Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

Possible role of each repeat structure of the microtubule-binding domain of the tau protein in in vitro aggregation

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.     

Linkage-dependent contribution of repeat peptides to self-aggregation of three- or four-repeat microtubule-binding domains in tau protein

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism for the tau protein, it is still unclear how it is transformed from a normal structure in a neuron. To examine the linkage-dependent contribution of each repeat peptide (R1-R4) to filament formation of the three- or four-repeat microtubule-binding domain (MBD) in the tau protein, four two-repeat peptides (R12, R13, R23 and R34) and two three-repeat peptides (R123 and R234) were prepared, and their in vitro self-aggregation was investigated by thioflavin S fluorescence and circular dichroism measurements, and by electron microscopy in neutral buffer (pH 7.6). Comparison of these aggregation behaviors with previous results for single-repeat peptides and wild-type 3RMBD (R134) and 4RMBD (R1234) indicated that (a) the two-repeat R23, not the R2 or R3 single repeat, forms the core structure in self-aggregation of 4RMBD, whereas that of 3RMBD comprises the R3 single repeat, (b) co-existence of R1 and R4 repeats is necessary for the aggregation behavior inherent in 3RMBD and 4RMBD, whereas the R1 or R4 repeat alone functions as a repressor or modifier of the filament formation, (c) 4RMBD aggregation is accompanied by R1-driven transition from random and alpha-helix structures to a beta-sheet structure, whereas 3RMBD aggregation involves three-repeat R134-specific transition from a random structure to an alpha-helix structure without the participation of a beta-sheet structure, and (d) the peptides that include the R1 repeat form a long filament irrespective of the absence or presence of the R4 repeat, whereas those that include the R4 repeat, but not the R1 repeat, form a relatively short filament. To the best of our knowledge, a systematic study of the linkage-dependent contribution of each repeat peptide to the paired helical filament formation of tau MBD has not been carried out previously, and thus the present information is useful for understanding the essence of the filament formation of tau MBD.     

Sites of tau important for aggregation populate {beta}-structure and bind to microtubules and polyanions

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual beta-structure for 8-10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-beta-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the beta-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.     

C-H ... π interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau

Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer's disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of β-sheet structures from a short hexapeptide (306)VQIVYK(311) in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C-H … π interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.     

Importance of local structures of second and third repeat fragments of microtubule-binding domain for tau filament formation

To investigate the importance of the seventh residue of the second and third repeat fragments (R2 and R3 peptides) of the microtubule-binding domain (MBD) for tau filamentous assembly, the residues Lys and Pro were substituted (R2-K7P and R3-P7K). The filament formations of the R2 and R3 peptides were almost lost due to their substitutions despite their overall conformational similarities. The NOE analyses showed the importance of the conformational flexibility for the R2 peptide and the coupled extended and helical conformations for the R3 peptide in their limited N-terminal regions around their seventh residues. The result shows that the filament formation of MBD is initiated from a short fragment region containing the minimal conformational or functional motif.     

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of beta-structures or alpha-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris-HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the alpha-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10-30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an alpha-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of < 10% or > 40%, the intermediate conformation between the random and alpha-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native alpha-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.     

Marked difference between self-aggregations of first and fourth repeat peptides on tau microtubule-binding domain in acidic solution

The heparin-induced self-aggregation behaviours of four repeat peptides (R1-R4) in an acidic solution (pH = 4.5) were investigated by fluorescence and circular dichroism (CD) measurements and compared with those in a neutral solution (pH = 7.5). In contrast with the self-aggregation-resistive behaviours of the R1 and R4 repeat peptides in the neutral solution, the R4 peptide formed a filament similarly to the R2 and R3 peptides in the acidic solution, whereas the R1 peptide still showed resistive behaviour for filament formation. This is the first report on the markedly different self-aggregation behaviours of the first and fourth repeat peptides on tau microtubule-binding domain.     

Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods

The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent years, several factors regulating tau assembly have come to light, yet some important questions remain to be answered. In this work, the His-tagged gene constructs of the four-repeat microtubule binding domain (4RMBD) in tau protein and its three mutants, 4RMBD S305N, N279K, and P301L, were expressed in E. coli and purified. Gel filtration chromatography and dynamic light scattering measurement yielded a Stokes radius of 3.1 nm, indicating that the His-tagged 4RMBD normally exists in buffer solution in a dimer state, which is formed by non-covalent intermolecular interactions. This non-covalent dimer can further polymerize to form filaments in the presence of polyanions such as heparin. The kinetics of the in vitro aggregation was monitored by thioflavine S dye fluorescence and CD measurements. The aggregation of 4RMBD was suggested to be a nucleation-dependent process, where the non-covalent dimer acts as an effective structural unit. The aggregation rate was strongly affected by the point mutation. Among the 4RMBD mutants, the rate of S305N was exceptionally fast, whereas N279K was the slowest, even slower than the wild-type. The aggregations were optimal in a weakly reducing environment for all the mutants and the wild type. However, the aggregations were affected differently by buffer pH, depending on the 4RMBD mutation.     

Structural evaluation of conformational transition state responsible for self-assembly of tau microtubule-binding domain

In the brains of Alzheimer's disease patients, the tau protein abnormally aggregates to form an insoluble paired helical filament (PHF). Since the third repeat structure (R3) of the tau microtubule-binding domain plays an essential role in PHF formation and self-aggregates most significantly in an aqueous solution of 20-40% trifluoroethanol (TFE), its possible conformation was estimated from the combination of (i) the TFE-dependent deviations of NH and CalphaH proton chemical shifts from those of the random structure in water and (ii) the TFE-dependent NOE effect connectivity diagrams between the neighboring protons. Consequently, it was indicated that the extended structure of the N-terminal VQIVYK moiety and the alpha-helical-like structure of the LSKVTSKC region provide a structural scaffold for initiating the self-assembled filament formation of the R3 structure. To the best of our knowledge, this is the first study that demonstrated the initial structural moiety and its structural feature necessary for starting the tau PHF formation.     

Post-translational modifications within tau paired helical filament nucleating motifs perturb microtubule interactions and oligomer formation

Post-translationally modified tau is the primary component of tau neurofibrillary tangles, a pathological hallmark of Alzheimer''s disease and other tauopathies. Post-translational modifications (PTMs) within the tau microtubule (MT)-binding domain (MBD), which encompasses two hexapeptide motifs that act as critical nucleating regions for tau aggregation, can potentially modulate tau aggregation as well as interactions with MTs and membranes. Here, we characterize the effects of a recently discovered tau PTM, lysine succinylation, on tau–tubulin interactions and compare these to the effects of two previously reported MBD modifications, lysine acetylation and tyrosine phosphorylation. As generation of site-specific PTMs in proteins is challenging, we used short synthetic peptides to quantify the effects on tubulin binding of three site-specific PTMs located within the PHF6^∗ (paired helical filament [PHF] residues 275–280) and PHF6 (residues 306–311) hexapeptide motifs: K280 acetylation, Y310 phosphorylation, and K311 succinylation. We compared these effects to those observed for MBD PTM-mimetic point mutations K280Q, Y310E, and K311E. Finally, we evaluated the effects of these PTM-mimetic mutations on MBD membrane binding and membrane-induced fibril and oligomer formation. We found that all three PTMs perturb tau MT binding, with Y310 phosphorylation exerting the strongest effect. PTM-mimetic mutations partially recapitulated the effects of the PTMs on MT binding and also disrupted tau membrane binding and membrane-induced oligomer and fibril formation. These results imply that these PTMs, including the novel and Alzheimer''s disease–specific succinylation of tau K311, may influence both the physiological and pathological interactions of tau and thus represent targets for therapeutic intervention.     

Variations in filament conformation dictate seeding barrier between three- and four-repeat tau

Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimer's disease. Six tau isoforms exist that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. Recent evidence suggests that tau filaments can transfer between cells and spread through the brain. Here we demonstrate in vitro that seeded filament growth, a prerequisite for tau spreading, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms. Seeds of 4R tau recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau filament. These findings provide fundamental mechanistic insights into the seeding, propagation, and diversification of tau filaments.     

Tau Binds to Lipid Membrane Surfaces via Short Amphipathic Helices Located in Its Microtubule-Binding Repeats

Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer’s disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau’s poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.     

Tau Binds to Lipid Membrane Surfaces via Short Amphipathic Helices Located in Its Microtubule-Binding Repeats

Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer’s disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau’s poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.     

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule‐binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule‐binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule‐binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N‐terminus of repeat 2 has a lower propensity to form strand‐like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle‐bound state. Interestingly, the behavior of the Module‐B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module‐B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand‐like structure and the resistance of these propensities to changes in tau's environment.     

