DNA methylation pathways and their crosstalk with histone methylation

Abstract

Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk.

Figure 1. A unidirectional pathway in Neurospora crassa

a | A cartoon model of the life cycle of Neurospora crassa. Neurospora grows from a single haploid spore into a multinucleated branched thread called mycelium. This vegetative stage continues as the mycelium expands and can originate new mycelia as colonies bud off and disperse (conidium stage). This cycle is asexual and continues until two colonies of opposite mating types (A and a) interact and give rise to a fruiting body. The fusion of the two haploid spores gives rise to a dikaryon that then proliferates within the fruiting body. After premeiotic DNA synthesis, nuclei of the opposite mating type fuse, and meiosis is initiated. Each of the meiotic spores then undergoes mitosis to give rise to an octad of haploid spores. This series of events constitutes the sexual cycle. b | A schematic representation of the unidirectional pathway from H3K9me3 to DNA methylation in N. crassa. The DCDC complex associates with H3K9 methyltransferase DIM-5 and targets it to certain chromatin loci to create the H3K9me3 mark. Once the H3K9me3 is established, the heterochromatin protein 1 (HP1) can specifically recognize the H3K9me3 mark to facilitate targeting of the associated DNA methyltransferase DIM-2 to methylate DNA at the same sites.

Figure 2. Structural basis underlying the cross-regulation of CHG DNA methylation and histone H3K9me2 in Arabidopsis thaliana by the self-reinforcing loop between CMT3 and KRYPTONITE

a | A cartoon representation of the self-reinforcing model between Chromomethylase 3 (CMT3) and CMT2 and KRYPTONITE (KYP) in CHG and CHH methylation, respectively, and H3K9me2 methylation. CMT3 and CMT2 are targeted by H3K9me2, which catalyses the transfer of a methyl group to CHG and CHH sites on the DNA in the corresponding region. Similarly, the CHG and CHH methylation mark can be captured by KYP, which then catalyses the transfer of a methyl group to H3K9 of nearby nucleosomes, creating the binding sites for CMT3 and CMT2 to establish the reinforcing loop. b | Domain architecture of CMT3 in a colour-coded representation. c | A superposed composite structural model of ZMET2 (the maize homologue of CMT3) in complex with H3K9me2 peptide and modelled DNA (based on the RCSB protein databank (PDB) codes: 4FSX, 4FT2, 4FT4 and 4DA4). The bromo adjacent homology (BAH), DNA methyltransferase, and chromodomains are coloured in green, purple, and cyan, respectively. The bound peptide and cofactor SAH are shown in space-filling representations, and modelled DNA is magenta ribbon representation. d | The aromatic residues Phe441, Trp466, and Tyr469 of the ZMET2 chromodomain form an aromatic cage for recognition of the H3K9me2 in a methyllysine-dependent manner. e | The aromatic residues Tyr203, Trp224, and Phe226 of the ZMET2 BAH domain form an aromatic cage for recognition of H3K9me2 in a methyllysine-dependent manner. f | Domain architecture of Arabidopsis thaliana KYP in a colour-coded representation. g | A ribbon representation of the crystal structure of KYP in complex with methylated non-CG DNA, cofactor SAH, and unmodified H3(1–15) substrate peptide (PDB codes: 4QEN, 4QEO, and 4QEP). The amino-terminal 2-helix bundle, SRA (SET- and RING-associated), pre-SET, SET, and post-SET domains of KYP are coloured in orange, cyan, green, magenta and brown, respectively. The bound methylated DNA, cofactor SAH, and peptide substrate are shown in salmon ribbon, lilac stick, and space-filling representation, respectively. h | Structural basis underlying specific recognition by the SRA domain of the flipped out 5mC base of the bound methylated DNA. The base is stacked between two tyrosine residues from the top and bottom directions. The Watson-Crick edge of the 5mC forms several hydrogen bonding interactions with the surrounding residues as indicated by dashed red lines. The methyl group of 5mC is accommodated within a small hydrophobic pocket. i | The peptide substrate and cofactor SAH are embedded in between the post-SET and SET domains. The to-be-methylated lysine forms several hydrogen-bonding interactions with important tyrosine residues as shown by dashed pink lines.

