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Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome1. The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine2, 3. Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners4, including transcriptional co-repressors (for example, the NCoR/SMRT complex5), transcriptional activators6, RNA7, chromatin remodellers8, 9, microRNA-processing proteins10 and splicing factors11. Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons12. At odds with the concept of broad functionality, missense mutations that cause Rett syndrome are concentrated in two discrete clusters coinciding with interaction sites for partner macromolecules: the methyl-CpG binding domain13 and the NCoR/SMRT interaction domain5. Here we test the hypothesis that the single dominant function of MeCP2 is to physically connect DNA with the NCoR/SMRT complex, by removing almost all amino-acid sequences except the methyl-CpG binding and NCoR/SMRT interaction domains. We find that mice expressing truncated MeCP2 lacking both the N- and C-terminal regions (approximately half of the native protein) are phenotypically near-normal; and those expressing a minimal MeCP2 additionally lacking a central domain survive for over one year with only mild symptoms. This minimal protein is able to prevent or reverse neurological symptoms when introduced into MeCP2-deficient mice by genetic activation or virus-mediated delivery to the brain. Thus, despite evolutionary conservation of the entire MeCP2 protein sequence, the DNA and co-repressor binding domains alone are sufficient to avoid Rett syndrome-like defects and may therefore have therapeutic utility.

The presence of selfish elements in the genome is accepted, but the remainder of genes are usually considered to be ‘law-abiding’.‘Law-abiding’ genes can also indulge in game playing to ensure their propagation, allowing nonessential processes to secure a genetic heritage.Appreciation of this aspect of gene ‘function’ can shed new light on the origin of molecular mechanisms that underlie biological processes.More generally, a bottom-up ‘gene-centric’ view of genome evolution may help to explain the complexity and robustness of biological entities.Molecular complexity of living organisms may therefore be driven from below by the opportunism of genes.Selfish genes were once controversial, but it is now accepted that the genome contains parasitic elements in addition to a complement of conventional genes. This opinion article argues that ‘law-abiding’ genes also indulge in game playing to ensure their propagation, so that initially nonessential processes secure a genetic heritage. A gene-centered view of this kind can help to explain otherwise puzzling aspects of biology, including the complexity and stability of living systems.

Lying at the intersection between neurobiology and epigenetics, Rett syndrome (RTT) has garnered intense interest in recent years, not only from a broad range of academic scientists, but also from the pharmaceutical and biotechnology industries. In addition to the critical need for treatments for this devastating disorder, optimism for developing RTT treatments derives from a unique convergence of factors, including a known monogenic cause, reversibility of symptoms in preclinical models, a strong clinical research infrastructure highlighted by an NIH-funded natural history study and well-established clinics with significant patient populations. Here, we review recent advances in understanding the biology of RTT, particularly promising preclinical findings, lessons from past clinical trials, and critical elements of trial design for rare disorders.

MeCP2 is an abundant protein in mature nerve cells, where it binds to DNA sequences containing methylated cytosine. Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome (RTT), provoking intensive study of the underlying molecular mechanisms. Multiple functions have been proposed, one of which involves a regulatory role in splicing. Here we leverage the recent availability of high-quality transcriptomic data sets to probe quantitatively the potential influence of MeCP2 on alternative splicing. Using a variety of machine learning approaches that can capture both linear and non-linear associations, we show that widely different levels of MeCP2 have a minimal effect on alternative splicing in three different systems. Alternative splicing was also apparently indifferent to developmental changes in DNA methylation levels. Our results suggest that regulation of splicing is not a major function of MeCP2. They also highlight the importance of multi-variate quantitative analyses in the formulation of biological hypotheses., Author summary Rett Syndrome (RTT) is a devastating neurological disorder affecting approximately 1 in 10,000 female births. Most cases of RTT are caused by mutations in the gene identified as methyl-CG binding protein 2 (MECP2) which is an epigenetic reader of DNA methylation. Although the primary function of MeCP2 is to recruit NCoR to methylated sites in the genome, the downstream effect on gene expression is subtle and multiple additional functions have been proposed. Here we focus on the influence of MeCP2 on one of these: alternative splicing to generate different messenger RNAs from a single primary transcript. Using machine learning approaches, we show that neither MeCP2 nor DNA methylation influence alternative splicing. Our results emphasize the importance of multi-variate quantitative analyses and they challenge the over-interpretation of causal relationships based on high-throughput data sets.

