SHH1 and Pol IV targeting in RNA-directed DNA methylation

Julie A. Law1,5*, Jiamu Du2*, Christopher J. Hale1*, Suhua Feng1, Ana Marie S. Palanca5, Krzysztof Krajewski3, Brian D. Strahl3, Dinshaw J. Patel2, Steven E. Jacobsen1,4

1Department of Molecular Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095; 2Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065; 3Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; 4Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, CA 90095. 5Plant Molecular and Cellular Biology, Salk Institute, La Jolla, CA 92037
In eukaryotic organisms DNA is wrapped around specialized packaging proteins, called histones, and organized into a highly dynamic structure called chromatin.  Both the DNA and histone components of chromatin can be modified through the covalent attachment of chemical groups and these modifications influence the expression of the underlying genes and play critical roles in development and the maintenance of genome integrity.   In the plant model Arabidopsis thaliana, DNA methylation is associated with the silencing of transposons and is targeted by 24 nt small RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. To better understand the mechanisms controlling RdDM, we affinity purified RNA polymerase IV (Pol IV), the polymerase that initiates siRNA biogenesis. This RNA polymerase associates with several accessory factors including SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), which we demonstrate plays a central role in the targeting of Pol IV to chromatin. Genome-wide investigation of SHH1 function revealed that this factor acts to enable siRNA production at loci undergoing active DNA methylation through the recruitment of Pol IV. This recruitment requires the histone binding activities of the SHH1 SAWADEE domain which adopts a tandem Tudor-like fold and recognizes H3K9 methylation as well as unmodified H3K4 residues. This dual binding specificity may be important for shaping the DNA methylation landscape at transposons and other repeat elements located near genes. Together, our findings connect DNA methylation and H3K9 methylation within the context of the RdDM pathway and provide the first mechanistic insight into a key initiating step in the RdDM pathway, the targeting of RNA Pol-IV. *authors contributed equally