• Array comparative genomic hybridization
• Case report
• Methyl-CpG-binding protein 2
• Neurodevelopmental Delay
• Xq28 duplication

• AMPK
• BDNF
• DNA methylation
• MeCP2 isoforms
• Rett Syndrome
• autophagy
• brain development
• brain metabolism
• cholesterol
• epigenetics
• glucose
• mTOR

• ADHD
• Alzheimer’s
• DNA methylation
• Depression
• Neurological disorders
• Rett syndrome

The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.

• Cell penetrating peptide
• MeCP2
• Protein structural characterization
• Rett Syndrome
• TAT-fusion proteins

• asthma
• dna methylation
• epigenesis, genetic
• single-cell analysis

• Asthma/genetics
• Asthma/metabolism
• DNA Methylation
• Epigenesis, Genetic
• Epigenomics
• Humans
• Sequence Analysis, DNA
• Single-Cell Analysis

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

• Methyl-CpG-binding protein 2 (MECP2)
• Mouse models
• Neuropsychiatric disorders
• Rett syndrome

• Animals
• Biomarkers
• Disease Models, Animal
• Disease Susceptibility
• Genetic Association Studies
• Genetic Predisposition to Disease
• Genotype
• Humans
• Mental Disorders/etiology
• Mental Disorders/metabolism
• Methyl-CpG-Binding Protein 2/genetics
• Methyl-CpG-Binding Protein 2/metabolism
• Mutation
• Nervous System Diseases/etiology
• Phenotype
• Rett Syndrome/etiology
• Rett Syndrome/metabolism

Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.

• GTPase
• aluminum fluoride
• guanylate binding protein
• oligomerization
• protein-protein interaction

Epigenetic mechanisms play a role in human disease but their involvement in pathologies from the central nervous system has been hampered by the complexity of the brain together with its unique cellular architecture and diversity. Until recently, disease targeted neural types were only available as postmortem materials after many years of disease evolution. Current in vitro systems of induced pluripotent stem cells (iPSCs) generated by cell reprogramming of somatic cells from patients have provided valuable disease models recapitulating key pathological molecular events. Yet whether cell reprogramming on itself implies a truly epigenetic reprogramming, the epigenetic mechanisms governing this process are only partially understood. Moreover, elucidating epigenetic regulation using patient-specific iPSC-derived neural models is expected to have a great impact to unravel the pathophysiology of neurodegenerative diseases and to hopefully expand future therapeutic possibilities. Here we will critically review current knowledge of epigenetic involvement in neurodegenerative disorders focusing on the potential of iPSCs as a promising tool for epigenetic research of these diseases.

• Cell Cycle
• Cell Proliferation
• Centrosome
• MeCP2
• Microtubule
• Mitotic Spindle
• Protein Phosphorylation

• DNA demethylation
• DNA methylation
• Histone modifications
• MicroRNAs

Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.

• MeCP2
• Rett syndrome
• chromatin
• mouse models
• phosphorylation
• post-translational modifications
• synaptic plasticity

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells. Ablation of Hdac1 or Hdac2 by Nestin-Cre had no obvious consequences on brain development and architecture owing to compensation by the paralog. By contrast, combined deletion of Hdac1 and Hdac2 resulted in impaired chromatin structure, DNA damage, apoptosis and embryonic lethality. To dissect the individual roles of HDAC1 and HDAC2, we expressed single alleles of either Hdac1 or Hdac2 in the absence of the respective paralog in neural cells. The DNA-damage phenotype observed in double knockout brains was prevented by expression of a single allele of either Hdac1 or Hdac2. Strikingly, Hdac1^−/−Hdac2^+/− brains showed normal development and no obvious phenotype, whereas Hdac1^+/−Hdac2^−/− mice displayed impaired brain development and perinatal lethality. Hdac1^+/−Hdac2^−/− neural precursor cells showed reduced proliferation and premature differentiation mediated by overexpression of protein kinase C, delta, which is a direct target of HDAC2. Importantly, chemical inhibition or knockdown of protein kinase C delta was sufficient to rescue the phenotype of neural progenitor cells in vitro. Our data indicate that HDAC1 and HDAC2 have a common function in maintaining proper chromatin structures and show that HDAC2 has a unique role by controlling the fate of neural progenitors during normal brain development.

