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New hydration sites in the methylated structures. The methylated CpG steps 2-3 (A) and 5-6 (B) of the hexagonal methylated decamer and 5-6 of the orthorhombic decamer (C). Additional water molecules are named wA and wB depending on their proximity to the wA and wB sites.
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The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG s...
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... major features of the hydration geometry of the CpG steps in the three structures are described in Tables II and III. In addition, new major groove hydration sites were found to be correlated with the presence of the methyl group of some m 5 CpG sequences ( Figure 3A and B). The water molecules were observed just in front of the methyl group at a distance too great for interacting with other donor or acceptor atoms of the DNA molecule. ...
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... water molecules were observed just in front of the methyl group at a distance too great for interacting with other donor or acceptor atoms of the DNA molecule. As indicated in Figure 3A and B, these water molecules are referred to wA or wB depending on their proximity to the wA and wB sites, respectively. For example, in the hexagonal form, the wA and wB water molecules are located in front of the methyl group of m 5 C5 and m 5 C18 residues, respectively ( Figure 3A and B). ...
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... indicated in Figure 3A and B, these water molecules are referred to wA or wB depending on their proximity to the wA and wB sites, respectively. For example, in the hexagonal form, the wA and wB water molecules are located in front of the methyl group of m 5 C5 and m 5 C18 residues, respectively ( Figure 3A and B). These additional solvent molecules are also stabilized through the formation of hydrogen bond networks with their neighboring wA and wB solvent molecules. ...
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... additional solvent molecules are also stabilized through the formation of hydrogen bond networks with their neighboring wA and wB solvent molecules. A particular helical conform- ation also contributes to organize solvent networks in front of the methylated bases: due to the high twist angle at step 2-3, the two wA water molecules bound to the N4 amino groups of m 5 C2 and m 5 C18 interact with each other ( Figure 3A). Since in all cases the geometry of hydration is suitable for establishing C-H···O hydrogen bonds between the water molecules and the methyl group, it can be concluded that C-H···O bonds and solvent- solvent interactions are mainly responsible for the stabilization of these water molecules. ...
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... these deviations from regularity cannot be attributed to the methylation and/or the change in hydration since the native duplex shares similar helical parameters at the corresponding steps, with a different hydration pattern. Note that the geometry of step 2-3 is reminiscent of that observed in the B-DNA dodecamer methylated at its central CpG step where the anionic oxygen atoms of the phosphate group of a symmetry-related molecule occupy a similar position in front of the alternate N4 amino groups ( Figure 3A; Figure 3 of Mayer- Jung et al., 1997). ...
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... these deviations from regularity cannot be attributed to the methylation and/or the change in hydration since the native duplex shares similar helical parameters at the corresponding steps, with a different hydration pattern. Note that the geometry of step 2-3 is reminiscent of that observed in the B-DNA dodecamer methylated at its central CpG step where the anionic oxygen atoms of the phosphate group of a symmetry-related molecule occupy a similar position in front of the alternate N4 amino groups ( Figure 3A; Figure 3 of Mayer- Jung et al., 1997). ...
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... methyl group of the m 5 C5 methylcytosine base seems to participate in the stabilization of the distorted conformation by binding two additional water molecules. On both sides of the cytosine base plane they form a solvent network which bridges the adjacent residues C4 and G7 ( Figure 3C). The methyl group points into a cone of three polar atoms including the two water molecules and the anionic oxygen atom of the phosphate group of the m 5 C5 residue. ...
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Citations
... A previous X-ray crystallo-graphic analysis suggested that the specificity of MECP2 to methylated CpG is mediated by the conserved hydrophilic region in the MBD, where water molecules had been proposed to enable the DNA-protein interaction as intermediates in earlier studies (Ho et al., 2008;Mayer-Jung et al., 1998;Schwabe, 1997). A dispersed pattern of MECP2 in the nuclei can be observed in MECP2 with mutated MBD or reduced level of DNA methylation (Guy et al., 2011;Nan et al., 1996), indicating failed localization of the protein. ...
