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![WT-GFP mice show minimal overt phenotype. (A) Schematic representation of MeCP2 with an EGFP tag. Missense mutations analysed in this study (T158M, R133C and R306C) are shown in relation to the MBD and NID. (B) Levels (mean ± SD) of Mecp2 transcripts in WT-GFP mouse brain (n = 9) compared with WT littermates (n = 9), expressed relative to Cyclophilin A transcript (CycA). (C) Representative western blot and quantification comparing MeCP2 protein abundance in WT-GFP mouse brain (double arrow-head, n = 6) versus WT littermates (single arrow-head, n = 6). Gamma tubulin (GT) served as an internal control. Mean ± SEM plotted. (D) The KaplanMeyer plots showing survival of WT-GFP mice (n = 8) compared with their WT littermates (n = 8) and Mecp2-null mice [Ref. (18)] (n = 24). (E) Growth curve showing average weight of WT-GFP mice (n = 8) compared with their WT littermates (n = 8). Using repeated measures ANOVA, the difference was consistent and significant](profile/Sabine-Lagger/publication/286445247/figure/fig8/AS:667909781192706@1536253509096/WT-GFP-mice-show-minimal-overt-phenotype-A-Schematic-representation-of-MeCP2-with-an.png)
WT-GFP mice show minimal overt phenotype. (A) Schematic representation of MeCP2 with an EGFP tag. Missense mutations analysed in this study (T158M, R133C and R306C) are shown in relation to the MBD and NID. (B) Levels (mean ± SD) of Mecp2 transcripts in WT-GFP mouse brain (n = 9) compared with WT littermates (n = 9), expressed relative to Cyclophilin A transcript (CycA). (C) Representative western blot and quantification comparing MeCP2 protein abundance in WT-GFP mouse brain (double arrow-head, n = 6) versus WT littermates (single arrow-head, n = 6). Gamma tubulin (GT) served as an internal control. Mean ± SEM plotted. (D) The KaplanMeyer plots showing survival of WT-GFP mice (n = 8) compared with their WT littermates (n = 8) and Mecp2-null mice [Ref. (18)] (n = 24). (E) Growth curve showing average weight of WT-GFP mice (n = 8) compared with their WT littermates (n = 8). Using repeated measures ANOVA, the difference was consistent and significant
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Rett syndrome is caused by mutations in the X-linked MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore
allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations,...
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... generated an allelic series in which endogenous Mecp2 or mutant Mecp2 genes were fused in frame with EGFP at their C-termini ( Fig. 1A; Supplementary Material, Fig. S1). Mice expres- sing knock-in WT MeCP2-EGFP fusion genes have been reported (13,17), but without extensive characterization. We initially looked for phenotypic defects due to the fusion of WT MeCP2 with EGFP by monitoring male mice at the molecular and whole organism levels. Analysis of hemizygous males has the advantage that phenotypic ...
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... tern of X chromosome inactivation, and so severity of individual mutations can be assessed in an unbiased fashion. Quantitative polymerase chain reaction (PCR) and western blots indicated that both mRNA and protein products of the Mecp2-GFP gene (WT-GFP) were expressed in brain, though at somewhat higher levels than the endogenous Mecp2 gene (WT) (Fig. 1B and C). Quantitative western blots suggested that the level of WT-GFP is ∼1.6-fold higher than in untagged WT littermates. At the whole organism level, we analysed cohorts of WT-GFP mice back-crossed for four generations to give a genetic background that is ∼94% C57BL/6J. WT-GFP knock-in mice were fertile and showed normal survival but tended ...
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... suggested that the level of WT-GFP is ∼1.6-fold higher than in untagged WT littermates. At the whole organism level, we analysed cohorts of WT-GFP mice back-crossed for four generations to give a genetic background that is ∼94% C57BL/6J. WT-GFP knock-in mice were fertile and showed normal survival but tended to be smaller than WT littermates ( Fig. 1D and E). Co- horts were monitored using a phenotypic scoring methodology that records breathing, tremor, gait, hindlimb clasping, mobility and general condition (18). This series of observational tests has the advantage that it is not affected by learning and can therefore be performed weekly over long periods, giving reprodu- cible results. ...
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... gait, hindlimb clasping, mobility and general condition (18). This series of observational tests has the advantage that it is not affected by learning and can therefore be performed weekly over long periods, giving reprodu- cible results. Using this method, WT-GFP mice showed no signifi- cant phenotypic deterioration compared with WT littermates (Fig. 1F), reinforcing the view that, despite the presence of the EGFP tag, they are essentially ...