Most missense mutations causing Rett syndrome affect domains of MeCP2 that have been shown to either bind methylated DNA or interact with a transcriptional co-repressor complex. Several mutations, however, including the C-terminal truncations that account for ~10% of cases, fall outside these characterised domains. We studied the molecular consequences of four of these “non-canonical” mutations in cultured neurons and mice to see if they reveal additional essential domains without affecting known properties of MeCP2. The results show that the mutations partially or strongly deplete the protein and also in some cases interfere with co-repressor recruitment. These mutations therefore impact the activity of known functional domains and do not invoke new molecular causes of Rett syndrome. The finding that a stable C-terminal truncation does not compromise MeCP2 function raises the possibility that small molecules which stabilise these mutant proteins may be of therapeutic value.

The discovery of CpG islands (CGIs) and the study of their structure and properties run parallel to the development of molecular biology in the last two decades of the twentieth century and to the development of high-throughput genomic technologies at the turn of the millennium. First identified as discrete G + C-rich regions of unmethylated DNA in several vertebrates, CGIs were soon found to display additional distinctive chromatin features from the rest of the genome in terms of accessibility and of the epigenetic modifications of their histones. These features, together with their colocalization with promoters and with origins of DNA replication in mammals, highlighted their relevance in the regulation of genomic processes. Recent approaches have shown with unprecedented detail the dynamics and diversity of the epigenetic landscape of CGIs during normal development and under pathological conditions. Also, comparative analyses across species have started revealing how CGIs evolve and contribute to the evolution of the vertebrate genome.

Item does not contain fulltext The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalog of high-resolution reference epigenomes of major primary human cell types. The studies now presented (see the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic datasets to gain insights in the epigenetic control of cell states relevant for human health and disease. PAPERCLIP.

Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the methyl-CpG–binding protein 2 ( MeCP2 ) gene. The majority of RTT missense mutations disrupt the interaction of the MeCP2 with DNA or the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid receptors (SMRT) corepressor complex. Here, we show that the “NCoR/SMRT interaction domain” (NID) of MeCP2 directly contacts transducin beta-like 1 (TBL1) and TBL1 related (TBLR1), two paralogs that are core components of NCoR/SMRT. We determine the cocrystal structure of the MeCP2 NID in complex with the WD40 domain of TBLR1 and confirm by in vitro and ex vivo assays that mutation of interacting residues of TBLR1 and TBL1 disrupts binding to MeCP2. Strikingly, the four MeCP2-NID residues mutated in RTT are those residues that make the most extensive contacts with TBLR1. Moreover, missense mutations in the gene for TBLR1 that are associated with intellectual disability also prevent MeCP2 binding. Our study therefore reveals the molecular basis of an interaction that is crucial for optimal brain function.

Methylation of DNA at position five of the cytosine ring occurs at most CpG dinucleotides in the mammalian genome and is essential for embryonic viability. With several of the key proteins now known, it has become possible to approach the biological significance of this epigenetic system through both biochemistry and genetics. As a result, advances have been made in our understanding of the mechanisms by which DNA methylation is targeted to specific regions of the genome and interpreted by methyl-CpG-binding proteins. Recent studies have illuminated the role of DNA methylation in controlling gene expression and have strengthened its links with histone modification and chromatin remodelling.

Methylation of cytosine in human DNA has been studied for over 60 years, but has only recently been confirmed as an important player in human disease. Rett syndrome is a neurological disorder caused by mutations in the MeCP2 protein, which has been shown to bind methylated DNA and repress transcription. This review will focus on experiments addressing the basic properties of MeCP2 and on mouse models of Rett syndrome that are starting to yield insights into this condition.

Mutations in the human methyl-CpG-binding protein gene MECP2 cause the neurological disorder Rett syndrome and some cases of X-linked mental retardation (XLMR). We report that MeCP2 interacts with ATRX, a SWI2/SNF2 DNA helicase/ATPase that is mutated in ATRX syndrome (α-thalassemia/mental retardation, X-linked). MeCP2 can recruit the helicase domain of ATRX to heterochromatic foci in living mouse cells in a DNA methylation-dependent manner. Also, ATRX localization is disrupted in neurons of Mecp2 -null mice. Point mutations within the methylated DNA-binding domain of MeCP2 that cause Rett syndrome or X-linked mental retardation inhibit its interaction with ATRX in vitro and its localization in vivo without affecting methyl-CpG binding. We propose that disruption of the MeCP2–ATRX interaction leads to pathological changes that contribute to mental retardation.

