[Show abstract][Hide abstract] ABSTRACT: Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransfearse 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1β and KAP1) in ES cells and orchestrates retroviral-silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney Murine Leukemia Virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1γ), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly-integrated retroviruses.
[Show abstract][Hide abstract] ABSTRACT: AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.
Nucleic Acids Research 06/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transcription factor Krüppel-like factor 4 (Klf4), one of the factors directing cellular reprogramming, recognizes the CpG dinucleotide (whether methylated or unmodified) within a specific G/C-rich sequence. The binding affinity of the mouse Klf4 DNA-binding domain for methylated DNA is only slightly stronger than that for an unmodified oligonucleotide. The structure of the C-terminal three Krüppel-like zinc fingers (ZnFs) of mouse Klf4, in complex with fully methylated DNA, was determined at 1.85 Å resolution. An arginine and a glutamate interact with the methyl group. By comparison with two other recently characterized structures of ZnF protein complexes with methylated DNA, we propose a common principle of recognition of methylated CpG by C2H2 ZnF proteins, which involves a spatially conserved Arg-Glu pair.
Nucleic Acids Research 02/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer including cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only few non-nucleoside DNMTi have been identified so far, and much less validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as potent non-nucleoside DNMTi, and selective towards other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit µM against a panel of cancer cells, and less toxic in peripheral blood mononuclear cells. In mouse medulloblastoma stem cells 5 inhibited cell growth while the related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.
Journal of Medicinal Chemistry 01/2014; · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.
[Show abstract][Hide abstract] ABSTRACT: Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency.
PLoS ONE 01/2014; 9(5):e96941. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(ii)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.
[Show abstract][Hide abstract] ABSTRACT: The Zfp57 gene encodes a KRAB (Krüppel-associated box) domain-containing C2H2 zinc finger (ZnF) transcription factor that is expressed in early development. Zfp57 protein recognizes methylated CpG dinucleotide within GCGGCA elements at multiple imprinting control regions. In the previously solved structure of mouse Zfp57 DNA binding domain in complex with DNA containing 5-methylcytosine (5mC), the side chains of Arg178 and Glu182 contact the methyl group via hydrophobic and van der Waals interactions. We examined the role of Glu182 in recognition of 5mC by mutagenesis. The majority of mutants examined lose selectivity of methylated (5mC) over unmodified (C) and oxidative derivatives, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine (5caC), suggesting that the side chain of Glu182 (the size and the charge) is dispensable for methyl group recognition but negatively impacts the binding of unmodified cytosine as well as oxidized derivatives of 5mC to achieve the 5mC selectivity. Substitution of Glu182 with its corresponding amide (E182Q) had no effect on methylated DNA binding but gained significant binding affinity to 5caC DNA, resulting in a comparable binding affinity for 5caC DNA to that of wild-type protein for 5mC. We show structurally that the uncharged amide group of E182Q interacts favorably with the carboxylate group of 5caC. Furthermore, introducing a positively charged arginine at the residue 182 resulted in a mutant (E182R) having higher selectivity for the negatively charged 5caC.
[Show abstract][Hide abstract] ABSTRACT: The mammalian thymine DNA glycosylase (TDG) excises 5-carboxylcytosine (5caC) when paired with a guanine in a CpG sequence, in addition to mismatched bases. Here we present a complex structure of the human TDG catalytic mutant, asparagine 140 to alanine (N140A), with a 28-base pair DNA containing a G:5caC pair at pH 4.6. TDG interacts with the carboxylate moiety of target nucleotide 5caC using the side chain of asparagine 230 (N230), instead of asparagine 157 (N157) as previously reported. Mutation of either N157 or N230 residues to aspartate has minimal effect on G:5caC activity while significantly reducing activity on G:U substrate. Combination of both the asparagine-to-aspartate mutations (N157D/N230D) resulted in complete loss of activity on G:5caC while retaining measurable activity on G:U, implying that 5caC can adopt alternative conformations (either N157-interacting or N230-interacting) in the TDG active site to interact with either of the two asparagine side chain for 5caC excision.