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... search for neurological phenotypes in more detail, we sub- jected WT-GFP mice to a series of motor coordination and behav- ioural tests (Fig. 1G-I). Performance on the elevated plus maze was indistinguishable from WT and on the accelerating rotarod was also not significantly different from WT littermates. The hanging-wire test showed a weak but reproducible reduction in the ability to engage hindlimbs with the wire. We noted in add- ition that there was a trend towards a mild ...
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... these analyses, WT littermates lacked the GFP tag on en- dogenous MeCP2, but as shown in Figure 1, the presence of this tag has minimal phenotypic consequences. We confirmed that the presence of C-terminal GFP was not contributing to pheno- type by comparing the performance of mutant-GFP mice with age-matched WT-GFP mice in the same tests (Fig. 3B, D and F). ...
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... and T158M-GFP, however, Figure 3. Behavioural analysis of the Mecp2-GFP allelic series indicates that R133C-GFP mice are less severely affected than R306C-GFP and T158M-GFP. Mutant males and WT male littermates underwent behavioural analysis as in Figure 1. (A) Hanging-wire test, (C) elevated plus maze and (E) accelerating rotarod. ...
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... vectors to create EGFP-tagged alleles for WT Mecp2, Mecp2R133C, Mecp2T158M and Mecp2R306C were constructed using a 7.2-kb plasmid subclone of mouse 129/Ola genomic DNA (3), including Mecp2 exons 3 and 4 (Supplementary Material, Fig. S1). The coding sequence of EGFP was fused in-frame to the end of the coding sequence of Mecp2 in exon 4 followed by a loxP- flanked NeoStop cassette as a selectable marker, retaining the first 1.8 kb of the Mecp2 3′ UTR as the 3′ homology arm. Point muta- tions R133C, T158M and R306C were introduced into the WT Mecp2- EGFP targeting ...
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... muta- tions R133C, T158M and R306C were introduced into the WT Mecp2- EGFP targeting vector using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). Linearized constructs were electroporated into 129/Ola E14 TG2a mouse ES cells and cor- rectly targeted clones identified by PCR screening and Southern blotting (Supplementary Material, Fig. S1). Mice were generated from ES cells by standard procedures (3 ...
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Background
Rett syndrome is best known due to its severe and devastating symptoms in the central nervous system. It is produced by mutations affecting the Mecp2 gene that codes for a transcription factor. Nevertheless, evidence for MECP2 activity has been reported for tissues other than those of the central nervous system. Patients affected by Rett...
ntroduction. New pharmacological agents and behavioral interventions designed to ease key symptoms of individuals with Rett syndrome are currently emerging. To carefully evaluate these interventions in this complex and rare disorder, it is paramount to develop robust and objective measures which can be utilized and compared across clinics and resea...
Rett syndrome is a rare neuropsychiatric disorder with a wide symptomatology including impaired communication and movement, cardio-respiratory abnormalities, and seizures. The clinical presentation is typically associated to mutations in the gene coding for the methyl-CpG-binding protein 2 (MECP2), which is a transcription factor. The gene is ubiqu...
Rett syndrome (RTT) is an early-onset neurodevelopmental disorder that almost exclusively affects girls and is totally disabling. Three genes have been identified that cause RTT: MECP2, CDKL5 and FOXG1. However, the etiology of some of RTT patients still remains unknown. Recently, next generation sequencing (NGS) has promoted genetic diagnoses beca...
LIN28A aberrant expression contributes to the development of human malignancies. However, the LIN28A expression profile remains to be clarified. Herein, we report that LIN28A expression is directly associated with the methylation status of its two CpG island sites in pancreatic cancer cells. First, Bisulfite sequencing reveals that PANC1 cells poss...
Citations
... It has been well documented that both overexpression and depletion of Mecp2 have deleterious effects on neuronal homeostasis (Na et al., 2013), and thus, tight regulation of Mecp2 protein levels is critical for its physiological functions. In addition, several studies have demonstrated that many Rett syndrome-causing mutations in the methyl-CpG binding domain not only compromise DNA binding capacity of Mecp2 but also reduce its protein stability, implying the relevance of protein stability in Mecp2 dysfunction (Tillotson and Bird, 2020;Chen et al., 2017;Brown et al., 2016). Mecp2 has been shown to undergo various post-translational modifications, including phosphorylation, acetylation, ubiquitination, and sumoylation, which may also affect protein stability (Ausió et al., 2014). ...
