Methylation of cytosines in DNA is a stable epigenetic mark that impacts on gene regulation, imprinting, X chromosome inactivation and transposon silencing. Given that only certain CpG sites are methylated, there is currently considerable room to improve our understanding of the observed tissue- and cell-type specificity of DNA methylation. DNA methylation plays a critical role in epigenetic regulation given that defects and abnormalities in writing, reading and erasing of methylation marks are embryonic lethal in mammals and can lead to pleiotropic defects in plants.
Our laboratory has published the following reviews on epigenetic regulation modulated by DNA methylation marks.
Du, J., Johnson, L. M., Jacobsen, S. E. and Patel, D. J. (2015). DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 16, 519-532.
Writers of DNA Methylation Marks
DNA methylation marks in mammals are written at CpG sites by the de novo DNA methyltransferase DNMT3A/3L and maintained by the maintenance DNA methyltransferase DNMT1. In plants, cytosine methylation occurs in CpG, CpHpG and CpHpH DNA sequence contexts.
A project championed by the David Allis (Rockefeller) and Haitao Li (Tsinghua University) laboratories on mutants within the ADD reader domain of DNMT3a highlight the biological significance of the precise interplay between histone H3 modifications and DNA methylation in mammalian pluripotent cells.
Noh, K. M., Wang, H., Kim, H. R., Wenderski, W., Fang, F., Li, C., Dewell, S., Ferris, A., Hughes, S. H., Zheng, D., Melnick, A, Patel, D. J., Li, H. and Allis, C. D. (2015). Engineering of a histone recognition domain in Dnmt3a alters the epigenetic landscape and phenotypic features of mouse ESCs. Mol. Cell 59, 89-103.
DNA methylation occurs in CG and non-CG sequence contexts. Non-CG methylation is abundant in plants and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however, its roles remain poorly understood. Here, in a project championed by the Steve Jacobsen laboratory (UCLA Medical School, CA), the studies characterized the roles of non-CG methylation in Arabidopsis thaliana. These studies showed that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 preferentially binds histone H3 Lys9 (H3K9) dimethylation and methylates non-CG cytosines that are regulated by H3K9 methylation. These studies revealed the contributions and redundancies between each non-CG methyltransferase in DNA methylation patterning and in regulating transcription. These studies also demonstrate extensive dependencies of small-RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the landscapes of histone modification and small noncoding RNA.
Stroud, H., Do, T., Du, J., Zhong, X., Feng, S., Johnson, L., Patel, D. J. and Jacobsen, S. E. (2014). Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64-72.
DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, as part of a collaborative effort with the Steve Jacobsen laboratory (UCLA Medical School, CA), we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the siRNA effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V mediated non-coding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.
Zhong, X., Du, J., Hale, C. J., Gallego-Bartolome, J., Feng, S., Vashisht, A. A., Chory, J., Wohlschlegel, J. A., Patel, D. J. and Jacobsen, S. E. (2014). Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157, 1050-1060.
DNMT1, the major maintenance DNA methyltransferase in animals, helps regulate gene expression, genome imprinting and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methyl-cytosine is positioned within a shallow hydrophobic concave surface, while the cytosine on the target strand is looped-out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensure the high fidelity of DNMT1-mediated maintenance DNA methylation.
Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. (2012). Structure-based mechanistic insights in DNMT1-mediated maintenance DNA methylation. Science 335, 709-712.
Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved crystal structures of mouse DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1 and BAH2) and methyltransferase domains in the free state and bound to duplex DNA containing unmethylated CpG sites. The CXXC domain specifically binds to the unmethylated CpG site and positions a segment within the CXXC-BAH1 linker into a position between the DNA and the active site of DNMT1, thereby preventing de novo methylation. In addition, a loop projecting from the BAH2 domain interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position, and preventing it from binding in the DNA major groove. Our structural and biochemical studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.
Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. (2011). Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331,1036-1040.
Readout of DNA Methylation Marks
The readout of the cytosine methylation mark in hemi-methylated CpG-containing DNA requires flipping out of the methylated cytosine from the DNA duplex and positioning it inside a pocket within the reader module. The SET and RING-associated (SRA) domain is one such reader module that inserts two loops from the major and minor grooves to fill the gap following extrusion of the methylated cytosine from the DNA duplex and at the same time monitors the status of unmodified CpG on the partner strand.
Cytosine DNA methylation is evolutionarily ancient and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals and the 5-methyl-cytosine (5mC) binding specificity of several SRA domains have been characterized and found to show a preference for DNA methylation in different sequence contexts. Here in a collaborative effort with the Steve Jacobsen laboratory (University of California, Los Angeles), we demonstrate from gel shift and calorimetric measurements that the SRA domain of SUVH5 differs from that of other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of SUVH5 SRA domains bound to 5mC-containing DNA identify a dual flip out mechanism, where both 5mC and a base (5mC, C or G) from the partner strand are simultaneously extruded from the duplex and positioned within binding pockets of individual SRA domains in fully- and hemi-methylated CG and methylated CHH sequence contexts. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9me2 modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci
Rajakumara, E., Law, J. A., Shimanshu, D. K., Voigt, P., Johnson, L. M., Reinberg, D., Patel, D. J. & Jacobsen, S. E. (2011). A dual flip out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo. Genes Dev. 25, 137-152.
Cross-talk Between Histone and DNA Methylation
Both histone modification and DNA methylation contribute to the establishment of patterns of gene repression during development. As an example, the maintenance of DNA methylation at CpHpG sites in plants requires the combined action of CMT3, a DNA methyltransferase and KRPTONITE, a histone lysine methyltransferase. The BAH domain and chromodomain of CMT3 target H3K9me2 marks, while KRPTONITE contains an SRA domain that binds methylated CpHpG DNA. Thus, CMT3 and KRYPTONITE bind to each others product to maintain feedback loop for CpHpG DNA methylation in plants.
