Hemi-methylated CpG Research Articles

Mechanistic insights into allosteric regulation of methylated DNA and histone H3 recognition by SRA and SET domains of SUVH5 and the basis for di-methylation of lysine residue.

Su-(var)3-9 homologue 5 (SUVH5), a member of SUVH family of histone lysine methyltransferase (HKMT) in Arabidopsis, is involved in epigenetic regulation of chromatin by recognizing 5-methyl-cytosine (5mC), in both CpG and non-CpG DNA context, through SRA domain and simultaneously performing the di-methylation of lysine 9 of histone H3 (H3K9) through SET domain. Here, we establish that the SET domain of SUVH5 allosterically restricts the SRA domain to the 5mC containing strand(s) of fully methylated CpG, hemi-methylated CpG and methylated CpHpH DNA. In addition, SET domain enhances the binding affinity of the SRA-SET dual domains to fully-mCpG but not to hemi-mCpG. Also, the recognition of methylated DNA by the SRA positively influences the recognition of H3K9 by the SET domain. Our further studies revealed that the SET domain recognizes the "A(R/K)KST" motif present in H3K9 and in other histone H2A variants. Further, computational analyses and quantum mechanics/molecular mechanics calculations explain the bases for robust mono-MTase but weak di-MTase activities of SUVH5. Given that the majority of eukaryotic proteins, including those involved in epigenetic gene regulation, contain more than one domain, our study suggests that understanding the allosteric regulation among multiple domains of proteins is relevant for unravelling biological outcomes.

Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases.

In mammals, three major DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains. For instance, Dnmt3a and Dnmt3b each contain a Pro-Trp-Trp-Pro (PWWP) domain that recognizes the histone H3K36me2/3 mark, an Atrx-Dnmt3-Dnmt3l (ADD) domain that recognizes unmodified histone H3 tail, and a catalytic domain that methylates CpG sites. Dnmt1 contains an N-terminal independently folded domain (NTD) that interacts with a variety of regulatory factors, a replication foci-targeting sequence (RFTS) domain that recognizes the histone H3K9me3 mark and H3 ubiquitylation, a CXXC domain that recognizes unmodified CpG DNA, two tandem Bromo-Adjacent-homology (BAH1 and BAH2) domains that read the H4K20me3 mark with BAH1, and a catalytic domain that preferentially methylates hemimethylated CpG sites. In this chapter, the structures and functions of these domains are described.

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads.

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein’s NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

Functional relevance of promoter CpG island of human Angiotensin II type 1 receptor (AT1R) gene.

Angiotensin II type 1 receptor can activate number of signalling pathways upon stimulation and consequently its involvement in cancer progression have also been revealed. But which epigenetic mechanisms are involved in its regulation, need to be further explored. In-silico analysis revealed a promoter CpG island (CGI) which was cloned and assayed for functional activity using reporter gene system. The effect of methylation on this CGI was analysed through varying degree of methyltransferase treatment of cloned fragment. Results thus obtained were validated by direct sequencing. To further establish the status of this effect, in-vivo analysis was done through screening of methylation pattern in the targeted region among hypertensives (HTN) and normotensives(NTN) using PCR-Bisulphite sequencing. Additionally, clinical details of all participants, biochemical parameters and lifestyle related information was also collected and statistically evaluated. Reporter gene assay assigned functional activity to the cloned promoter CpG island. Increased dose and durations of methyltransferase treatment decreased the expression of reporter gene indicating the role of promoter DNA methylation. Among all the human samples screened, only one of the HTN individual was found to have single hemi-methylated CpG site at a position which happens to be a part of Sp1 transcription factor binding site. To conclude, CpG island in the promoter region of AT1R (CpG.P.AT1R) gets regulated through epigenetic mechanism of DNA methylation.

Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

The relationship between active DNA demethylation induced by overexpressing Tet1 and passive DNA demethylation induced by suppressing Dnmt1 remains unclear. Here, we found that DNMT1 preferentially methylated, but TET1 preferentially demethylated, hemi-methylated CpG sites. These phenomena resulted in a significant overlap in the targets of these two types of DNA demethylation and the counteractions of Dnmt1 and Tet1 during somatic cell reprogramming. Since the hemi-methylated CpG sites generated during cell proliferation were enriched at core pluripotency loci, DNA demethylation induced by Tet1 or sh-RNA against Dnmt1 (sh-Dnmt1) was enriched in these loci, which, in combination with Yamanaka factors, led to the up-regulation of these genes and promoted somatic cell reprogramming. In addition, since sh-Dnmt1 induces DNA demethylation by impairing the further methylation of hemi-methylated CpG sites generated during cell proliferation, while Tet1 induced DNA demethylation by demethylating these hemi-methylated CpG sites, Tet1-induced DNA demethylation, compared with sh-Dnmt1-induced DNA demethylation, exhibited a higher ability to open the chromatin structure and up-regulate gene expression. Thus, Tet1-induced but not sh-Dnmt1-induced DNA demethylation led to the up-regulation of an additional set of genes that can promote the epithelial-mesenchymal transition and impair reprogramming. When vitamin C was used to further increase the demethylation ability of TET1 during reprogramming, Tet1 induced a larger up-regulation of these genes and significantly impaired reprogramming. Therefore, the current studies provide additional information regarding DNA demethylation during somatic cell reprogramming.

Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter.

Enzymatic oxidation of 5-methylcytosine (5-mC) in the CpG dinucleotides to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) has central role in the process of active DNA demethylation and epigenetic reprogramming in mammals. However, it is not known whether the 5-mC oxidation products have autonomous epigenetic or regulatory functions in the genome. We used an artificial upstream promoter constituted of one cAMP response element (CRE) to measure the impact of 5-mC in a hemi-methylated CpG on the promoter activity and further explored the consequences of 5-hmC, 5-fC, and 5-caC in the same system. All modifications induced mild impairment of the CREB transcription factor binding to the consensus 5′-TGACGTCA-3′ CRE sequence. The decrease of the gene expression by 5-mC or 5-hmC was proportional to the impairment of CREB binding and had a steady character over at least 48 h. In contrast, promoters containing single 5-fC or 5-caC underwent further progressive loss of activity, up to an almost complete repression. This decline was dependent on the thymine-DNA glycosylase (TDG). The results thus indicate that 5-fC and 5-caC can provide a signal for perpetuation and enhancement of the repressed transcriptional state by a mechanism that requires base excision repair.

Conserved threonine 1505 in the catalytic domain stabilizes mouse DNA methyltransferase 1.

In mammals, DNA methyltransferase 1 (DNMT1) is responsible for propagating the DNA methylation pattern into the next generation through selective methylation of hemi-methylated CpG that emerges just after replication, a process known as maintenance methylation. The T1505, which is conserved among DNMT1s of vertebrates, in the catalytic domain of mouse DNMT1 forms the hydrogen bond with the W1512, which is also conserved among vertebrates and one of the essential residues in recognition of the 5-methylcytosine in hemi-methylated CpGs. However, importance of the hydrogen bond between T1505 and W1512 is unknown. In this study, we determined the crystal structure of mouse DNMT1(291-1620) that replaced T1505 with alanine (DNMT1(291-1620)T1505A) and examined its DNA methylation activity in vitro. Although the mutation lost the hydrogen bond between T1505 and W1512, the overall structure of DNMT1(291-1620)T1505A remained almost identical with that of the wild type. Structural stability and DNA methylation activity of DNMT1(291-1620)T1505A under physiological temperature were lower than those of DNMT1(291-1620). T1505 is crucial on the DNA methylation activity of DNMT1 through stabilizing its structure during ongoing round of DNA methylation.

Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-mCpG-Np95-Dnmt1 Axis.

