Function Of Methyl-CpG Binding Protein 2 Research Articles

A Role for MeCP2 in Switching Gene Activity via Chromatin Unfolding and HP1γ Displacement

Methyl-CpG-binding protein 2 (MeCP2) is generally considered to act as a transcriptional repressor, whereas recent studies suggest that MeCP2 is also involved in transcription activation. To gain insight into this dual function of MeCP2, we assessed the impact of MeCP2 on higher-order chromatin structure in living cells using mammalian cell systems harbouring a lactose operator and reporter gene-containing chromosomal domain to assess the effect of lactose repressor-tagged MeCP2 (and separate MeCP2 domains) binding in living cells. Our data reveal that targeted binding of MeCP2 elicits extensive chromatin unfolding. MeCP2-induced chromatin unfolding is triggered independently of the methyl-cytosine-binding domain. Interestingly, MeCP2 binding triggers the loss of HP1γ at the chromosomal domain and an increased HP1γ mobility, which is not observed for HP1α and HP1β. Surprisingly, MeCP2-induced chromatin unfolding is not associated with transcriptional activation. Our study suggests a novel role for MeCP2 in reorganizing chromatin to facilitate a switch in gene activity.

Mechanisms and therapeutic challenges in autism spectrum disorders

A major challenge for understanding neurodevelopmental disorders, including autism spectrum disorders (ASDs), is to advance the findings from gene discovery to an exposition of neurobiological mechanisms that underlie these disorders and subsequently translate this knowledge into mechanism-based therapeutics. A promising way to proceed is revealed by the recent studies of rare subsets of ASDs. In this review, we summarize the latest advances in the mechanisms and emerging therapeutics for a rare single-gene ASD, Rett syndrome. Rett syndrome is caused by mutations in the gene coding for methyl CpG-binding protein 2 (MeCP2). Although MeCP2 has diverse functions, examination of MeCP2 mutant mice suggests the hypothesis that MeCP2 deficiency leads to aberrant maturation and maintenance of synapses and circuits in multiple brain systems. Some of the deficits arise from alterations in specific intracellular pathways such as the PI3K/Akt signaling pathway. These abnormalities can be at least partially rescued in MeCP2 mutant mice by treatment with therapeutic agents. Mechanism-based therapeutics are emerging for single-gene neurodevelopmental disorders such as Rett syndrome. Given the complexity of MeCP2 function, future directions include combination therapeutics that target multiple molecules and pathways. Such approaches will likely be applicable to other ASDs as well.

A Critical and Cell-Autonomous Role for MeCP2 in Synaptic Scaling Up

Rett syndrome (Rett) is the leading genetic cause of mental retardation in females. Most cases of Rett are caused by loss-of-function mutations in the gene coding for the transcriptional regulator methyl-CpG binding protein 2 (MeCP2), but despite much effort, it remains unclear how a loss of MeCP2 function generates the neurological deficits of Rett. Here we show that MeCP2 plays an essential and cell-autonomous role in homeostatic synaptic scaling up in response to reduced firing or reduced sensory drive in rat visual cortical pyramidal neurons. We found that acute RNAi knockdown of MeCP2 blocked synaptic scaling within targeted neocortical pyramidal neurons. Furthermore, MeCP2 knockdown decreased excitatory synapse number without affecting basal mEPSC amplitude or AMPAR accumulation at spared synapses, demonstrating that MeCP2 acts cell-autonomously to maintain both excitatory synapse number and synaptic scaling in individual neocortical neurons. Finally, we used a mouse model of Rett to show that MeCP2 loss prevents homeostatic synaptic scaling up in response to visual deprivation in vivo, demonstrating for the first time that MeCP2 loss disrupts homeostatic plasticity within the intact developing neocortex. Our results establish MeCP2 as a critical mediator of synaptic scaling and raise the possibility that some of the neurological defects of Rett arise from a disruption of homeostatic plasticity.

MeCP2 Phosphorylation Is Required for Modulating Synaptic Scaling through mGluR5

MeCP2 (methyl CpG binding protein 2) is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. The functional significance of MeCP2 to the mammalian nervous system is highlighted by the discovery that mutations in the MECP2 gene cause Rett syndrome (RTT), a devastating neurological disease that shares many features with autism. Synaptic scaling is a form of non-Hebbian homeostatic plasticity that allows neurons to regulate overall excitability in response to changes in network neuronal activity levels. While it is known that neuronal activity can induce phosphorylation of MeCP2 and that MeCP2 can regulate synaptic scaling, the molecular link between MeCP2 phosphorylation and synaptic scaling remains undefined. We show here that MeCP2 phosphorylation is specifically required for bicuculline-induced synaptic scaling down in mouse hippocampal neurons and this phenotype is mediated by mGluR5 (metabotropic glutamate receptor 5). Our results reveal an important function of MeCP2 in regulating neuronal homeostasis and may eventually help us understand how MECP2 mutations cause RTT.

