV. Consistent with the current model for Pol IV, tgr1-1 mutants are deficient in 24nt siRNA biosynthesis. Identification of Tgr1 as a subunit of Pol IV agrees with previous reports that determined that BTG silencing is mediated by an RNA-dependent silencing pathway. Mutants affecting additional RNA polymerases that are part of the RdDM pathway—--Pol IV (Mop2-1 and mop3-1), Pol V (Mop2-1) and an RNA dependent RNA polymerase (mop1-1)—--were used in conjunction with inverted repeat transgenes complementary to two maize loci to ascertain the requirements for these RNA polymerases in the establishment of silencing at endogenous loci. The a1 locus requires functional MOP2, but is not dependent on the function of MOP1 or MOP3 to establish silencing. A second transgene complementary to the enhancer of the b1 locus does not require functional MOP1, MOP2, or MOP3 to establish silencing of the b1 enhancer. This indicates that Pol V is required at some, but not all genomic loci for silencing to be established. Thus the genetic requirements for establishment of silencing at three genetic loci vary with respect to these three RNA polymerases.