SHH1 Is Critical For RNA Polymerase-IV Recruitment To RNA-Directed DNA Methylation Targets

DNA methylation is an epigenetic modification that plays critical roles in gene silencing, development, and the maintenance of genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and is targeted by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM).  This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions in the upstream siRNA generating phase to initiate siRNA biogenesis and Pol-V, which functions in the downstream DNA methyltransferase targeting phase to generate scaffold transcripts that recruit downstream RdDM factors.  To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a Pol-IV interacting protein.  Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets and that SHH1 is required for Pol-IV occupancy at these same loci.  We also show that the SHH1 SAWADEE domain is a novel chromatin binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone H3 tail.  In addition, key residues within both lysine binding pockets of SHH1 are required to maintain DNA methylation in vivo.  These findings suggest that SHH1 recruits Pol-IV to genomic loci that contain this combination of histone marks, providing the first insights into the mechanism of Pol-IV targeting.  Given the parallels between methylation systems in plants and mammals a further understanding of this early targeting step may aid in our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.