MeCP2在Rett综合征中的调控机制

Rett综合征(Rett syndrome, RTT)是一种X连锁的神经发育障碍性遗传病, 是导致女性严重智力障碍的主要原因之一。编码甲基化CpG结合蛋白2(Methyl-CpG-binding protein 2, MeCP2)基因突变是RTT主要的遗传病理学改变, MeCP2作为转录抑制因子调控基因表达。在RTT发病机制中, 由于缺乏MeCP2与甲基化DNA的正确结合, 阻碍了它对下游靶基因表达的正常调控, 最终导致脑功能障碍。目前, 对MeCP2在脑发育过程中的作用以及如何导致RTT的发生, 其机制尚不清楚。文章从MECP2基因和MeCP2蛋白两个方面, 对基因结构、蛋白质功能以及在分子水平上的调控机制进行了综述, 以期为RTT的发病机制研究提供新思路。     

MeCP2与神经发育性疾病

作为一种转录抑制因子,甲基化CpG结合蛋白2（MeCP2）含有结合甲基化DNA和转录抑制两个特征性的结构域,具有调节转录激活、调节染色体构象、参与RNA剪切等多种功能,在神经发育过程中起着重要的作用。近来的研究表明,MeCP2基因突变与Rett综合征、孤独症等多种神经发育性疾病相关,已成为研究基因型与人类神经发育性疾病关系的一个热点。就MeCP2在Rett综合征、孤独症及药物成瘾方面的进展作一综述。     

雷特综合征与多巴胺系统功能障碍

雷特综合征(Rett syndrome)属于神经发育障碍类疾病,主要由X性染色体上mecp2基因突变所致,患者多数为女孩。临床症状于出生后6~18个月逐渐显现,主要表现为头部发育缓慢,已获得的语言及手部目的性运动技能消退,智力障碍,呼吸功能障碍及自闭倾向等。多巴胺系统的功能包括运动调节、奖赏学习、情感、内分泌调控以及药物成瘾等多个方面。由于多巴胺系统在运动和精神方面与雷特综合征部分临床症状存在表面相关性,早期有学者根据临床特征提出雷特综合征患者可能存在多巴胺系统功能障碍,但两者之间是否具有实质性的内在联系以及mecp2基因是否会通过影响多巴胺系统导致相关临床症状是目前雷特综合征研究的一个热点。本文将针对雷特综合征与多巴胺系统功能障碍的相关研究进展作一综述。     

Profiling beneficial and potential adverse effects of MeCP2 overexpression in a hypomorphic Rett syndrome mouse model

De novo loss-of-function mutations in methyl-CpG-binding protein 2 (MeCP2) lead to the neurodevelopmental disorder Rett syndrome (RTT). Despite promising results from strategies aimed at increasing MeCP2 levels, additional studies exploring how hypomorphic MeCP2 mutations impact the therapeutic window are needed. Here, we investigated the consequences of genetically introducing a wild-type MECP2 transgene in the Mecp2 R133C mouse model of RTT. The MECP2 transgene reversed the majority of RTT-like phenotypes exhibited by male and female Mecp2 R133C mice. However, three core symptom domains were adversely affected in female Mecp2^R133C/+ animals; these phenotypes resemble those observed in disease contexts of excess MeCP2. Parallel control experiments in Mecp2^Null/+ mice linked these adverse effects to the hypomorphic R133C mutation. Collectively, these data provide evidence regarding the safety and efficacy of genetically overexpressing functional MeCP2 in Mecp2 R133C mice and suggest that personalized approaches may warrant consideration for the clinical assessment of MeCP2-targeted therapies.     

Binding of the Rett syndrome protein, MeCP2, to methylated and unmethylated DNA and chromatin

Methylated CpG Binding Protein 2 (MeCP2) is a nuclear protein named for its ability to selectively recognize methylated DNA. Much attention has been focused on understanding MeCP2 structure and function in the context of its role in Rett syndrome, a severe neurodevelopmental disorder that afflicts one in 10,000-15,000 girls. Early studies suggested a connection between DNA methylation, MeCP2, and establishment of a repressive chromatin structure at specific gene promoters. However, it is now recognized that MeCP2 can both activate and repress specific genes depending on the context. Likewise, in the cell, MeCP2 is bound to unmethylated DNA and chromatin in addition to methylated DNA. Thus, to understand the molecular basis of MeCP2 functionality, it is necessary to unravel the complex interrelationships between MeCP2 binding to unmethylated and methylated regions of the genome. MeCP2 is unusual and interesting in that it is an intrinsically disordered protein, that is, much of its primary sequence fails to fold into secondary structure and yet is functional. The unique structure of MeCP2 is the subject of the first section of this article. We then discuss recent investigations of the in vitro binding of MeCP2 to unmethylated and methylated DNA, and the potential ramifications of this work for in vivo function. We close by focusing on mechanistic studies indicating that the binding of MeCP2 to chromatin results in compaction into local (secondary) and global (tertiary) higher order structures. MeCP2 also competes with histone H1 for nucleosomal binding sites. The recent finding that MeCP2 is found at near stoichiometric levels with nucleosomes in neuronal cells underscores the multiple modes of engagement of MeCP2 with the genome, which include the cooperative tracking of methylation density.     