In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase Chromomethylase3 (CMT3) and H3K9 histone methyltransferase Kryptonite/SUVH4 (KYP). Here, in a collaborative effort with the Steve Jacobsen laboratory (UCLA Medical School, CA), we report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the e-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation.
Du, J., Johnson, L. M., Groth, M., Feng, S., Hale, C. J., Li, S., Vashisht, A. A., Gallego-Bartolome, J., Wohlschlegel, J. A., Patel, D. J. and Jacobsen, S. E. (2014). Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Mol. Cell 55, 495-504.
Dual Binding of Chromomethylase Domains to H3K9me2-containing Nucleosomes Mediates DNA Methylation in Plants
DNA methylation and histone modification exert epigenetic control over gene expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. Here, in a collaborative effort with the Steve Jacobsen laboratory (University of California, Los Angeles), we report multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2 and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homologue, ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both BAH and chromo domains. The structures reveal an aromatic cage within both BAH and chromo domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes, and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH and chromo domains, and reveals a novel mechanism of interplay between DNA methylation and histone modification.
Du, J., Zhong, X., Barnatavichute, Y. V., Stroud, H., Feng, S., Caro, E., Vashisht, A. A., Terragni, J., Chin, H. G., Tu, J., Hetzel, J., Wohlschlegel, J. A., Pradhan, S., Patel, D. J. & Jacobsen, S. E. (2012). Dual binding of chromomethylase BAH and chromo domains to H3K9me2-containing nucleosomes in the targeting of DNA methylation. Cell 151,167-180.
In a collaborative project championed by the Stephen Jane laboratory (Melbourne, Australia), it has been shown that PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, thereby coupling histone and DNA methylation in gene silencing.
Zhao, Q., Rank, G., Tan, Y. T., Li, H., Moritz, R. L., Simpson, R. J., Cerruti, L., Curtis, D. J., Patel, D. J., Allis, C. D., Cunningham, J. M. & Jane, S. M. (2009). PRMT5-mediated methylation of histone H4R3 recruits DNMT3A coupling histone and DNA methylation in gene silencing. Nat. Struct. Mol. Biol. 16, 304-311. [PubMed Abstract]
RNA-Directed DNA Methylation
In Arabidopsis thaliana, 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). 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. Current efforts address the identification and characterization of protein modules in controlling Pol-IV and Pol-V occupancy at RNA-directed DNA methylation sites.
RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the synthesis of non-coding RNAs by DNA-dependent RNA polymerase V (Pol V). Previously, we have shown that the SRA/SET domain proteins SUVH2 and SUVH9 are required for RdDM. Here, as part of a collaboration with the Steve Jacobsen laboratory (UCLA Medical School, CA), we report on the crystal structure of SUVH9 which reveals a two-helix bundle that is sandwiched between the SRA and pre-SET/SET domains and an incomplete SAM binding site, as well as the absence of a peptide substrate-binding cleft within the structure of SUVH9, reflecting the absence of a POST-SET domain in this enzyme, that explains the lack of methyltransferase activity observed for these proteins in vitro. We also show that these SRA-domain proteins are required for Pol V association with chromatin and provide a link between DNA methylation and RdDM. Through tethering SUVH2 with a zinc finger to an unmethylated epiallele of the FWA gene, we show that SUVH2 is sufficient to both recruit Pol V and establish DNA methylation at FWA. This DNA methylation causes gene silencing that results in early flowering plants. Our results suggest that the primary function of SUVH2/SUVH9 is in recruitment of Pol V to chromatin through its methyl-DNA binding SRA domain, providing a self-reinforcing mechanism through which DNA methylation promotes the transcription of non-coding RNAs that in turn targets further DNA methylation.
Johnson, L. M., Du, J., Hale, C. J., Bischof, S., Feng, S., Chodavarapu, R. K., Zhong, X., Marson, G., Pellergrini, M., Segal, D. J., Patel, D. J. and Jacobsen, S. E. (2014). SRA/SET domain proteins link RNA polymerase V occupancy to DNA methylation. Nature 507, 124-128.
To understand the mechanisms controlling Pol-IV targeting, the laboratory of our collaborator Steve Jacobsen (University of California, Los Angeles) 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 RdRM targets, and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that 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. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation level, as well as Pol-IV occupancy at RdRM 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.
Law, J. A., Du, J., Hale, C. J., Feng, S., Krajewski, K., Strahl, B. D., Patel, D. J. & Jacobsen, S. E. (2013). SHH1 recruits RNA polymerase-IV to RNA-directed DNA methylation targets. Nature 498, 385-389.
At least three pathways control maintenance DNA cytosine methylation in Arabidopsis thaliana. However, RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IND2) as being an RNA-binding RdRM component that is required for DNA methylation establishment. In this study, the laboratory of our collaborator Steve Jacobsen (University of California, Los Angeles) describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE-1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis conforms the placement of the IDN proteins in an RdRM pathway that affects DNA methylation and transcription control at many sites in the genome. Results from this study identify and describe two unique components of the RdRM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.
Ausin, I., Greenburg, M. V., Simanshu, D. K., Hale, C. J., Vashisht, A., Lee, Y-F., Simon, S. A., Feng, S., Espanola, S., Meyers, B., Wohlschlegel, J. A., Patel, D. J. & Jacobsen, S. E. (2012). An IDN2-containing complex involved in RNA-directed DNA methylation in Arabidopsis. Proc. Natl. Acad. Scis. USA. 109, 8374-8381.