Rett Syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants in the MECP2 gene. While the majority of RTT-causing variants are clustered in the methyl-CpG binding domain and NCoR/SMRT interaction domain, we report a female patient with a functionally uncharacterized MECP2 variant in the C-terminal domain, c.1030C>T (R344W). We functionally characterized MECP2-R344W in terms of protein stability, NCoR/SMRT complex interaction, and protein nuclear localization in vitro. MECP2-R344W cells showed an increased protein degradation rate without significant change in NCoR/SMRT complex interaction and nuclear localization pattern, suggesting that enhanced MECP2 degradation is sufficient to cause a Rett Syndrome-like phenotype. This study highlights the pathogenicity of the C-terminal domain in Rett Syndrome, and demonstrates the potential of targeting MECP2 protein stability as a therapeutic approach.
... Structural studies with NMR and X-ray crystallography have demonstrated that methylated cytosine reduces the dynamics of the backbone of DNA duplexes [24][25][26][27][28][29]. In agreement with these experimental studies, molecular dynamics (MD) studies have also indicated that DNA flexibility is reduced by steric repulsion and hydrophobicity of the methyl groups [30,31]. Moreover, the effects of methylated cytosine on the thermal stability of DNA structures have also been reported. ...
... The Tm value increased from 51.4 °C to 59.5 °C as the number of methylated cytosines increased from zero to eight. Previously, it was reported that methylation of cytosine increases the Tm value of a B-form duplex [30]. The increment of around 1 °C per one methylation observed here is consistent with previous reports [54]. ...
Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.
... Second, all the ZF MBPs exhibit asymmetric binding at the mCpG palindrome, where the majority of arginine/lysine and glutamate interactions predominate at one mCpG (Fig. 2) [224]. In most cases, the remaining cross-strand mC has a hydration shell of ordered water molecules [252,253] that is believed to contribute to the overall binding energetics [132,254,255]. Notably, each of the ZF MBPs have similar binding affinities for symmetrically methylated and strand-specific hemi-methylated DNA [57,58,132,159,190], though the cellular significance of this asymmetric binding capability for each protein is not yet fully understood. ...
DNA methylation is an essential epigenetic modification involved in the maintenance of genomic stability, preservation of cellular identity, and regulation of the transcriptional landscape needed to maintain cellular function. In an increasing number of disease conditions, DNA methylation patterns are inappropriately distributed in a manner that supports the disease phenotype. Methyl-CpG binding proteins (MBPs) are specialized transcription factors that read and translate methylated DNA signals into recruitment of protein assemblies that can alter local chromatin architecture and transcription. MBPs thus play a key intermediary role in gene regulation for both normal and diseased cells. Here, we highlight established and potential structure-function relationships for the best characterized members of the zinc finger (ZF) family of MBPs proteins in propagating DNA methylation signals into downstream cellular responses. Current and future investigations aimed toward expanding our understanding of ZF MBP cellular roles will provide needed mechanistic insight into normal and disease state functions, as well as afford evaluation for the potential of these proteins as epigenetic-based therapeutic targets.
... In each of these cases, the remaining palindromic mC is surrounded by an ordered water layer. It has been established that the methyls of mCs are significantly hydrated [112,113], and that this hydration can contribute to the overall binding energetics for MBP recognition of mCpG sites [99,114,115]. In the case of ZBTB33, the glutamate makes cross-strand interactions with both mCs displacing any ordered water molecules around the mC methyl groups. ...
DNA methylation is a prevalent epigenetic modification involved in regulating a number of essential cellular processes, including genomic accessibility and transcriptional outcomes. As such, aberrant alterations in global DNA methylation patterns have been associated with a growing number of disease conditions. Nevertheless, the full mechanisms by which DNA methylation information is interpreted and translated into genomic responses is not yet fully understood. Methyl-CpG binding proteins (MBPs) function as important mediators of this essential process by selectively reading DNA methylation signals and translating this information into down-stream cellular outcomes. The Cys2His2 zinc finger scaffold is one of the most abundant DNA binding motifs found within human transcription factors, yet only a few zinc finger containing proteins capable of conferring selectivity for mCpG over CpG sites have been characterized. This review summarizes our current structural understanding for the mechanisms by which the zinc finger MBPs evaluated to date read this essential epigenetic mark. Further, some of the biological implications for mCpG readout elicited by this family of MBPs are discussed.