DNA methylation at C of CpG dyads (mCpG) in vertebrate genomes is essential for gene regulation, genome stability and development. We show in this study that proper functioning of post-replicative DNA mismatch repair (MMR) in mammalian cells relies on the presence of genomic mCpG, as well as on the maintenance DNA methyltransferase Dnmt1 independently of its catalytic activity. More importantly, high efficiency of mammalian MMR surveillance is achieved through a hemi-mCpG-Np95(Uhrf1)-Dnmt1 axis, in which the MMR surveillance complex(es) is recruited to post-replicative DNA by Dnmt1, requiring its interactions with MutSα, as well as with Np95 bound at the hemi-methylated CpG sites. Thus, efficiency of MMR surveillance over the mammalian genome in vivo is enhanced at the epigenetic level. This synergy endows vertebrate CpG methylation with a new biological significance and, consequently, an additional mechanism for the maintenance of vertebrate genome stability.

BioSpotlight / Citations

BioTechniquesVol. 61, No. 5 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114469AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit An enriching experienceAccurate detection and quantification of low-abundance proteins by immunoblotting can be difficult since the amount of sample that can be loaded onto SDS-polyacrylamide gels is limited. However, methods to enrich for specific classes of proteins prior to gel electrophoresis can help overcome this problem. In this issue of BioTechniques, Azamat Karginov and Michael Agaphonov at the Russian Academy of Sciences describe a simple enrichment technique that greatly improves the immunoblotting of low-abundance yeast membrane proteins and is also superior to the standard method of boiling yeast cell lysates in Laemmli SDS gel-electrophoresis sample buffer. In the new approach, yeast cells are lysed in a buffer containing ammonium acetate, which protects proteins from degradation and facilitates membrane precipitation. The membrane fraction is obtained by a 10-minute centrifugation in a benchtop microcentrifuge and then resuspended in an alternative sample buffer containing SDS and a high concentration of urea, eliminating the boiling step that can cause protein degradation.See “A simple enrichment procedure improves detection of membrane proteins by immunoblotting” on page 260.Double-fish for frog embryosIn situ hybridization (ISH) for the simultaneous detection of two different transcripts in Xenopus embryos has been tricky to carry out using conventional chromogenic substrates of alkaline phosphatase (AP) since the darker signal of substrates such as nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/ BCIP) obscures the lighter signal from other substrates such as Vector Red. In addition, the opaque yolk platelets in the endodermal cells of Xenopus embryos interfere with signal detection. In this month's issue, Zachary Agricola and Sang-Wook Cha at Cincinnati Children's Hospital Medical Center describe their protocol for sequential double-fluorescence ISH (FISH) in Xenopus that resolves both of these issues. As substrates for AP development, they used Vector Red and NBT/BCIP, but these were visualized fluorescently by excitation with red and far-red light, respectively, which allowed the signal from each substrate to be detected without interference from the other. Coupled with treatment of the embryos by a clearing agent to render them transparent, this new approach enabled single-cell resolution of the expression of each target transcript.See “Two-color fluorescence in situ hybridization using chromogenic substrates in Xenopus” on page 263.Deciphering cytosine methylationThe epigenetic modifications methylcytosine (mC) and hydroxymethylcytosine (hmC) play important roles in modulating gene expression. Although techniques have emerged to identify cytosine methylation patterns, they are limited in resolution, which makes distinguishing these marks experimentally challenging for researchers. Now, Kawasaki et al. present a method to simultaneously identify cytosine, mC and hmC at single-base resolution. Their approach relies on the enzyme DNMT1, which methylates the unmethylated cytosine in a hemi-methylated CpG but not in a hemi-hydroxymethylated CpG. Using DNMT1 alongside standard bisulfite treatment, the authors were able to identify mC, hmC, and cytosine sites in single assay. They validated the new EnIGMA (Enzyme-assisted Identification of Genome Modification Assay) technique by assaying a 200-bp sequence containing multiple cytosine modifications. This new approach should help researchers better catalog cytosine modifications and their effects on gene expression patterns.Kawasaki et al. 2016. “A novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution.”Nucleic Acid Research. doi:10.1093/nar/gkw994Single-cell, single-tube transcriptomics and proteomicsA fundamental mystery of molecular biology is the relationship between gene expression and protein production. Attempts to understand how RNA and protein levels co-vary within cells have led to the development of new techniques for assaying these molecules simultaneously. Genshaft et al. now offer a method for profiling single-cell transcriptomes and proteomes in the same reaction. The key to their approach is the use of the reverse transcriptase activity of DNA polymerase for both complementary DNA synthesis and proximity extension assays. Proximity extension uses two antibodies attached to short DNA sequences. When the antibodies are close, the DNA sequences bind together and are extended by DNA polymerase to generate a protein-indexed DNA readout, which can be detected by sequencing. Using this method, the authors were able to profile changes in the transcriptome and proteome of a human breast cancer cell line in response to different chemical perturbations. This multiplexed profiling technique provides a new option for scientists exploring gene—protein expression relationships.Genshaft et al. 2016. “Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction.” Genome Biology. 17:188. doi:10.1186/s13059-016-1045-6FiguresReferencesRelatedDetails Vol. 61, No. 5 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print November 2016 Information© 2016 Author(s)PDF download