Figure 3. Structures of regulators of Arabidopsis thaliana RdDM pathway

a | An updated scheme of the RNA-directed DNA methylation (RdDM) pathway. The RdDM is initiated when RNA POLYMERASE IV (Pol IV) is targeted by SAWADEE HOMEODOMAIN HOMOLOGUE 1(SHH1)-bound H3K4me0K9me2 (orange circles) to produce single RNA transcripts. The H3K4me0K9me2 state is regulated by the histone modification enzymes JMJ14, LDL1 and LDL2, KRYPTONITE (KYP) and HDA6. Pol IV produced RNA is replicated by Pol IV-associated RNA-DIRECTED RNA POLYMERASE 2 (RDR2) to generate double-stranded RNA, which is further processed by DICER-LIKE 3 (DCL3) and ARGONAUTE 4 (AGO4) to produce 24-nucleotide (nt) siRNAs which are loaded onto AGO4. Meanwhile, the DRD1, DMS3, RDM1 (DDR) complex is directed to the methylated DNA (green circle) region by its associated SUVH2 and SUVH9 and targets Pol V to produce scaffold non-coding RNA. siRNA-bound AGO4 can interact with Pol V either directly or indirectly through SPT5L and through base-pairing between siRNA and non-coding RNA to further target DNA methylase 2 (DRM2) to methylate target DNA. b | Domain architecture of Arabidopsis thaliana SHH1. c | Ribbon representation of the crystal structure of the SAWADEE domain of SHH1 in complex with H3K9me2 peptide (PDB code: 4IUT). The first tudor domain, second tudor domain and the bound peptide are coloured in green, purple and yellow, respectively. The peptide is shown in a space-filling representation. d | The structural basis underlying specific recognition of H3K9me2. Three aromatic residues of SHH1, Tyr140, Phe162, and Phe165, form an aromatic cage to accommodate the methyllysine side chain, involving stabilization by cation-π interactions. e | The structural basis underlying specific recognition of unmodified H3K4. Two acidic residues, Glu130 and Asp141, form salt bridges and hydrogen bonding interactions with the amide protons of unmodified H3K4, with hydrogen bonding alignments shown as dashed pink lines. f | Domain architecture of Arabidopsis thaliana SUVH9, which possesses SET- and RING-associated (SRA), pre-SET and SET domains but lacks the post-SET domain. g | Crystal structure of SUVH9 (PDB code: 4NJ5) with a modelled DNA in ribbon representation. The amino-terminal 2-helix bundle, SRA domain, pre-SET domain, SET domain, and the modelled DNA are coloured in orange, cyan, yellow, green and salmon, respectively. h | Domain architecture of Arabidopsis thaliana de novo DNA methyltransferase DRM2. i | Ribbon representation of the structure of the symmetric dimer (coloured in green) formed by the catalytic domain of Nicotiana tabacum DRM (PDB code: 4ONJ). The cofactor analogue sinefungin is shown in a stick representation. j | A superposition of N. tabacum DRM monomer (in green) with DNA methyltransferase 3A (DNMT3A) catalytic domain (in silver, PDB code: 2QRV) reveals NtDRM possesses classic type I methyltransferase fold like DNMT3A. Mol, molecule. Part a is from REF. 3, Nature Publishing Group.

