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

Publication Type:

Journal Article


Nature, Volume 498, Issue 7454, p.385-9 (2013)


Arabidopsis, Arabidopsis Proteins, Binding Sites, Chromatin, Crystallography, X-Ray, DNA Methylation, DNA-Directed RNA Polymerases, Epigenesis, Genetic, Histones, Homeodomain Proteins, Lysine, Methyltransferases, Models, Molecular, Mutation, Protein Folding, Protein Structure, Tertiary, RNA, Small Interfering


<p>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.</p>

4IUP for the Se–SAWADEE (L200M, L218M) complex and 4IUQ for the wild-type SAWADEE domain in the free state, 4IUR for the H3(1–15)K9me3–SAWADEE complex, 4IUT for the H3(1–15)K9me2– SAWADEE complex, 4IUU for the H3(1–15)K9me1–SAWADEE complex, and 4IUV for the H3(1–15)K4me1K9me1–SAWADEE complex.