Nuclear Calcium Signaling Controls Methyl-CpG-binding Protein 2 (MeCP2) Phosphorylation on Serine 421 following Synaptic Activity

The function of MeCP2, a methylated DNA-interacting protein that may act as a global chromatin modifier, is controlled by its phosphorylation on serine 421. Here we show that in hippocampal neurons, nuclear calcium signaling controls synaptic activity-induced phosphorylation of MeCP2 on serine 421. Pharmacological inhibition of calcium/calmodulin-dependent protein (CaM)kinases blocked activity-induced MeCP2 serine 421 phosphorylation. CaM kinase II (CaMKII) but not CaMKIV, the major nuclear CaM kinase in hippocampal neurons, appeared to mediate this phosphorylation event. Biochemical subcellular fractionations and immunolocalization studies revealed that several isoforms of CaMKII (i.e. CaMKIIα, -β, -γ, and -δ) are expressed in the cytosol but are also detectable in the cell nucleus of hippocampal neurons, suggesting that nuclear CaMKII catalyzes MeCP2 serine 421 phosphorylation. Thus, in addition to the classical nuclear calcium-CaMKIV-CREB/CBP (cAMP-response element-binding protein/CREB-binding protein) pathway that regulates transcription of specific target genes, nuclear calcium may also modulate genome-wide the chromatin state in response to synaptic activity via nuclear CaMKII-MeCP2 signaling.

Elevated IKKα Accelerates the Differentiation of Human Neuronal Progenitor Cells and Induces MeCP2-Dependent BDNF Expression

The IκB kinase α (IKKα) is implicated in the differentiation of epithelial and immune cells. We examined whether IKKα also plays a role in the differentiation and maturation of embryonic human neuronal progenitor cells (NPCs). We find that expression of an extra copy of IKKα (IKKα+) blocks self-renewal and accelerates the differentiation of NPCs. This coincides with reduced expression of the Repressor Element Silencing Transcription Factor/Neuron-Restrictive Silencing Factor (REST/NRSF), which is a prominent inhibitor of neurogenesis, and subsequent induction of the pro-differentiation non-coding RNA, miR-124a. However, the effects of IKKα on REST/NRSF and miR-124a expression are likely to be indirect. IKKα+ neurons display extensive neurite outgrowth and accumulate protein markers of neuronal maturation such as SCG10/stathmin-2, postsynaptic density 95 (PSD95), syntaxin, and methyl-CpG binding protein 2 (MeCP2). Interestingly, IKKα associates with MeCP2 in the nuclei of human neurons and can phosphorylate MeCP2 in vitro. Using chromatin immunoprecipitation assays, we find that IKKα is recruited to the exon-IV brain-derived neurotrophic factor (BDNF) promoter, which is a well-characterized target of MeCP2 activity. Moreover, IKKα induces the transcription of BDNF and knockdown expression of MeCP2 interferes with this event. These studies highlight a role for IKKα in accelerating the differentiation of human NPCs and identify IKKα as a potential regulator of MeCP2 function and BDNF expression.

Coil‐to‐helix transitions in intrinsically disordered methyl CpG binding protein 2 and its isolated domains

Methyl CpG binding protein 2 (MeCP2) is a canonical intrinsically disordered protein (IDP), that is, it lacks stable secondary structure throughout its entire polypeptide chain. Because IDPs often have the propensity to become locally ordered, we tested whether full-length MeCP2 and its constituent domains would gain secondary structure in 2,2,2-trifluoroethanol (TFE), a cosolvent that stabilizes intramolecular hydrogen bonding in proteins. The α-helix, β-strand/turn, and unstructured content were determined as a function of TFE concentration by deconvolution of circular dichroism data. Results indicate that approximately two-thirds of the unstructured residues present in full-length MeCP2 were converted to α-helix in 70% TFE without a change in β-strand/turn. Thus, much of the MeCP2 polypeptide chain undergoes coil-to-helix transitions under conditions that favor intrachain hydrogen bond formation. The unstructured residues of the N-terminal (NTD) and C-terminal (CTD) domains were partially converted to α-helix in 70% TFE. In contrast, the central transcription regulation domain (TRD) became almost completely α-helical in 70% TFE. Unlike the NTD, CTD, and TRD, the unstructured content of the methyl DNA binding domain and the intervening domain did not change with increasing TFE concentration. These results indicate that the coil-to-helix transitions that occur in full-length MeCP2 are localized to the NTD, CTD, and TRD, with the TRD showing the greatest tendency for helix formation. The potential relationships between intrinsic disorder, coil-to-helix transitions, and MeCP2 structure and function are discussed.