Neuroepigenetics is a newly emerging field in neurobiology that addresses the epigenetic mechanism of gene expression regulation in various postmitotic neurons, both over time and in response to environmental stimuli. In addition to its fundamental contribution to our understanding of basic neuronal physiology, alterations in these neuroepigenetic mechanisms have been recently linked to numerous neurodevelopmental, psychiatric, and neurodegenerative disorders. This article provides a selective review of the role of DNA and histone modifications in neuronal signal-induced gene expression regulation, plasticity, and survival and how targeting these mechanisms could advance the development of future therapies. In addition, we discuss a recent discovery on how double-strand breaks of genomic DNA mediate the rapid induction of activity-dependent gene expression in neurons.

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.

Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate significantly from the average genomic pattern by being GC-rich, CpG-rich, and predominantly nonmethylated. Most, perhaps all, CGIs are sites of transcription initiation, including thousands that are remote from currently annotated promoters. Shared DNA sequence features adapt CGIs for promoter function by destabilizing nucleosomes and attracting proteins that create a transcriptionally permissive chromatin state. Silencing of CGI promoters is achieved through dense CpG methylation or polycomb recruitment, again using their distinctive DNA sequence composition. CGIs are therefore generically equipped to influence local chromatin structure and simplify regulation of gene activity.

Mutations in the X-linked MECP2 gene are the primary cause of the severe autism spectrum disorder RTT (Rett syndrome). Deletion of Mecp2 in mice recapitulates many of the overt neurological features seen in humans, and the delayed onset of symptoms is accompanied by deficits in neuronal morphology and synaptic physiology. Recent evidence suggests that reactivation of endogenous Mecp2 in young and adult mice can reverse aspects of RTT-like pathology. In the current perspective, we discuss these findings as well as other genetic, pharmacological and environmental interventions that attempt phenotypic rescue in RTT. We believe these studies provide valuable insights into the tractability of RTT and related conditions and are useful pointers for the development of future therapeutic strategies.

Mammalian genomes are punctuated by DNA sequences containing an atypically high frequency of CpG sites termed CpG islands (CGIs). CGIs generally lack DNA methylation and associate with the majority of annotated gene promoters. Many studies, however, have identified examples of CGI methylation in malignant cells, leading to improper gene silencing. CGI methylation also occurs in normal tissues and is known to function in X-inactivation and genomic imprinting. More recently, differential methylation has been shown between tissues, suggesting a potential role in transcriptional regulation during cell specification. Many of these tissue-specific methylated CGIs localise to regions distal to promoters, the regulatory function of which remains to be determined.

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, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions.

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Two decades of research have fostered the view that MeCP2 is a multifunctional chromatin protein that integrates diverse aspects of neuronal biology. More recently, studies have focused on specific RTT-associated mutations within the protein. This work has yielded molecular insights into the critical functions of MeCP2 that promise to simplify our understanding of RTT pathology.

DNA methylation is an epigenetic mechanism involved in transcriptional silencing of imprinted genes, genes located on the inactive X chromosome, and a number of tumour suppressor genes in cancer. MBD (methyl-CpG-binding domain) proteins selectively bind to methylated DNA and recruit chromatin remodelling and transcriptional repressor complexes, thereby establishing a repressive chromatin state. MBD2, a member of the MBD protein family, binds to methylated promoter CpG islands (clusters of high-density CpG dinucleotides) and acts as a methylation-dependent transcriptional repressor. Previous work has demonstrated that decreased CpG island methylation in mice lacking the DNA methyltransferase DNMT1 is associated with impaired tumorigenesis when crossed on the tumour-susceptible Apc Min/+ background. Mbd2 deficiency also dramatically reduces adenoma burden and extends life span in a gene dosage-dependent manner in this mouse model. Mbd2 is therefore essential for tumorigenesis in the murine intestine, although it is dispensable for the viability of the host animals. These findings validate MBD2 as a potential target for therapeutic intervention in colorectal cancer. Abbreviations: ChIP, chromatin immunoprecipitation; DNMT, DNA methyltransferase; GSTP1, glutathione S-transferase pi; HDAC, histone deacetylase; IFN-γ, interferon-γ; IL-4, interleukin-4; MBD, methyl-CpG-binding domain; MIZF, MBD2 interacting zinc finger; TRD, transcriptional repressor domain

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.