小鼠围着床期子宫内膜甲基化CpG结合域蛋白2的表达规律

为研究甲基化CpG结合域蛋白2（methyl-CpG binding domain protein 2,MBD2）在围植入期小鼠子宫内膜的表达规律,通过采用实时荧光定量PCR（Real-time fluorescence quantitative PCR,qPCR）、Western blot和免疫组化技术检测未孕小鼠（d0）和不同孕天小鼠子宫MBD2的表达情况。qPCR结果显示,d0至d7的小鼠子宫内膜组织均有MBD2 mRNA表达,在d5至d7高表达。MBD2蛋白在子宫内膜的表达规律与qPCR结果相符。MBD2蛋白在孕d1到d4中度表达于腔上皮、腺上皮和基质细胞,在d5至d7基质细胞表达增强,主要表达于蜕膜区。假孕小鼠子宫内膜中,MBD2在腔上皮、腺上皮和基质细胞中中度表达,d5至d7基质细胞表达明显减弱。动物模型中,宫角注射MBD2基因反义寡聚脱氧核苷酸,可抑制MBD2的表达,降低人工诱导蜕膜化反应和蜕膜化标志物PRL的表达。MBD2在早孕小鼠子宫内膜的表达模式提示其可能参与了蜕膜化过程。     

Synergistic interactions between repeats in tau protein and Aβ amyloids may be responsible for accelerated aggregation via polymorphic states

Amyloid plaques and neurofibrillary tangles simultaneously accumulate in Alzheimer's disease (AD). It is known that Aβ and tau exist together in the mitochondria; however, the interactions between Aβ oligomers and tau are controversial. Moreover, it is still unclear which specific domains in the tau protein can interact with Aβ oligomers and what could be the effect of these interactions. Herein, we examine three different Aβ-tau oligomeric complexes. These complexes present interactions of Aβ with three domains in the tau protein; all contain high β-structure propensity in their R2, R3, and R4 repeats. Our results show that, among these, Aβ oligomers are likely to interact with the R2 domain to form a stable complex with better alignment in the turn region and the β-structure domain. We therefore propose that the R2 domain can interact with soluble Aβ oligomers and consequently promote aggregation. EM and AFM images and dimensions revealed highly polymorphic tau aggregates. We suggest that the polymorphic tau and Aβ-tau aggregates may be largely due to repeat sequences which are prone to variable turn locations along the tau repeats.     

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation-associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full-length four-repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild-type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild-type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.     

Probing the Dynamic Distribution of Bound States for Methylcytosine-binding Domains on DNA

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.     

Functional conservation of MBD proteins: MeCP2 and Drosophila MBD proteins alter sleep

Proteins containing a methyl‐CpG‐binding domain (MBD) bind 5mC and convert the methylation pattern information into appropriate functional cellular states. The correct readout of epigenetic marks is of particular importance in the nervous system where abnormal expression or compromised MBD protein function, can lead to disease and developmental disorders. Recent evidence indicates that the genome of Drosophila melanogaster is methylated and two MBD proteins, dMBD2/3 and dMBD‐R2, are present. Are Drosophila MBD proteins required for neuronal function, and as MBD‐containing proteins have diverged and evolved, does the MBD domain retain the molecular properties required for conserved cellular function across species? To address these questions, we expressed the human MBD‐containing protein, hMeCP2, in distinct amine neurons and quantified functional changes in sleep circuitry output using a high throughput assay in Drosophila. hMeCP2 expression resulted in phase‐specific sleep loss and sleep fragmentation with the hMeCP2‐mediated sleep deficits requiring an intact MBD domain. Reducing endogenous dMBD2/3 and dMBD‐R2 levels also generated sleep fragmentation, with an increase in sleep occurring upon dMBD‐R2 reduction. To examine if hMeCP2 and dMBD‐R2 are targeting common neuronal functions, we reduced dMBD‐R2 levels in combination with hMeCP2 expression and observed a complete rescue of sleep deficits. Furthermore, chromosomal binding experiments indicate MBD‐R2 and MeCP2 associate on shared genomic loci. Our results provide the first demonstration that Drosophila MBD‐containing family members are required for neuronal function and suggest that the MBD domain retains considerable functional conservation at the whole organism level across species.