Drosophila as a model for MECP2 gain of function in neurons.

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model.

The Many Faces of MeCP2

Methyl-CpG-binding protein 2 (MeCP2) is an X-linked transcription factor that binds to methylated CpG dinucleotides in the genome. Its primary function was originally thought to be limited to repression of gene transcription in keeping with the transcriptional silence of methylated genes. CpG methylation is a hallmark of epigenetic regulation of genome function because it is copied to replicating DNA strands during cell division and is maintained across development as a form of epigenetic memory. Cytosine methylation in CpG clusters (islands) in the genome can modify chromatin structure by inhibiting activator DNA-binding proteins from binding to recognition sequences in DNA while allowing access by repressor proteins to methyl-CpP binding domains (MBD) that prevent the activation of gene transcription. MeCP2 is a member of the MBD family of proteins that associate with histone deacetylases (HDACs) and histone metyltransferases (HMTs) to form stable repressor complexes. MeCP2 point mutations cause Rett syndrome, the most common inherited form of mental retardation in girls. However, exactly which protein partners are affected by different MeCP2 mutations is controversial. For example, MeCP2 has been shown to associate with the Sin3a–HDAC chromatin remodeling complex in the brain-derived neurotrophic factor (Bdnf) gene to modulate its expression, and loss of MeCP2 from the Bdnf promoter causes loss of local histone methylation (Chen et al, 2003; Martinowich et al, 2003). However, the function of MeCP2 may be determined by its multifaceted interactions with many different protein partners leading to different functional outcomes. Indeed, MeCP2 appears to associate with many different complexes in brain nuclear extracts some of which repress, but others, like CREB, activate, gene transcription (Chahrour et al, 2008).

The Role of MeCP2 in the Brain

Methyl-CpG binding protein 2 (MeCP2) was first identified in 1992 as a protein that binds specifically to methylated DNA. Mutations in the MECP2 gene were later found to be the cause of an autism spectrum disorder, Rett syndrome. Despite almost 20 years of research into the molecular mechanisms of MeCP2 function, many questions are yet to be answered conclusively. This review considers several key questions and attempts to evaluate the current state of evidence. For example, is MeCP2 just a methyl-CpG binding protein? Is it a multifunctional protein or primarily a transcriptional repressor? We also consider whether MeCP2, as a chromosome-binding protein, acts at specific sites within the genome or more globally, and in which cell types it is functionally important. Finally, we consider two alternative views of MeCP2 in the brain: as a regulator of brain development or as a factor that helps maintain neuronal/glial function.

A role for glia in the progression of Rett’s syndrome

Rett's syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.

In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system

The array of specialized neuronal and glial cell types that characterize the adult central nervous system originates from neuroepithelial proliferating precursor cells. The transition from proliferating neuroepithelial precursor cells to neuronal lineages is accompanied by rapid global changes in gene expression in coordination with epigenetic modifications at the level of the chromatin structure. A number of genetic studies have begun to reveal how epigenetic deregulation results in neurodevelopmental disorders such as mental retardation, autism, Rubinstein–Taybi syndrome and Rett syndrome. In this review we focus on the role of the methyl-CpG binding protein 2 (MeCP2) during development of the central nervous system and its involvement in Rett syndrome. First, we present recent findings that indicate a previously unconsidered role of glial cells in the development of Rett syndrome. Next, we discuss evidence of how MeCP2 deficiency or loss of function results in aberrant gene expression leading to Rett syndrome. We also discuss MeCP2's function as a repressor and activator of gene expression and the role of its different target genes, including microRNAs, during neuronal development. Finally, we address different signaling pathways that regulate MeCP2 expression at both the post-transcriptional and post-translational level, and discuss how mutations in MeCP2 may result in lack of responsiveness to environmental signals.