Quiescence (G0) maintenance and exit are crucial for tissue homeostasis and regeneration in mammals. Here, we show that methyl-CpG binding protein 2 (Mecp2) expression is cell cycle-dependent and negatively regulates quiescence exit in cultured cells and in an injury-induced liver regeneration mouse model. Specifically, acute reduction of Mecp2 is required for efficient quiescence exit as deletion of Mecp2 accelerates, while overexpression of Mecp2 delays quiescence exit, and forced expression of Mecp2 after Mecp2 conditional knockout rescues cell cycle reentry. The E3 ligase Nedd4 mediates the ubiquitination and degradation of Mecp2, and thus facilitates quiescence exit. A genome-wide study uncovered the dual role of Mecp2 in preventing quiescence exit by transcriptionally activating metabolic genes while repressing proliferation-associated genes. Particularly disruption of two nuclear receptors, Rara or Nr1h3, accelerates quiescence exit, mimicking the Mecp2 depletion phenotype. Our studies unravel a previously unrecognized role for Mecp2 as an essential regulator of quiescence exit and tissue regeneration.
... It remains unclear how the disruption of a DNA methylation-binding protein that is present in every tissue and cell type produces the neurologic deficits observed in RTT. Mouse models carrying knockout [44,45] and knockin [30,35,[46][47][48][49][50][51] mutations of MeCP2 have been generated and recapitulate multiple features of RTT. Even though most of the patient population are heterozygous females, mechanistic studies have often been limited to hemizygous male mice, largely to avoid the confounding effects of random XCI. ...
X-linked genetic causes of intellectual disability (ID) account for a substantial proportion of cases and remain poorly understood, in part due to the heterogeneous expression of X-linked genes in females. This is because most genes on the X chromosome are subject to random X chromosome inactivation (XCI) during early embryonic development, which results in a mosaic pattern of gene expression for a given X-linked mutant allele. This mosaic expression produces substantial complexity, especially when attempting to study the already complicated neural circuits that underly behavior, thus impeding the understanding of disease-related pathophysiology and the development of therapeutics. Here, we review a few selected X-linked forms of ID that predominantly affect heterozygous females and the current obstacles for developing effective therapies for such disorders. We also propose a genetic strategy to overcome the complexity presented by mosaicism in heterozygous females and highlight specific tools for studying synaptic and circuit mechanisms, many of which could be shared across multiple forms of intellectual disability.
... A few such models include the MeCP2 knock out mouse model developed in Adrian Bird's lab [17] and a mouse strain lacking a substantial part of MBD developed in Rudolf Jaenisch's lab [18]. As well, a few knock in mice strains expressing MeCP2 consisting of individual Rett syndrome relevant mutations, such as T158M [19,20] and R306C [19,21], and a mouse model containing C-terminal MeCP2 truncation, R294X [22] are being used. ...
... A few such models include the MeCP2 knock out mouse model developed in Adrian Bird's lab [17] and a mouse strain lacking a substantial part of MBD developed in Rudolf Jaenisch's lab [18]. As well, a few knock in mice strains expressing MeCP2 consisting of individual Rett syndrome relevant mutations, such as T158M [19,20] and R306C [19,21], and a mouse model containing C-terminal MeCP2 truncation, R294X [22] are being used. ...
An immortalized neural cell line derived from the human ventral mesencephalon, called ReNCell, and its MeCP2 knock out were used. With it, we characterized the chromatin compositional transitions undergone during differentiation, with special emphasis on linker histones. While the WT cells displayed the development of dendrites and axons the KO cells did not, despite undergoing differentiation as monitored by NeuN. ReNCell expressed minimal amounts of histone H1.0 and their linker histone complement consisted mainly of histone H1.2, H1.4 and H1.5. The overall level of histone H1 exhibited a trend to increase during the differentiation of MeCP2 KO cells. The phosphorylation levels of histone H1 proteins decreased dramatically during ReNCell’s cell differentiation independently of the presence of MeCP2. Immunofluorescence analysis showed that MeCP2 exhibits an extensive co-localization with linker histones. Interestingly, the average size of the nucleus decreased during differentiation but in the MeCP2 KO cells, the smaller size of the nuclei at the start of differentiation increased by almost 40% after differentiation by 8 days (8 DIV). In summary, our data provide a compelling perspective on the dynamic changes of H1 histones during neural differentiation, coupled with the intricate interplay between H1 variants and MeCP2.