... 10 Moreover, Mayer-Jung et al. obtained crystal structures to show that epigenetic modifications (e.g., methylation of cytosines) on DNA can change the hydration patterns in the major groove. 11 In addition, protein-mediated shape change of DNA by histones and other large DNA−protein complexes, like CRISPR/Cas9, involves hydrating waters around DNA. 12 At large, evolution of our biological world hinges on the fidelity of DNA functions. The capacity to characterize the structures and dynamics of chiral water in DNA by optical methods should prove fruitful in uncovering the detailed mechanisms behind the biomolecular machineries for propagating genetic information. ...
Chiral vibrational sum frequency generation spectroscopy reveals a chiral water superstructure surrounding DNA and suggests that new biophysical insights are just around the bend.
... The key role of water molecules involved in the mCpG-dsDNA recognition by MBD has been postulated to be a general mechanism associated with mCpG recognition 50 , and, thus, the thermodynamic signature found for MeCP2 MBD should be a common general feature among mCpG recognition proteins. It is increasingly apparent that water is not just a passive matrix were physiological reactions take place, but water molecules are key active elements in many biomolecular processes such as protein folding 51,52 , nucleic acid assembly 53 , enzyme catalysis 54 , and molecular recognition [55][56][57][58][59][60] . ...
Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.
... When comparisons between duplex structures with C or mC were studied, a significant difference in base pair geometry or helical parameters between them was not observed; 50 in contrast, mC induces a change in the ordered waters found in the major groove. 51 Whether the difference in observed current results from the methyl groups altering the duplex hydration, or if the difference results from a steric effect altering the current, cannot be determined from these initial results. ...
The α-hemolysin (α-HL) nanopore can detect DNA strands under an electrophoretic force via many regions of the channel. Our laboratories previously demonstrated that trapping duplex DNA in the vestibule of wild-type α-HL under force could distinguish the presence of an abasic site compared to a G:C base pair positioned in the latch zone at the top of the vestibule. Herein, a series of duplexes were probed in the latch zone to establish if this region can detect more subtle features of base pairs beyond the complete absence of a base. The results of these studies demonstrate that the most sensitive region of the latch can readily discriminate duplexes in which one G:C base pair is replaced by an A:T. Additional experiments determined that while neither 8-oxo-7,8-dihydroguanine nor 7-deazaguanine opposite C could be differentiated from a G:C base pair, in contrast, the epigenetic marker 5-methylcytosine, when present in both strands of the duplex, yielded new blocking currents when compared to strands with unmodified cytosine. The results are discussed with respect to experimental design for utilization of the latch zone of α-HL to probe specific regions of genomic samples.
... The addition of a methyl group in the major groove of cytosine bases alters the hydrophobicity of DNA at the base-pair step. In crystallographic studies, it was found that a methylated A-form DNA oligomer (Mayer-Jung, Moras, and Timsit, 1998) is well hydrated, thereby allowing for the possibility of specific recognition of methylated DNA sequences, through the interaction with the tightly bound water at the methyl group. ...