• Chromatin
• Epigenetics
• Histone deacetylase
• Mouse

• 5-aza-dc
• 5′-aza-2′-deoxycytidine
• CPHD
• Chromatin
• CpG
• DNA methyltransferase
• DNMT
• FSHβ
• GnRH-R
• HDAC
• LHX3
• LIM-homeodomain protein 3
• MBP
• NF1
• PRL
• Promoter
• SP1
• TSA
• TSS
• Transcription
• alpha glycoprotein subunit
• combined pituitary hormone deficiency
• cytosine-phosphate bond-guanine
• follicle-stimulating hormone
• gonadotropin-releasing hormone receptor
• histone deacetylase
• methyl-CpG binding protein
• nuclear factor 1
• prolactin
• specificity protein 1
• transcription start site
• trichostatin A
• αGSU

• Animals
• Cell Line
• DNA Methylation
• Gene Expression/genetics
• Histones/genetics
• Histones/metabolism
• LIM-Homeodomain Proteins/genetics
• Methyl-CpG-Binding Protein 2/genetics
• Methyl-CpG-Binding Protein 2/metabolism
• Mice
• Pituitary Gland/metabolism
• Pituitary Gland/physiology
• Promoter Regions, Genetic
• RNA, Messenger/genetics
• Transcription Factors/genetics

• R01 HD042024 NICHD NIH HHS
• HD42024 NICHD NIH HHS

• Animals
• Brain/metabolism
• Brain/pathology
• Cells, Cultured
• Disease Models, Animal
• Exploratory Behavior/physiology
• Green Fluorescent Proteins/genetics
• Histone Deacetylases/genetics
• Histone Deacetylases/metabolism
• Immunoprecipitation
• Methyl-CpG-Binding Protein 2/genetics
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic
• Models, Molecular
• Mutation/genetics
• Nuclear Receptor Co-Repressor 1/genetics
• Nuclear Receptor Co-Repressor 1/metabolism
• Nuclear Receptor Co-Repressor 2/genetics
• Nuclear Receptor Co-Repressor 2/metabolism
• Rett Syndrome/genetics
• Rett Syndrome/pathology
• Rett Syndrome/physiopathology

• K08MH90306 NIMH NIH HHS
• 084229 Wellcome Trust
• 091580 Wellcome Trust
• P30 HD018655 NICHD NIH HHS
• Wellcome Trust
• R01 NS048276 NINDS NIH HHS
• P30-HD 18655 NICHD NIH HHS
• K08 MH090306 NIMH NIH HHS
• 092076 Wellcome Trust
• R01NS048276 NINDS NIH HHS
• 091020 Wellcome Trust

Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163–309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.

• Animals
• Cell Line
• Chromatin/metabolism
• DNA-Binding Proteins/chemistry
• DNA-Binding Proteins/genetics
• DNA-Binding Proteins/metabolism
• Gene Expression
• Humans
• Methyl-CpG-Binding Protein 2/chemistry
• Methyl-CpG-Binding Protein 2/genetics
• Methyl-CpG-Binding Protein 2/metabolism
• Mice
• Protein Binding
• Protein Interaction Domains and Motifs
• Protein Interaction Mapping

• Alzheimer’s disease
• Huntington’s disease
• Parkinson’s disease
• epigenetics
• imprinting disorders
• neurodevelopment

• Chicken
• Chromatin
• CpG island
• Demethylase
• Epigenetics
• Human
• Methylation
• Mouse
• Transcription
• Xenopus
• Zebrafish

• DNA methylation
• MECP2
• Rett syndrome
• chromatin
• epigenetics

• MeCP2
• Rett syndrome
• X chromosome inactivation
• autism
• chromatin
• epigenetic
• imprinting
• methylation

• Binding Sites
• DNA/chemistry
• Humans
• Methyl-CpG-Binding Protein 2/chemistry
• Methyl-CpG-Binding Protein 2/metabolism
• Models, Molecular
• Nucleic Acid Conformation
• Nucleosomes/chemistry
• Nucleosomes/metabolism
• Scattering, Small Angle
• X-Ray Diffraction

• R01GM096192 NIGMS NIH HHS
• CA92584 NCI NIH HHS
• Howard Hughes Medical Institute
• R01GM066834 NIGMS NIH HHS
• P01 CA092584 NCI NIH HHS
• R01 GM061909 NIGMS NIH HHS
• R01 GM066834-09 NIGMS NIH HHS
• R01GM061909 NIGMS NIH HHS
• R01 GM096192 NIGMS NIH HHS
• R01 GM066834 NIGMS NIH HHS

• Alleles
• Animals
• Brain/cytology
• Cell Line
• Chromatin/genetics
• Chromatin/metabolism
• Chromatin Assembly and Disassembly
• Chromatin Immunoprecipitation
• CpG Islands/genetics
• DNA Methylation
• Genome/genetics
• Histone-Lysine N-Methyltransferase/metabolism
• Histones/chemistry
• Histones/metabolism
• Methylation
• Mice
• NIH 3T3 Cells
• Promoter Regions, Genetic
• Trans-Activators/chemistry
• Trans-Activators/deficiency
• Trans-Activators/genetics
• Trans-Activators/metabolism
• Zinc Fingers