Age-dependent expression of MeCP2 in a heterozygous mosaic mouse model

Functional deficiency of the X-linked methyl-CPG binding protein 2 (MeCP2) leads to the neurodevelopmental disorder Rett syndrome (RTT). Due to random X-chromosome inactivation (XCI), most RTT patients are females who are heterozygous for the MECP2 mutation and therefore mosaic in MeCP2 deficiency. Some MECP2 heterozygote females are found to have unbalanced XCI, which may affect the severity of neurological symptoms seen in these patients; however, whether MeCP2 deficiency affects XCI in the postnatal and adult brain is unclear. Here we developed a novel MeCP2 mosaic mouse model in which the X chromosome containing the wild-type Mecp2 expresses a green fluorescent protein (GFP) transgene, while the X chromosome harboring the mutant Mecp2 does not. Due to random XCI, the neurons in the female MeCP2 mosaic mice express either wild-type MeCP2 (GFP+) or mutant MeCP2 (GFP-), and the two can be distinguished by GFP fluorescence. Using this mouse model, we evaluated XCI in female heterozygote mice from 3 to 9 months after birth. We found that MeCP2 deficiency does not affect XCI at 3 months of age, but does alter the proportion of wild-type MeCP2-expressing neurons at later ages, suggesting that MeCP2 impacts XCI patterns in an age-dependent manner. Given the important function of MeCP2 in neuronal development, our data could shed light on how MeCP2 deficiency affects postnatal brain functions and the dynamic changes in the neurological symptoms of RTT.

The Role of MeCP2 in Brain Development and Neurodevelopmental Disorders

Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Rett syndrome, the primary disorder caused by mutations in the X-linked MECP2 gene, is characterized by a period of cognitive decline and development of hand stereotypies and seizures following an apparently normal early infancy. In addition, MECP2 mutations and duplications are observed in a spectrum of neurodevelopmental disorders, including severe neonatal encephalopathy, X-linked mental retardation, and autism, implicating MeCP2 as an essential regulator of postnatal brain development. In this review, we compare the mutation types and inheritance patterns of the human disorders associated with MECP2. In addition, we summarize the current understanding of MeCP2 as a central epigenetic regulator of activity-dependent synaptic maturation. As MeCP2 occupies a central role in the pathogenesis of multiple neurodevelopmental disorders, continued investigation into MeCP2 function and regulatory pathways may show promise for developing broad-spectrum therapies.

Neuronal differentiation of neural precursor cells is promoted by the methyl-CpG-binding protein MeCP2

Methyl-CpG-binding protein 2 (MeCP2), a methyl-CpG-binding domain protein family member which is expressed predominantly in neurons in the nervous system, acts as a transcriptional repressor by binding to methylated genes, and mutations in mecp2 cause the neurological disorder known as Rett syndrome (RTT). Although MeCP2 has been reported to regulate neuronal maturation rather than fate specification of neural precursor cells (NPCs), we have previously shown that it inhibits astrocyte differentiation of NPCs when ectopically expressed. Here, we show that expression of MeCP2 in NPCs not only suppresses astrocytic differentiation but actually promotes neuronal differentiation, even in the presence of well-known astrocyte-inducing cytokines. This dual function of MeCP2 was abolished by the MEK inhibitor U0126. Moreover, we observed that a truncated form of MeCP2 found in RTT patients fails to promote neuronal differentiation. We further demonstrate that transplanted MeCP2-expressing NPCs differentiate in vivo into neurons in two non-neurogenic regions, striatum and spinal cord. These results suggest a possible therapeutic application for MeCP2 in neurodegenerative diseases and injuries to the central nervous system.

The yin and yang of MeCP2 phosphorylation

Studies of the childhood neurological disorder Rett syndrome and methyl-CpG-binding protein 2 (MeCP2) taught us that MeCP2 performs a balancing act in modulating neurological functions. Rett syndrome, characterized by cognitive deficits, motor impairments, autistic-like features, seizures, and stereotyped repetitive hand movements is caused by loss of MeCP2 function (1). On the other hand, a syndrome with overlapping features including autism, mental retardation, seizures, motor impairments, and repetitive movements results from gain of MeCP2 function due to doubling or tripling the protein level (2–4). In animal models, neuronal loss of MeCP2 leads to reduced glutamatergic synaptic strength due to reduced synapse numbers and gain of MeCP2 in neurons results in increased synaptic strength due to increased synapse numbers (5). Until recently it was believed that MeCP2 was exclusively expressed in neurons in the central nervous system (6, 7), we now know MeCP2 is also expressed in astrocytes and that MeCP2 deficient astrocytes cannot support neuronal dendritic arborization (8). At the molecular level, several studies have shown that MeCP2 functions as a transcriptional repressor by binding to methylated CpG dinucleotides and recruiting co-repressor proteins to silence gene expression (9, 10). However, in vivo studies showed that loss of MeCP2 leads to reduced expression of thousands of genes suggesting that MeCP2 may be a transcriptional modulator important for decreasing the expression of some genes and enhancing the expression of others (11). In this issue of PNAS, we learn about yet other balancing forces in modulating MeCP2 function: the differential phosphorylation of MeCP2 in response to neuronal activity (12). Such phosphorylation events may be one key mechanism by which MeCP2 modulates gene expression. Fig. 1. MeCP2 phosphorylation: a balancing act in neurons. A. MeCP2 in resting neurons … 1To whom correspondence should be addressed. E-mail: hzoghbi{at}bcm.edu