Engineering affinity agents for the detection of hemi-methylated CpG sites in DNA.

Wild-type methyl-CpG-binding domain (MBD) proteins specifically bind symmetrically methylated DNA sequences, and assays have been developed that use these proteins for profiling DNA methylation. Here, we use directed evolution in the yeast surface display format to identify a new protein variant that binds hemi-methylated CpG dinucleotides.

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells.

The USFDA approved “epigenetic drug”, Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC.

Selective impairment of methylation maintenance is the major cause of DNA methylation reprogramming in the early embryo.

BackgroundDNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine (5mC). Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive (replication-dependent) or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing (DHBS) maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications.ResultsWe compared DHBS maps of repetitive elements in the developing zygote, the early embryo, and primordial germ cells (PGCs) at defined stages of development. In the zygote, we observed distinct effects in paternal and maternal chromosomes. A significant loss of paternal DNA methylation was linked to replication and to an increase in continuous and dispersed hemimethylated CpG dyad patterns. Overall methylation levels at maternal copies remained largely unchanged, but showed an increased level of dispersed hemi-methylated CpG dyads. After the first cell cycle, the combined DHBS patterns of paternal and maternal chromosomes remained unchanged over the next three cell divisions. By contrast, in PGCs the DNA demethylation process was continuous, as seen by a consistent decrease in fully methylated CpG dyads over consecutive cell divisions.ConclusionsThe main driver of DNA demethylation in germ cells and in the zygote is partial impairment of maintenance of symmetric DNA methylation at CpG dyads. In the embryo, this passive demethylation is restricted to the first cell division, whereas it continues over several cell divisions in germ cells. The dispersed patterns of CpG dyads in the early-cleavage embryo suggest a continuous partial (and to a low extent active) loss of methylation apparently compensated for by selective de novo methylation. We conclude that a combination of passive and active demethylation events counteracted by de novo methylation are involved in the distinct reprogramming dynamics of DNA methylomes in the zygote, the early embryo, and PGCs.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-8935-8-1) contains supplementary material, which is available to authorized users.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.

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.

Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

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.

3P119 Characterization of the hemi-methylated CpG methylation process using fluorescent labeled SRA(04. Nucleic acid binding proteins,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

3P119 Characterization of the hemi-methylated CpG methylation process using fluorescent labeled SRA(04. Nucleic acid binding proteins,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Dual base-flipping of cytosines in a CpG dinucleotide sequence

Simultaneous flipped-out conformation of two neighboring bases on opposite strands of DNAs has been observed in several X-ray structures. It has also been detected for two cytosines on opposite strands in different contexts of CpG sites. In this paper, we study by MD simulations the dual base flipping of the two cytosines in hemi-methylated CpG site. We calculate the potential of mean force of flipping-out the unmethylated cytosine in three model systems. The first is for DNA bound to the regulatory protein UHRF1. In this case, the methyl-cytosine on the complementary strand is flipped-out into the binding pocket of the SRA domain of the protein. The other two systems are for unbound DNAs in which the methyl-cytosine is either intra-helical or extra-helical. We find that when the methyl-cytosine is flipped-out it is easier to flip-out the other (unmethylated) cytosine on the opposite strand by about 14–16kJ/mol. This lower penalty for dual-base flipping is observed for both the bound and unbound states of the DNA. Analyses of the hydrogen bond network and stacking interactions within the CpG site indicate that the lower penalty is due to stabilization of the dual-base flipped-out conformation via interactions involving the orphan guanines. The results presented in this paper suggest that the extra-helical conformation of the methyl-cytosine recognized by UHRF1 can facilitate the base-flipping process of the target cytosine to be methylated by Dnmt1.

