No abstract

DNA methylation is an epigenetic modification that plays 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 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. 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 factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV interacting protein3. 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 mammals1,5, 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.

A successful structure-based design of a class of non-peptide small-molecule MDM2 inhibitors targeting the p53-MDM2 protein-protein interaction is reported. The most potent compound 1d binds to MDM2 protein with a Ki value of 86 nM and is 18 times more potent than a natural p53 peptide (residues 16-27). Compound 1d is potent in inhibition of cell growth in LNCaP prostate cancer cells with wild-type p53 and shows only a weak activity in PC-3 prostate cancer cells with a deleted p53. Importantly, 1d has a minimal toxicity to normal prostate epithelial cells. Our studies provide a convincing example that structure-based strategy can be employed to design highly potent, non-peptide, cell-permeable, small-molecule inhibitors to target protein-protein interaction, which remains a very challenging area in chemical biology and drug design.

Potent, specific, non-peptide small-molecule inhibitors of the MDM2-p53 interaction were successfully designed. The most potent inhibitor (MI-63) has a K(i) value of 3 nM binding to MDM2 and greater than 10,000-fold selectivity over Bcl-2/Bcl-xL proteins. MI-63 is highly effective in activation of p53 function and in inhibition of cell growth in cancer cells with wild-type p53 status. MI-63 has excellent specificity over cancer cells with deleted p53 and shows a minimal toxicity to normal cells.