Genetic Modifiers of MeCP2 Function in Drosophila

The levels of methyl-CpG–binding protein 2 (MeCP2) are critical for normal post-natal development and function of the nervous system. Loss of function of MeCP2, a transcriptional regulator involved in chromatin remodeling, causes classic Rett syndrome (RTT) as well as other related conditions characterized by autism, learning disabilities, or mental retardation. Increased dosage of MeCP2 also leads to clinically similar neurological disorders and mental retardation. To identify molecular mechanisms capable of compensating for altered MeCP2 levels, we generated transgenic Drosophila overexpressing human MeCP2. We find that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is found in mammals. MeCP2 overexpression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. We used a candidate gene approach to identify genes that are able to compensate for abnormal phenotypes caused by MeCP2 increased activity. These genetic modifiers include other chromatin remodeling genes (Additional sex combs, corto, osa, Sex combs on midleg, and trithorax), the kinase tricornered, the UBE3A target pebble, and Drosophila homologues of the MeCP2 physical interactors Sin3a, REST, and N-CoR. These findings demonstrate that anatomical and behavioral phenotypes caused by MeCP2 activity can be ameliorated by altering other factors that might be more amenable to manipulation than MeCP2 itself.

Deletion of Mecp2 in Sim1-Expressing Neurons Reveals a Critical Role for MeCP2 in Feeding Behavior, Aggression, and the Response to Stress

Rett Syndrome (RTT) is an autism spectrum disorder caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). In order to map the neuroanatomic origins of the complex neuropsychiatric behaviors observed in patients with RTT and to uncover endogenous functions of MeCP2 in the hypothalamus, we removed Mecp2 from Sim1-expressing neurons in the hypothalamus using Cre-loxP technology. Loss of MeCP2 in Sim1-expressing neurons resulted in mice that recapitulated the abnormal physiological stress response that is seen upon MeCP2 dysfunction in the entire brain. Surprisingly, we also uncovered a role for MeCP2 in the regulation of social and feeding behaviors since the Mecp2 conditional knockout (CKO) mice were aggressive, hyperphagic, and obese. This study demonstrates that deleting Mecp2 in a defined brain region is an excellent approach to map the neuronal origins of complex behaviors and provides new insight about the function of MeCP2 in specific neurons.

Analysis of protein domains and Rett syndrome mutations indicate that multiple regions influence chromatin-binding dynamics of the chromatin-associated protein MECP2 in vivo

The methyl-CpG-binding protein 2 (MECP2) serves both organizational and transcriptional functions in the nucleus, with two well-characterized domains integrally related to these functions. The recognition of methylated CpG dinucleotides is accomplished by the methyl-binding domain (MBD), and the transcriptional repression domain (TRD) facilitates protein-protein interactions with chromatin remodeling proteins. For each known function of MECP2, chromatin binding is a crucial activity. Here, we apply photobleaching strategies within the nucleus using domain-deleted MECP2 proteins as well as naturally occurring point mutations identified in individuals with the neurodevelopmental disorder Rett syndrome (RTT). These studies reveal that MECP2 is transiently associated with chromatin in vivo and confirm a central role for the MBD in directing the protein to heterochromatin. In addition, we report for the first time that the small region between the MBD and the TRD, known as the interdomain region (ID), stabilizes chromatin binding by MECP2 independently of the MBD. The TRD of MECP2 also contributes towards chromatin binding, whereas the N- and C-termini do not. Some common RTT missense and nonsense mutations significantly affect binding kinetics, suggesting that alterations in chromatin binding can result in protein dysfunction and hence a disease phenotype.