Cytosine DNA methylation is an epigenetic modification of DNA that is usually associated with the stable and heritable repression of gene transcription. Many methylated sequences found in genomes are transposable elements, indicating the likely ancestral role of DNA methylation in genome defense. DNA methylation is also important in imprinting, X chromosome inactivation, and the epigenetic regulation of genes. While DNA methylation is widespread in plants, fungi and animals, it has been curiously lost in some well-studied model organisms including S. cerevisiae, S. pombe, C. elegans and Drosophila.
We study DNA methylation in the model plant Arabidopsis thaliana because of its facile genetics, small size, and trim genome. Furthermore, unlike other organisms like mouse, where DNA methylation mutants are inviable, Arabidopsis can tolerate mutations that virtually eliminate methylation, allowing for further study. Arabidopsis methylation mutants display developmental abnormalities because of defects in the methylation of several key genes that regulate development. We have taken advantage of the stable methylation present several of these developmental genes (epigenetic alleles), in order to perform genetic screens for mutants affecting DNA methylation. Our studies have revealed that DNA methylation is controlled by 1) the specificity of several different DNA methyltransferases, 2) targeting by other chromatin modifications such as the methylation of histone tails, and 3) targeting of specific DNA sequences by small interfering RNAs (siRNAs) and long non-coding RNAs. These screens have also uncovered novel pathways by which DNA methylated sequences are read and silenced. In addition to genetics, we rely heavily on computational biology and biochemistry in our work. We also have a small mammalian program, focused on studies of conserved regulators of DNA methylation.
Epigenetic mutations at SUPERMAN and FWA.
At the heart of our work is the use of so-called epigenetic mutations. These are alleles of developmentally important genes that are stably silent as a result of DNA methylation. Unlike real genetic alleles, epigenetic alleles have a DNA sequence that is identical to wild type. Nonetheless, these alleles are inherited in a Mendelian fashion and can be used for classical genetic studies. SUPERMAN loss-of-function mutants show an altered floral structure, and thus the phenotype can be easily monitored by the naked eye. SUPERMAN epigenetic alleles (called the clark kent alleles) are caused by dense hypermethylation and silencing of the SUPERMAN gene, which is otherwise unmethylated in wild type plants. This hypermethylation effect is meiotically heritable and causes a recessive loss-of-function phenotype.
A hypermethylated superman epimutation.
A second set of epigenetic mutants we study are at the imprinted FWA locus. In wild type, FWA is methylated and silent in all adult tissues of the plant. fwa hypomethylated mutant strains show a dominant late flowering phenotype due to a permanent loss of methylation present within two direct repeats in the FWA promoter, which causes ectopic expression of the gene.
A hypomethylated fwa epimutation.
Thus, SUPERMAN and FWA can adopt two heritable epigenetic states, either methylated and silent, or unmethylated and active. We have also taken advantage of other methylated genes in our work, such as the SDC gene that is normally methylated in wild type plants.
Mechanisms of DNA methylation control.
DNA methylation is found at cytosine residues in three different sequence contexts, CG, CHG, and asymmetric CHH sites (H = A, T, or C). Furthermore DNA methylation can be classified as the initial establishment of methylation (de novo methylation) or maintenance of preexisting methylation. The main enzyme that maintains preexisting CG methylation is MET1, a homolog of mammalian DNMT1. MET1 works along with a highly conserved co-factor called VIM in plants and UHRF1 in mammals, which assists in the maintenance of methylation. Our work showed that VIM/UHRF1 works by recognizing hemimethylated DNA that is produced at the DNA replication fork, which facilitates methylation by MET1/DNMT1 to restore fully methylated CG sites.
Our work also helped to define the enzymes involved in non-CG methylation and the enzymes controlling de novo methylation.
First, we performed a mutant screen to identify genes required for the maintenance of SUPERMAN DNA methylation and silencing. This screen uncovered loss-of-function alleles of the CHROMOMETHYLASE3 gene, which encodes a novel type of CHG specific DNA methyltransferase. CHROMOMETHYLASE3 mutants cause a genome wide reduction of CHG DNA methylation and result in the reactivation of SUPERMAN as well as many previously silent retrotransposons. These findings showed that CHG methylation is important for gene silencing, and clearly define CHROMOMETHYLASE3 as the main enzyme controlling this modification.
Second, we used reverse genetics, to show that the DOMAINS REARRANGED METHYLASE2 (DRM2) gene (the plant ortholog of mammalian DNMT3) encodes the major de novo methyltransferase in Arabidopsis. To do this, we first needed to develop in vivo assays for de novo methylation. First, we discovered that FWA is an efficient substrate for de novo methylation and transgene silencing when it is transformed into wild type plants. However, when transformed into drm2 mutants, FWAde novo methylation is blocked. Second, we discovered that a transgenic inverted repeat of the SUPERMAN locus causes de novo methylation of the endogenous SUPERMAN gene. This de novo methylation and gene silencing is also blocked in the drm2 mutant. Importantly, drm2 does not block gene silencing of preexisting silent FWA or SUPERMAN alleles, demonstrating that the DRM2 is important for de novo methylation, but is dispensable for the maintenance of much of the preexisting methylation.