UHRF1 targets DNMT1 for DNA methylation through cooperative binding of hemi-methylated DNA and methylated H3K9

Epigenetic inheritance of DNA methylation in mammals requires a multifunctional protein UHRF1, which is believed to recruit DNMT1 to DNA replication forks through a unique hemi-methylated CpG-binding activity. Here we demonstrate that the UHRF1 mutants deficient in binding either hemi-methylated CpG or H3K9me2/3, but not both, are able to associate with pericentric heterochromatin, recruit Dnmt1 and partially rescue DNA methylation defects in mouse Uhrf1 null ES cells. Furthermore, we present evidence that the flip out of the methylated cytosine induced by UHRF1 binding is unlikely essential for subsequent DNA methylation by DNMT1. Together, our study demonstrates that UHRF1 can target DNMT1 for DNA maintenance methylation through binding either H3K9me2/3 or hemi-methylated CpG, and that the presence of both binding activities ensures high fidelity DNA maintenance methylation. In addition, our study indicates that UHRF1 mediates cross-talk between H3K9 methylation and DNA methylation at the level of DNA methylation maintenance.

Characterization of SRA-Methylated DNA Complexes Dynamics Related to Chromatin Structure Regulation

Eukaryotic gene expression is regulated by chromatin structures and/or DNA modification such as CpG methylation. The basic unit of eukaryotic chromatin structure is a nucleosome consisting of approximately 150 bp DNA wrapped in 1.7 superhelical turns around a histone octamer. The histone octamer consists of two copies each of H2A, H2B, H3 and H4. Posttranslational histone modifications such as acetylation, methylation, phosphorylation and ubiquitylation regulate chromatin structure, resulting in activation or repression of gene expression. On the other hand, CpG methylation represses gene expression and is essential for silencing of parasitic DNA, genomic imprinting and embryogenesis. During DNA replication, methylated CpGs are converted into hemi-methylated CpGs and newly replicated CpGs should be methylated to inherit methylation pattern. DNA methyltransferase 1 (Dnmt1) is the enzyme to methylate hemi-methylated CpG regions. Uhrf1 is methylated CpG binding protein and interacts with Dnmt1, followed by recruitment of Dnmt1 to hemi-methylated CpG regions. SRA domain of Uhrf1 is responsible for hemi-methylated CpG binding activity. We characterize the process of hemi-methylated CpG recognition by SRA domain using Single-Molecule technique, and in this symposium, we will show our present data.

How to Distinguish Methyl-Cytosine from Cytosine with High Fidelity

Methylation of cytosines in the DNA is central to the epigenetic code. The patterns along the DNA formed by these chemical marks instruct the cell which proteins to express and their faithful maintenance after replication are vital to the organism's life. Although Dnmt1 is the enzyme catalyzing the methylation reaction, it was found that UHRF1 (ubiquitin-like, containing PHD and RING finger domain 1) is the protein that actually recognizes hemi-methylated CpG sites. Nevertheless, the physical mechanism driving the strikingly robust distinction between hemi-methylated and unmethylated sites is not known. In this paper, we show that the large difference in the binding affinities of UHRF1 to these sites is possible not due to the presence of the methyl group itself but is a result of the accompanying changes in the distribution of the electrons around the cytosine ring. In particular, methylation reduces the dipole moment of cytosine and, as a consequence, unmethylated DNA in its unbound state in water is more stable than hemi-methylated DNA. Furthermore, the interaction energy of hemi-methylated DNA bound to UHRF1 with its surrounding is stronger than that of unmethylated DNA. Thus, the change in the electronic structure of cytosine upon methylation destabilizes the unbound state and stabilizes the bound state rendering discrimination with high fidelity possible.