[Show abstract][Hide abstract] ABSTRACT: Much is known about vertebrate DNA methylation, however it is not known how methylated CpG within particular sequences is recognized. Two recent structures of C2H2 zinc finger (ZnF) proteins in complex with methylated DNA reveal a common recognition mode for 5-methylcytosine (5mC) that involves a 5mC-Arg-G triad. In the two ZnF proteins, an arginine that precedes the first Zn-binding histidine (RH motif) can interact with a 5mCpG or TpG dinucleotide. Among a family of >300 human Krüppel-associated box (KRAB) domain containing ZnF proteins examined, two-thirds contained at least one ZnF that included an RH motif. We propose that the RH-ZnF motifs provide specificity for 5mCpG, whereas the neighboring Zn fingers recognize the surrounding DNA sequence context.
Trends in Biochemical Sciences 01/2013; · 13.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe the use of a unique DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5-hydroxymethylcytosine (5hmC) at low-occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follow the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the monomethylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.
[Show abstract][Hide abstract] ABSTRACT: The mammalian thymine DNA glycosylase (TDG) excises the mismatched base, uracil, thymine, or 5-hydroxymethyluracil (5hmU), as well as removes 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) when paired with a guanine. In the previously solved structure of TDG in complex with DNA containing 5caC, the side chain of asparagine 157 (N157) contacts the 5-carboxyl moiety of 5caC via a weak hydrogen bond. We examined the role of N157 in recognition of 5caC by mutagenesis. The asparagine-to-alanine (N157A) mutant has no detectable base excision activity for a G:T mismatch, and its excision activity is reduced for other substrates including G:5caC. Unexpectedly, the asparagine-to-aspartate (N157D) mutant has a comparable base excision rate for G:5caC substrate to that of wild type, but it only has residual activity for G:U and no detectable activity for other substrates. We further show that the N157D mutant has higher activity for 5caC at a lower pH (6.0), suggesting that increased protonation of the carboxylate of 5caC and the aspartate facilitates base excision. The N157D mutant remains highly specific for 5caC even in the presence of large excess of genomic DNA, a property that can potentially be used for mapping the very low amount of 5caC in genomes.
Journal of Molecular Biology 01/2013; · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc finger transcription factor Zfp57 recognizes the methylated CpG within the TGCCGC element. We determined the structure of the DNA-binding domain of Zfp57, consisting of two adjacent zinc fingers, in complex with fully methylated DNA at 1.0 Å resolution. The first zinc finger contacts the 5' half (TGC), and the second recognizes the 3' half (CGC) of the recognition sequence. Zfp57 recognizes the two 5-methylcytosines (5mCs) asymmetrically: One involves hydrophobic interactions with Arg178, which also interacts with the neighboring 3' guanine and forms a 5mC-Arg-G interaction, while the other involves a layer of ordered water molecules. Two point mutations in patients with transient neonatal diabetes abolish DNA-binding activity. Zfp57 has reduced binding affinity for unmodified DNA and the oxidative products of 5mC.
Genes & development 10/2012; · 12.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1' hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.
Nucleic Acids Research 09/2012; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3'-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N(12) cleavage in the same strand and then the second distal N(16) cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans.
Nucleic Acids Research 07/2012; 40(19):9763-73. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian DNA glycosylase--methyl-CpG binding domain protein 4 (MBD4)--is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA containing a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson-Crick polar edge of thymine or 5hmU via the O2, N3 and O4 atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivatives. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C1' of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases.
Nucleic Acids Research 06/2012; 40(17):8276–8284. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BIX-01294 and its analogs were originally identified and subsequently designed as potent inhibitors against histone H3 lysine 9 (H3K9) methyltransferases G9a and G9a-like protein. Here, we show that BIX-01294 and its analog E67 can also inhibit H3K9 Jumonji demethylase KIAA1718 with half-maximal inhibitory concentrations in low micromolar range. Crystallographic analysis of KIAA1718 Jumonji domain in complex with E67 indicated that the benzylated six-membered piperidine ring was disordered and exposed to solvent. Removing the moiety (generating compound E67-2) has no effect on the potency against KIAA1718 but, unexpectedly, lost inhibition against G9a-like protein by a factor of 1500. Furthermore, E67 and E67-2 have no effect on the activity against histone H3 lysine 4 (H3K4) demethylase JARID1C. Thus, our study provides a new avenue for designing and improving the potency and selectivity of inhibitors against H3K9 Jumonji demethylases over H3K9 methyltransferases and H3K4 demethylases.
Journal of Molecular Biology 02/2012; 416(3):319-27. · 3.91 Impact Factor