The role of chromatin modifications in DNA methylation control. Our screen for suppressors of SUPERMAN gene silencing uncovered a second gene with a silencing phenotype remarkably similar to that of the CHG specific DNA methyltransferase CHROMOMETHYLASE3. This gene, named KRYPTONITE, encodes a member of the Su(var)3-9 class of histone methyltransferases, and like other members of this group, methylates lysine 9 of histone H3 (H3K9me). Because CHG DNA methylation is lost in kyp mutants, this suggests that CHG methylation is controlled by histone methylation. Further work on CMT3 and KRYPTONITE showed that they act in a clear feedback loop to maintain both CHG methylation and H3K9 methylation. The feedback works because CMT3 binds directly to H3K9 methylation through its chromodomain and BAH domain, and because KRYTPONITE binds directly to CHG DNA methylation.
Later work showed that the relationship between H3K9 methylation and DNA methylation is more general. KRYPTONITE has two homologs, SUVH5 and SUVH6 that also methylate H3K9, and together, the KRYTPONITE, SUVH5 and SUVH6 proteins can bind to all types of DNA methylation. In addition, CMT3 has a homolog called CMT2, which appears to act in a similar manner but contributes to the maintenance of CHH methylation, especially in the dense blocks of heterochromatin near centromeres. Thus, regions of DNA methylation generally attract H3K9 methylation, and H3K9 methylation promotes both CHG and CHH DNA methylation.
3) The role of small RNAs and the RNA-directed DNA methylation pathway. Our screen for suppressors of SUPERMAN gene silencing uncovered a third gene called, ARGONAUTE4(AGO4). ago4 mutants reduce both non-CG DNA methylation and histone H3 lysine 9 methylation at SUPERMAN and other affected loci. At the time, AGO proteins were only known to be involved in RNA interference and microRNA pathways that target mRNAs post-transcriptionally, so it was initially surprising to find an AGO required for SUPERMANtranscriptional gene silencing. However, it is now clear from many experimental systems such as S. pombe, Tetrahymena, and Drosophila, as well as our work in Arabidopsis, that AGO proteins and small RNAs are central to the targeting of chromatin modifications such as DNA methylation.
Subsequent work from our lab and several other labs have uncovered many additional components of this so called RNA-directed DNA methylation system. The basic outline of this pathway is shown below. The “upstream” siRNA biogenesis part of the pathway starts with the action of the RNA Polymerase IV (Pol IV) together with RDR2 to make double stranded RNAs, which are diced by DCL3 into 24nt siRNAs that then load into the effector protein AGO4. The “downstream” DNA methylation-targeting step is driven by non-coding RNAs made by RNA polymerase Pol V and accessory proteins. Pol V RNAs then act as a scaffold to recruit AGO4/siRNA complexes, which finally recruit DRM2 to methylate the DNA.
It has recently become clear that the RNA-directed DNA methylation pathway acts as a self-reinforcing loop mechanism. First, Pol IV is recruited (through SHH1) to chromatin that is marked with H3K9 methylation, a mark that is associated with DNA methylation. SHH1 is also repelled by H3K4 methylation, a mark of active Pol II genes, which likely helps prevent methylation of genes. Second, Pol V is recruited (through SUVH2/SUVH9) to DNA methylation itself. Thus, Pol IV and Pol V are recruited back to silent regions of chromatin, reinforcing silencing at sites of pre-existing DNA methylation. Many of the precise mechanisms involved in RNA-directed DNA methylation are still poorly understood, and several factors important in this pathway have not been characterized.
We are also very interested in proteins and processes that act downstream of DNA methylation to cause gene silencing. Recent mutant screens have uncovered mutations in two genes, AtMORC1 and AtMORC6, which cause derepression of gene silencing, but do not cause changes in the patterns of DNA methylation, histone methylation, or small RNAs. Using a variety of techniques we showed that morc mutants show decondensation of pericentromeric heterochromatin (chromocenters), and that MORC proteins form heterodimers that are localized to punctate bodies adjacent to chromocenters (see figure below), suggesting that MORCs act to compartmentalize pericentromeric heterochromatin to maintain gene silencing. MORCs likely regulate gene silencing in many other organisms as well. With John Kim’s lab, we showed that the single C. elegans MORC gene regulates transgene silencing. In our lab, we also showed that the mouse MORC1 gene is a critical regulator of transposon silencing in the male germ line.
The mechanisms by which MORC proteins function is as yet completely unknown. In addition, our mutant screens have also uncovered other genes that appear to act in related processes downstream of DNA methylation.