Abbreviations: ACN, acetonitrile; A230, absorbance at 230 nm; bFGF, basic fibroblast growth factor; CM, chicken erythrocyte histone marker; CNS, central nervous system; CRISPR, clustered regulated interspaced short palindromic repeatsDAPI, 4,’6-diaminidino-2-phenylindole; DIV, days in vitro (days after differentiation is induced); DMEM, Dulbecco’s modified Eagle medium; EGF, epidermal growth factor; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic proteinHPLC, high-performance liquid chromatography; IF, immunofluorescence; iPSCs, induced pluripotent stem cells; MAP2, microtubule-associated protein 2; MBD, methyl-binding domain; MeCP2, methyl-CpG binding protein 2; MS, mass spectrometry; NCP, nucleosome core particle; NeuN, neuron nuclear antigen; NPC, neural progenitor cellPAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PFA, paraformaldehyde; PTM, posttranslational modification; RP-HPLC, reversed phase HPLC; ReNCells, ReNCells VM; RPLP0, ribosomal protein lateral stalk subunit P0; RT-qPCR, reverse transcription quantitative polymerase-chain reaction; RTT, Rett Syndrome; SDS, sodium dodecyl sulphate; TAD, topologically associating domain; Triple KO, triple knockout.
... Many severe RTT-causing missense mutations in MECP2, such as R111G and R106W, cluster in the methyl-CpG binding domain (MBD) (Nan et al. 1993) and abolish MeCP2 binding to methylated cytosines (Goffin et al. 2012;Heckman et al. 2014;Brown et al. 2016;Johnson et al. 2017). Mouse models carrying these severe mutations have highlighted the importance of MeCP2 levels and its DNA binding for normal brain function (Guy et al. 2001;Goffin et al. 2012;Heckman et al. 2014;Brown et al. 2016;Johnson et al. 2017). ...
... Many severe RTT-causing missense mutations in MECP2, such as R111G and R106W, cluster in the methyl-CpG binding domain (MBD) (Nan et al. 1993) and abolish MeCP2 binding to methylated cytosines (Goffin et al. 2012;Heckman et al. 2014;Brown et al. 2016;Johnson et al. 2017). Mouse models carrying these severe mutations have highlighted the importance of MeCP2 levels and its DNA binding for normal brain function (Guy et al. 2001;Goffin et al. 2012;Heckman et al. 2014;Brown et al. 2016;Johnson et al. 2017). Importantly, the majority (∼75%) of RTT cases are not caused by severe mutations that totally abolish MeCP2 function but rather by mutations that either reduce MeCP2 levels or DNA binding, leading to retention of some partial function of the protein. ...
... Two-way repeated measures ANOVA revealed significant main effects of population spike amplitudes between the two groups on day 0 (genotype: F 1,22 = 12.39, P = 0.002; time: F 11,242 = 9.51, P < 0.001; genotype × time interaction: F 11,242 = 6.72, P < 0.001), day 1 (genotype: They also had a reduced life span, with a median survival of ∼6 mo (Fig. 4D), which is quite longer than the Mecp2 null , Mecp2 T158M , and Mecp2 R111G mice but similar to the Mecp2 R306C mice and shorter than the Mecp2 R133C mice (Guy et al. 2007;Heckman et al. 2014;Brown et al. 2016). Furthermore, since we observed hippocampus-dependent learning and memory deficits in these mice, we examined in vivo long-term potentiation (LTP) to measure hippocampal synaptic plasticity, which serves as a neural substrate of learning and memory (Moser et al. 1998;Malenka and Bear 2004;Whitlock et al. 2006). ...
Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2 , in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.
... MECP2 protein instability-led MECP2 deficiency has been previously proposed to be an underlying cause of RTT . Several mutations reported to affect the functional domains of MECP2, including T158A, T158M, R133C, R168 * and R255 * , were also found to destabilize the MECP2 protein (Brown et al., 2016;Guy et al., 2018). This raises an interesting question of whether the pathological mechanisms behind these recurring mutations are caused by the conventional functional domains (MBD and NID) alone. ...
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.
... The 'core' MBD family proteins share a conserved domain of 70-80 residues and include the proteins MBD1-4 and methyl-CpG-binding protein 2 (MeCP2) (12). The latter represents a largely disordered DNA-binding protein for which loss-of-function mutations are associated with the neurological developmental disease Rett syndrome (RTT) (14,15). Its high-affinity interaction with mC/mC DNA hinges on two Arg fingers that both form two H-bonds to the guanosine of the CpG ( Figure 1C) (16). ...
... T 1 measurements employed eight recovery delays between 50 and 1100 ms. 15 N T 2 measurements were carried out using a CPMG pulse sequence (24) with relaxation delays of 5, 20, 35, 50, 70 and 90 ms. Steady-state [ 15 N, 1 H] heteronuclear-NOE measurements were carried out with and without proton saturation during the relaxation delay, using either 5 s of relaxation delay and 3 s of proton saturation or 8 s of relaxation delay only, respectively. ...