Figure 4. Structure of mammalian de novo DNA methyltransferases DNMT3A and DNMT3L

a | A cartoon model showing the DNA methyltransferase 3A (DNMT3A)–DNMT3L tetramer binds to unmodified H3K4 and then catalyses CG DNA methylation. b | Domain architecture of mammalian de novo DNA methyltransferase DNMT3A and its catalytically inactive cofactor DNMT3L in a colour-coded representation. c | Ribbon representation of the structure of the DNMT3L–DNMT3A–DNMT3A–DNMT3L functional tetramer with the catalytic domain of DNMT3L coloured in magenta and the catalytic domain of DNMT3a in green (RCSB protein databank (PDB) code: 2QRV). The cofactor SAH is shown in yellow in a stick representation. d | Structure of the DNMT3L–DNMT3A–DNMT3A–DNMT3L tetramer catalytic domains including the ADD (ATRX–DNMT3–DNMT3L) domain of DNMT3A (in light brown) (PDB code: 4U7P). e | Structure of the autoinhibitory conformation with a superposed model of bound DNA (PDB code: 4U7P, the DNA is modelled from Methyltransferase HhaI (M.HhaI)–DNA complex with a PDB code: 1MHT). The ADD domain, catalytic domain, and the modelled DNA are coloured in light brown, green, and red, respectively. The ADD domain interacts with the catalytic domain and blocks access to modelled DNA along one face. f | The structure of DNMT3a with captured H3K4me0 peptide together with a superposed model of bound DNA (PDB code: 4U7T). The bound peptide is shown in space-filling representation. Upon H3 peptide binding, the ADD domain interacts with the catalytic domain along another face that is positioned further away from the catalytic site, thereby releasing autoinhibition and providing access to modelled DNA. Panels e and f are aligned in the same orientation.

Figure 5. Structures of DNMT1 and its epigenetic regulator UHRF1 in maintenance DNA methylation

a | Domain architecture of DNA methyltransferase 1 (DNMT1). b | Structure of DNMT1 in complex with unmethylated CpG DNA in an autoinhibitory conformation (RCSB protein databank (PDB) code: 3PT6). The CXXC, bromo adjacent homology 1 (BAH1), BAH2 and DNA methyltransferase domains are coloured in yellow, magenta, cyan, and green, respectively. The unmethylated CpG DNA is shown in a purple ribbon representation, with the DNA interacting with the CXXC domain. The linker between the CXXC and BAH domain is positioned between the DNA and the catalytic pocket, thereby blocking access to the catalytic site. c | Structure of DNMT1 in complex with a hemimethylated CpG DNA in a productive conformation (PDB code: 4DA4). The to-be-methylated cytosine is flipped out from the DNA duplex and inserts into the active site of the methyltransferase domain. d | The base-flipping mechanism of DNMT1. The to-be-methylated fC (this cytosine analogue was used to covalently trap a productive complex) is highlighted in red and forms a covalent bond with a cysteine residue of the active site. Lysine and methionine residues insert into the space vacated by the flipped-out 5fC, with the alignment buttressed by a tryptophan residue. e | A superposition of the unmethylated DNA bound in an autoinhibitory conformation of DNMT1 (protein in light blue and DNA in dark blue) and hemimethylated DNA bound productive conformation of DNMT1 (protein in light red and DNA in dark red). f | The structure of a replication foci domain (RFD)-containing DNMT1 (free state, with RFD domain in orange) in an autoinhibitory conformation (PDB code: 3AV5). g | A model proposing that UHRF1 could target DNMT1 to hemimethylated CG (hmCG) DNA by recognizing and potentially binding to both H3K9me3 and hmCG DNA. h | Domain architecture of UHRF1. i | Structure of the tandem tudor-plant homeodomain (PHD) cassette of UHRF1 in complex with H3K9me3 peptide (PDB code: 4GY5). The tandem tudor and PHD domains are coloured in cyan and magenta, respectively. The unmodified R2 is specifically recognized by the acidic residues Asp334 and Asp337 of the PHD finger through salt bridges and hydrogen-bonding interactions, which are highlighted by dashed red lines. The trimethylated H3K9 is accommodated within an aromatic cage formed by Phe152, Tyr188 and Tyr191 of the tandem tudor domain. j | The SET- and RING-associated (SRA) domain of UHRF1 can specifically recognize hemimethylated CpG DNA (PDB code: 3CLZ). The SRA domain and DNA are coloured in green and yellow, respectively. The flipped out 5mC is highlighted in a space-filling representation. UBL, ubiquitin-like domain