• 091580 Wellcome Trust
• 079643 Wellcome Trust
• Wellcome Trust
• Cancer Research UK
• G0800026 Medical Research Council
• 098051 Wellcome Trust

• Animals
• Brain/cytology
• Brain/metabolism
• Cell Nucleus/metabolism
• Chromatin/metabolism
• CpG Islands
• DNA Methylation
• Genome
• Histones/metabolism
• Methyl-CpG-Binding Protein 2/deficiency
• Methyl-CpG-Binding Protein 2/metabolism
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Neurons/cytology
• Neurons/metabolism
• Nucleosomes/metabolism
• Protein Binding
• Protein Multimerization
• Transcription, Genetic

• Wellcome Trust
• 077224 Wellcome Trust
• 079643 Wellcome Trust

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function.

Formaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.

Using a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.

Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.

• Animals
• Cells, Cultured
• Chromatin Immunoprecipitation
• Formaldehyde/analysis
• Formaldehyde/metabolism
• Methyl-CpG-Binding Protein 2/genetics
• Methyl-CpG-Binding Protein 2/metabolism
• Mice
• Mutation

• Wellcome Trust

• dna methylation
• methyl-cpg binding proteins
• mbd
• chromatin

• Animals
• Chromatin/chemistry
• Chromatin/metabolism
• Chromosomal Proteins, Non-Histone/physiology
• CpG Islands
• DNA Methylation
• DNA-Binding Proteins/physiology
• Gene Silencing/physiology
• Humans
• Models, Biological
• Models, Molecular
• Repressor Proteins/physiology
• Transcription Factors/physiology

• 077691 Wellcome Trust
• 8983 Cancer Research UK
• A8983 Cancer Research UK

CpG islands (CGIs) are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%–8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.

The human genome contains about 22,000 genes, each encoding one of the proteins required for human life. A particular cell type (e.g., blood, skin, etc.) expresses a specific subset of protein genes and silences the remainder. To shed light on the mechanisms that cause genes to be activated or shut down, we studied DNA sequences called “CpG islands” (CGIs). These sequences are found at over half of all human genes and can exist in either the active or silent state depending on the presence or absence of methyl groups on the DNA. We devised a method for purifying all CGIs and showed that, unexpectedly, only half occur at the beginning of genes near the promoter, the rest occurring within or between genes. Notably, methylation of CGIs causes stable gene silencing. We tested 17,000 CGIs in four human tissues and found that 6%–8% were methylated in each. Genes whose protein products play an essential role during embryonic development were preferentially methylated, suggesting that gene expression during development could be regulated by CGI methylation.

CpG island methylation, an epigenetic phenomenon usually associated with abnormality in disease, is little characterised in the context of "normal" human cells. Here we highlight tissue-specific CpG Island methylation, which frequently associates with developmental genes.

• Animals
• Brain-Derived Neurotrophic Factor/therapeutic use
• Disease Models, Animal
• Female
• Humans
• Male
• Methyl-CpG-Binding Protein 2/deficiency
• Methyl-CpG-Binding Protein 2/genetics
• Mice
• Models, Biological
• Rett Syndrome/drug therapy
• Rett Syndrome/etiology
• Rett Syndrome/metabolism
• Rett Syndrome/pathology
• Sex Factors

• Animals
• Gene Expression Regulation/physiology
• Immediate-Early Proteins/blood
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/physiology
• Inflammation Mediators/blood
• Inflammation Mediators/metabolism
• Inflammation Mediators/physiology
• Male
• Methyl-CpG-Binding Protein 2/biosynthesis
• Methyl-CpG-Binding Protein 2/genetics
• Methyl-CpG-Binding Protein 2/physiology
• Neuronal Plasticity/genetics
• Pain/enzymology
• Pain/genetics
• Pain/metabolism
• Pain/pathology
• Pain Measurement/methods
• Phosphorylation
• Posterior Horn Cells/enzymology
• Posterior Horn Cells/metabolism
• Posterior Horn Cells/pathology
• Protein Serine-Threonine Kinases/blood
• Protein Serine-Threonine Kinases/genetics
• Protein Serine-Threonine Kinases/physiology
• Rats
• Rats, Sprague-Dawley
• Transcription, Genetic/physiology

• Wellcome Trust
• G0801381 Medical Research Council
• 065374 Wellcome Trust

• Animals
• Binding Sites
• DNA Methylation
• Epigenesis, Genetic
• Genome
• Methyl-CpG-Binding Protein 2/chemistry
• Methyl-CpG-Binding Protein 2/genetics
• Methyl-CpG-Binding Protein 2/metabolism
• Multigene Family
• Protein Binding
• Protein Structure, Tertiary

• Intramural NIH HHS