... For TAN, we combined the following residues for global exchange parameters: G103, T105, T109, Q110, R111, K112, S113, G114, S116, A117, G118, Y120, D121, Y123, I125, G129, K130, A131, F132, R133, N134, E137, L138, A140, Y141, F142, V145, G146, T148, S149 and N153. 15 N CEST experiments were implemented as described previously (28) at temperatures of 291, 283 and 278 K, using two sets of experiments at nominal 15 N B 1 fields of 15 Hz and 30 Hz and scanning 64 points across the 15 N spectral width from 134 to 99 ppm. CEST profiles were fit with the software Dynamics Center (version 2.7.2) to extract the exchange parameters. ...
5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel 'CpG duplex marks' could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.
... We also prepared two tMeCP2 variants with substitutions that alter function. The first is T158 M tMeCP2, with a methyl-CpGbinding domain (MBD) mutation that reduces specific DNA binding 43,44 and is seen commonly in RTT patients. 23 The second is P302L tMeCP2, which has a diminished ability to engage the TBLR1 subunit of the NCoR/SMRT repressor complex. ...
... At 1 μM, the fraction of ZF-tMeCP2 diffuses slowly (F slow = 26.3%) is higher than that of ZF-tMeCP2(T158M) (16.3%) in accord with relative methylated DNA affinities determined in vitro ( Figure 1B) and in cells. 44 Thus, the higher nuclear concentration of ZF-tMeCP2(T158M) is counterbalanced by the low DNA binding population; the result is no significant transcriptional repression ( Figure S12). At 2 μM, ZF-tMeCP2, Tat-tMeCP2, as well as ZF-tMeCP2(T158M) all reached the nucleus at significantly higher levels than tMeCP2 ( Figure 2D) and show higher levels of DNA binding, with values of F slow of 24.9%, 33.7%, and 25.6%, respectively (Table S3); the result is observable transcriptional repression. ...
The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-tMeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13-71, 313-484), binds DNA in a methylation-dependent manner in vitro, and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-tMeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.
... 12 The latter represents a largely disordered DNA-binding protein for which loss-of-function mutations are associated with the neurological developmental disease Rett syndrome (RTT). [15][16] Its high-affinity interaction with mC/mC DNA hinges on two Arg fingers that both form two Hbonds to the guanosine of the CpG (Fig. 1C). 14 Previous work on MBDs has shown that the stability of the three-dimensional fold is exceptionally susceptible to simple point mutations in the center of the hydrophobic core. ...
5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel "CpG duplex marks" could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.
... Whole-Body Plethysmography of the Mecp2 311G > A Mice. Mice were back-crossed to C57BL/6J mice for at least seven generations prior to behavioral testing, consistent with previous studies (53,54). Mice (8-wk old; 4-wk postinjection) were acclimated to the behavioral room for a minimum of 30 min in their home cage. ...
Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editase wt ) and either Mecp2 -targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2 -targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2 -targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editase wt . Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.
... Isoform-specific mutant mice have also been generated for E1 (Yasui et al., 2014) and E2 (Itoh et al., 2012). A number of Mecp2 knock-in mutant mouse models have been produced to represent Rett Syndrome patients more accurately by introducing mutations commonly occurring in patients such as R168X (Schaevitz et al., 2013), T308A, R306C (Ebert et al., 2013;Lyst et al., 2013a), T158A (Goffin et al., 2011), and T158M (Brown et al., 2016). ...
Eukaryotic gene expression is controlled at multiple steps that work in harmony to ensure proper maintenance of cellular morphology and function. Such regulatory mechanisms would include transcriptional gene regulation, which is in turn controlled by chromatin remodeling, distinct topologically associating domains of the chromatin structure, cis-regulatory elements such as enhancers and promoters, action of trans-acting factors, DNA methylation, RNA modifications, and post-translational modification of histones. These guiding mechanisms of gene expression play critical roles in the epigenetic setting of individual cells within the eukaryotic systems. Some epigenetic factors may play multiple functional roles in guarding the accurate gene expression program of the eukaryotic cells, especially within the central nervous system. A well-studied example of such multi-functional factors is the methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that is encoded by the X-linked MECP2 gene. Here, we aim to provide an overview of eukaryotic gene regulation, the three-dimensional chromatin organization, standard techniques to study newly synthesized transcripts, and the role of MeCP2 as an important transcriptional regulator in eukaryotes.
