Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1

Abstract

DNA methylation is an epigenetic modification that has critical roles in gene silencing, development and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24-nucleotide 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 to initiate siRNA biogenesis, and Pol-V, which functions 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 3 (H3) tail. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and 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 our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.

Figure 1. Epigenetic profile of siRNA clusters affected in RdDM mutants

a, Pie chart showing the abundance of 24 nt siRNA reads in wild-type (ecotype Col) sequencing libraries (5,967,213 uniquely mapping reads total). b, Schematic Venn diagram showing approximate relationships of 24 nt siRNA clusters reduced in each genotype and the subclasses used for downstream analysis. c, Pie charts showing the chromosomal distribution (based on previously described definitions of pericentromeric heterochromatin and euchromatin) of affected siRNA clusters in the indicated subclasses. d and e, Boxplots of siRNA and CHH methylation levels at the subclasses shown in (b) for various RdDM mutants (* indicates significant reduction; P<1e-10 Mann-Whitney U test). f, Metaplots showing CMT3 and Pol-V enrichment at affected siRNA clusters (+/− 5000 bp from the siRNA cluster midpoint).

Figure 2. Pol-IV levels at defined siRNA clusters

Metaplots of Pol-IV enrichment over the defined siRNA clusters in the indicated genetic backgrounds. Metaplots extend +/− 5000 bp from the midpiont of the siRNA cluster.

Figure 3. The SHH1 SAWADEE domain recognizes H3K9 methylation and adopts a unique tandem Tudor domain-like fold

a and b, ITC-based measurements of the SAWADEE domain binding to the modified or unmodified histone peptides as indicated. K_d values are listed. NDB means no detectable binding. c, The overall structure of the SHH1 SAWADEE domain in the free form. The zinc-binding motif is shown as an enlarged ball-and-stick model, highlighting the details of the metal coordination. A bound detergent molecule 4-Cyclohexyl-1-Butyl-β-D-Maltoside moiety from the crystallization condition is shown in a stick representation.

Figure 4. Structural basis for recognition of H3(1–15)K9me2 peptide by the SHH1 SAWADEE domain and the functional impact of mutations of residues lining the K4 and K9me2 pockets

a, Overall structure of the H3(1–15)K9me2-SAWADEE complex with the SAWADEE domain as a ribbon diagram and the peptide as a stick representation. The simulated annealing composite omit map at 1σ level of the bound peptide is also shown. b, Stereo view highlighting the intermolecular interactions between the SAWADEE domain and the bound peptide. Intermolecular hydrogen-bonding interactions are designated by dashed red lines. c, d and e, Close-up views of H3 lysine residues in their respective binding pockets. f and g, Boxplots of genome-wide % CHH methylation and siRNA levels in wild-type, shh1 mutants, and shh1 mutants transformed with SHH1 constructs (shh1 + SHH1) that encode wild-type SHH1 or K9 (F162AF165A and Y140A) or K4 (D141A and Y212A) binding pocket mutants. h, qPCR of Pol-IV enrichment in the backgrounds described in f at a defined Pol IV binding site. Bars are the average of two biological replicates normalized to input and actin levels (+/− SE).