MeCP2-chromatin interactions include the formation of chromatosome-like structures and are altered in mutations causing Rett syndrome

hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects approximately 11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the approximately 146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.     

Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3

Mutations in the methyl-CpG binding protein 2 (MECP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder characterized by the loss of language and motor skills during early childhood. We generated mice with a truncating mutation similar to those found in RTT patients. These mice appeared normal and exhibited normal motor function for about 6 weeks, but then developed a progressive neurological disease that includes many features of RTT: tremors, motor impairments, hypoactivity, increased anxiety-related behavior, seizures, kyphosis, and stereotypic forelimb motions. Additionally, we show that although the truncated MeCP2 protein in these mice localizes normally to heterochromatic domains in vivo, histone H3 is hyperacetylated, providing evidence that the chromatin architecture is abnormal and that gene expression may be misregulated in this model of Rett syndrome.     

Rett综合征相关基因MeCP2敲除大鼠模型的构建及分析

MeCP2(Methyl CpG binding protein 2)基因突变可导致Rett综合征(Rett syndrome, RTT)。目前已报道的MeCP2敲除小鼠表型与RTT病人症状存在显著差异。为探索MeCP2在脑发育中的作用及其导致RTT的机制,本研究利用CRISPR/Cas9技术构建了MeCP2基因敲除大鼠模型。通过构建靶向敲除MeCP2基因的载体,体外将Cas9 mRNA和sgRNA显微注射到SD大鼠受精卵中,在MeCP2基因exon2中造成移码突变,从而获得MeCP2基因敲除大鼠。利用测序和Western blotting方法鉴定MeCP2敲除大鼠,并对其表型和行为学特征进行分析,发现MeCP2敲除大鼠体重降低,存在焦虑倾向和认知缺陷。本研究成功构建了MeCP2基因敲除大鼠模型,其表型类似人类RTT患者的症状,为后续MeCP2功能研究提供了更好的动物模型。     

Methyl CpG-binding protein isoform MeCP2_e2 is dispensable for Rett syndrome phenotypes but essential for embryo viability and placenta development

Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.     

Graded and pan-neural disease phenotypes of Rett Syndrome linked with dosage of functional MeCP2

Rett syndrome (RTT) is a progressive neurodevelop-mental disorder,mainly caused by mutations in MeCP2 and currently with no cure.We report here that neurons from R106W MeCP2 RTT human iPSCs as well as human embryonic stem cells after MeCP2 knockdown exhibit consistent and long-lasting impairment in maturation as indicated by impaired action potentials and passive membrane properties as well as reduced soma size and spine density.Moreover,RTT-inherent defects in neu-ronal maturation could be pan-neuronal and occurred in neurons with both dorsal and ventral forebrain features.Knockdown of MeCP2 led to more severe neuronal deficits as compared to RTT iPSC-derived neurons,which appeared to retain partial function.Strikingly,consistent deficits in nuclear size,dendritic complexity and circuitry-dependent spontaneous postsynaptic currents could only be observed in MeCP2 knockdown neurons but not RTT iPSC-derived neurons.Both neu-ron-intrinsic and circuitry-dependent deficits of MeCP2-deficient neurons could be fully or partially rescued by re-expression of wild type or T158M MeCP2,strengthening the dosage dependency of MeCP2 on disease phenotypes and also the partial function of the mutant.Our findings thus reveal stable neuronal matu-ration deficits and unexpectedly,graded sensitivities of neuron-inherent and neural transmission phenotypes towards the extent of MeCP2 deficiency,which is infor-mative for future therapeutic development.     

Targeted manipulation of heterochromatin rescues MeCP2 Rett mutants and re-establishes higher order chromatin organization

Heterochromatic regions represent a significant portion of the mammalian genome and have been implied in several important cellular processes, including cell division and genomic stability. However, its composition and dynamics remain largely unknown. To better understand how heterochromatin functions and how it is organized within the context of the cell nucleus, we have developed molecular tools allowing the targeting of virtually any nuclear factor specifically to heterochromatic regions and, thereby, the manipulation, also in a temporally controlled manner, of its composition. To validate our approach, we have ectopically targeted MeCP2 chromatin binding deficient Rett mutants to constitutive heterochromatic regions and analyze its functional consequences. We could show that, once bound to their endogenous target regions, their ability to re-organize higher order chromatin structure is restored. Furthermore, a temporally controlled targeting strategy allowed us to monitor MeCP2-mediated chromatin rearrangements in vivo and to visualize large-scale chromatin movements over several micrometers, as well as heterochromatic foci fusion events. This novel strategy enables specific tethering of any protein to heterochromatin and lays the ground for controlled manipulation of its composition and organization.