MeCP2 is a methyl-CpG binding protein that can repress transcription of nearby genes. In humans, mutations in the MECP2 gene are the major cause of Rett syndrome. By searching expressed sequence tag (EST) databases we have found a novel MeCP2 splice isoform (MeCP2alpha) which encodes a distinct N-terminus. We demonstrate that the MeCP2alpha mRNA splice variant is more abundant than the previously annotated MeCP2 mRNA (MeCP2beta) in mouse tissues and human brain. Furthermore, MeCP2beta mRNA has an upstream open reading frame that inhibits its translation. As a result of these differences, >90% of MeCP2 in mouse brain is MeCP2alpha. Both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci in mouse cells. The presence of a previously unknown MeCP2 isoform has implications for the genetic screening of Rett syndrome patients and for studies of the functional significance of MeCP2.

The genes MBD1 and MBD2 encode methyl-CpG binding proteins that suppress transcription from methylated promoters. In contrast, CGBP encodes a protein that binds promoters containing unmethylated CpG and stimulates transcription. All three are located on human chromosome 18q21, a region of frequent loss of heterozygosity in several cancers. These genes therefore represent candidate tumour suppressor genes, whose loss of function could affect the normal regulation of gene expression, whether by lack of complete suppression of genes normally silenced (via loss of MBD1 and MBD2) or by some loss of activation of genes normally expressed (via loss of CGBP), either way contributing to the tumorigenic phenotype. We have confirmed by fluorescent in situ hybridization that MBD1 and MBD2 bracket the DCC locus giving a gene order of MBD1/CGBP-DCC 5'-DCC 3'-MBD2. Mutation analyses by single-stranded conformation polymorphism in colon and lung cancer cell lines and primary tumours revealed a small number of mutations, suggesting only a limited role of these genes in human tumorigenesis.

Fas-associated death domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector domain of FADD and a region of MBD4 adjacent to the glycosylase domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome. DOI: http://dx.doi.org/10.7554/eLife.03058.001

Methylation of DNA is essential for development in the mouse and plays an important role in inactivation of the X-chromosome, genomic imprinting and gene silencing. The properties of the methyl-CpG binding proteins (MeCPs) are being proved to be the key to interpreting the connection between DNA methylation and transcriptional repression. The founder member of the family, MeCP2, consists of a single polypeptide that contains both a methyl-CpG binding domain (MBD) and transcriptional repression domain (TRD). MBD binds to a single symmetrically methylated CpG site and is responsible for chromatin localization of the protein. NMR studies have revealed that the MBD adopts a wedge-shaped molecular structure. The TRD interacts with Sin3, which is known to form complexes with histone deacetylases. MeCP2-mediated transcriptional repression may involve two distinct mechanisms, one being dependent on chromatin modification by histone deacetylation and the other being chromatin independent. Mutations in MeCP2 gene cause the X-linked neurodevelopmental disease Rett syndrome. The spectrum of mutations reflects the importance of the MBD and TRD domains. We speculate that abnormal gene expression in Rett patients leads to dysfunction of the central nervous system. We propose a genetic therapeutic approach based on activation of the wild type copy of the MeCP2 gene located in the inactive X chromosome.

Rett syndrome (RTT) is an inherited neurodevelopmental disorder of females that occurs once in 10,000–15,000 births1,2. Affected females develop normally for 6–18 months, but then lose voluntary movements, including speech and hand skills. Most RTT patients are heterozygous for mutations in the Xlinked gene MECP2 (refs. 3–12), encoding a protein that binds to methylated sites in genomic DNA and facilitates gene silencing13– 17. Previous work with Mecp2-null embryonic stem cells indicated that MeCP2 is essential for mouse embryogenesis18. Here we generate mice lacking Mecp2 using Cre-loxP technology. Both Mecp2-null mice and mice in which Mecp2 was deleted in brain showed severe neurological symptoms at approximately six weeks of age. Compensation for absence of MeCP2 in other tissues by MeCP1 (refs. 19,20) was not apparent in genetic or biochemical tests. After several months, heterozygous female mice also showed behavioral symptoms. The overlapping delay before symptom onset in humans and mice, despite their profoundly different rates of development, raises the possibility that stability of brain function, not brain development per se, is compromised by the absence of MeCP2.