In higher organisms, all cells share the same genome, but every cell expresses only a limited and specific set of genes that defines the cell type. During cell division, not only the genome, but also the cell type is inherited by the daughter cells. This intriguing phenomenon is achieved by a variety of processes that have been collectively termed epigenetics: the stable and inheritable changes in gene expression patterns. This article reviews the extremely rich and exquisitely multi-scale physical mechanisms that govern the biological processes behind the initiation, spreading and inheritance of epigenetic states. These include not only the change in the molecular properties associated with the chemical modifications of DNA and histone proteins - such as methylation and acetylation - but also less conventional ones, such as the physics that governs the three-dimensional organization of the genome in cell nuclei. Strikingly, to achieve stability and heritability of epigenetic states, cells take advantage of many different physical principles, such as the universal behavior of polymers and copolymers, the general features of non-equilibrium dynamical systems, and the electrostatic and mechanical properties related to chemical modifications of DNA and histones. By putting the complex biological literature under this new light, the emerging picture is that a limited set of general physical rules play a key role in initiating, shaping and transmitting this crucial "epigenetic landscape". This new perspective not only allows to rationalize the normal cellular functions, but
also helps to understand the emergence of pathological states, in which the epigenetic landscape becomes disfunctional.
... In general, methyl groups of methyl-CpGs are well hydrated [36]. Recent crystallographic studies suggest that hydration around the 5mC methyl groups plays important roles in DNA recognition by methyl-CpG-binding domain (MBD) proteins such as MeCP2 and MBD4 [37,38]. ...
... Taking advantage of the high-resolution structures in the three different methylation states, we examined how the hydration water molecules are involved in methylation-insensitive recognition of target DNA by Egr-1. Around cytosine bases in DNA, there are two major hydration sites, which Mayer-Jung et al. referred to as wA and wB [36]. Upon CpG methylation, the position of wB is shifted toward DNA phosphate while the position of wA is virtually unaffected [36]. ...
... Around cytosine bases in DNA, there are two major hydration sites, which Mayer-Jung et al. referred to as wA and wB [36]. Upon CpG methylation, the position of wB is shifted toward DNA phosphate while the position of wA is virtually unaffected [36]. Fig. 2 shows wA and wB water molecules around CpG cytosine bases in the three Egr-1 zinc-finger-DNA complexes. ...
The inducible transcription factor Egr-1 binds specifically to 9-bp target sequences containing two CpG sites that can potentially be methylated at four cytosine bases. Although complete CpG methylation appears to make an unfavorable steric clash in the previous crystal structures of the complexes with unmethylated or partially methylated DNA, our affinity data suggest that Egr-1 is insensitive to CpG methylation. We have determined, at a 1.4-Å resolution, the crystal structure of the Egr-1 zinc-finger complex with completely methylated target DNA. Structural comparison of the three different methylation states reveals why Egr-1 can recognize the target sequences regardless of CpG methylation.
Copyright © 2015. Published by Elsevier B.V.
... Few structures containing 5mC bases have been solved by X-ray crystallography or nuclear magnetic resonance spectroscopy. Several structural studies have demonstrated the importance of the 5mC methyl group for contacts with hydrophobic patches on the protein surface of MBDs [87,88] or with water molecules [89,90]. Understanding the role of hydrophobic contacts with the 5mC methyl group in the major groove for methylation state-specific binding ideally requires 3D structures of a protein bound to methylated and unmethylated copies of its DNA target. ...
Many anecdotal observations exist of a regulatory effect of DNA methylation on gene expression. However, in general, the underlying
mechanisms of this effect are poorly understood. In this review, we summarize what is currently known about how this important,
but mysterious, epigenetic mark impacts cellular functions. Cytosine methylation can abrogate or enhance interactions with
DNA-binding proteins, or it may have no effect, depending on the context. Despite being only a small chemical change, the
addition of a methyl group to cytosine can affect base readout via hydrophobic contacts in the major groove and shape readout
via electrostatic contacts in the minor groove. We discuss the recent discovery that CpG methylation increases DNase I cleavage
at adjacent positions by an order of magnitude through altering the local 3D DNA shape and the possible implications of this
structural insight for understanding the methylation sensitivity of transcription factors (TFs). Additionally, 5-methylcytosines
change the stability of nucleosomes and, thus, affect the local chromatin structure and access of TFs to genomic DNA. Given
these complexities, it seems unlikely that the influence of DNA methylation on protein–DNA binding can be captured in a small
set of general rules. Hence, data-driven approaches may be essential to gain a better understanding of these mechanisms.