We have identified new members of the histone deacetylase enzyme family in Drosophila melanogaster. dHDAC6 is a class II deacetylase with two active sites, and dSIR2 is an NAD-dependent histone deacetylase. These proteins, together with two class I histone deacetylases, dHDAC1 and dHDAC3, have been expressed and characterized as epitope-tagged recombinant proteins in Schneider SL2 cells. All these proteins have in vitro deacetylase activity and are able to deacetylate core histone H4 at all four acetylatable lysine residues (5, 8, 12, and 16). Recombinant dHDAC6 and dSIR2 are both insensitive to TSA and HC toxin and resistant, relative to dHDAC1 and dHDAC3, to inhibition by sodium butyrate. Indirect immunofluorescence microscopy of stably transfected SL2 lines reveals that dHDAC1 and dSIR2 are nuclear, dHDAC6 is cytosolic, and dHDAC3 is detectable in both cytosol and nucleus. dHDAC6 and dSIR2 elute from Superose 6 columns with apparent molecular weights of 90 and 200 kDa, respectively. In contrast, dHDAC1 and dHDAC3elute at 800 and 700 kDa, respectively, suggesting that they are components of multiprotein complexes. Consistent with this, recombinant dHDAC1 coimmunoprecipitates with components of the Drosophila NuRD complex and dHDAC3 with an as yet unknown 45-kDa protein.

CpG islands are found at the 5' end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5' end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5' end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5' gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5' gene sequences from specific human chromosomes.

Over the past few years, the long-standing idea that covalent modification of chromatin can play a role in determining states of gene activity has been confirmed. Eukaryotic genes can be silenced by deacetylation of acetyl-lysine moieties in the N-terminal tails of histones. Recent work links histone deacetylases with an increasing number of repressors, suggesting that deacetylation might be a rather pervasive feature of transcriptional repression systems.

MBD1 belongs to a family of mammalian proteins that share a methyl-CpG binding domain. Previous work has shown that MBD1 binds to methylated sites in vivo and in vitro and can repress transcription from methylated templates in transcription extracts and in cultured cells. In the present study we established by several experimental criteria that, contrary to a previous report, MBD1 is not a component of the MeCP1 repressor complex. We identified a powerful transcriptional repression domain (TRD) at the C terminus of MBD1 that can actively repress transcription at a distance. Methylation-dependent repression in vivo depends on the presence of both the TRD and the methyl-CpG binding domain. The mechanism is likely to involve deacetylation, since the deacetylase inhibitor trichostatin A can overcome MBD1-mediated repression. Accordingly, we found that endogenous MBD1 is particularly concentrated at sites of centromeric heterochromatin, where acetylated histone H4 is deficient. Unlike MBD2 and MeCP2, MBD1 is not depleted by antibodies to the histone deacetylase HDAC1. Thus, the deacetylase-dependent pathway by which MBD1 actively silences methylated genes is likely to be different from that utilized by the methylation-dependent repressors MeCP1 and MeCP2.

Defects of mismatch repair are thought to be responsible for carcinogenesis in hereditary non-polyposis colorectal cancer and about 15% of sporadic colon cancers. The phenotype is seen as microsatellite instability and is known to be caused either by mutations in mismatch repair genes or by aberrant methylation of these genes stabilizing their downregulation. Lack of repair of microsatellite sequence errors, created during replication, leads to a mutation-prone phenotype. Where mutations occur within mononucleotide tracts within exons they cause translation frameshifts, premature cessation of translation and abnormal protein expression. Such mutations have been observed in the TGFbetaRII, BAX, IGFIIR, MSH3 and MSH6 genes in colon and other cancers. We describe here frameshift mutations affecting the gene for the methyl-CpG binding thymine glycosylase, MBD4, in over 40% of microsatellite unstable sporadic colon cancers. The mutations all appear heterozygous but their location would ensure truncation of the protein between the methyl-CpG binding and glycosylase domains, thus potentially generating a dominant negative effect. It is thus possible that such mutations enhance mutation frequency at other sites in these tumours. A suggestion has been made that MBD4 (MED1) mutations may lead to an increased rate of microsatellite instability but this mechanism appears unlikely due to the nature of mutations we have found.

The primary target for DNA methylation in mammalian genomes is cytosine in the dinucleotide CpG. High densities of CpG dinucleotides are found in CpG islands, but paradoxically CpG islands are normally in a non-methylated state. Here, we speculate why CpG islands are immune to methylation and why they are so rich in guanine and cytosine relative to the surrounding DNA. We propose that CpG islands are associated with promoters that are transcriptionally active at totipotent stages of development and can also act as origins of DNA replication. CpG islands may be 'footprints' caused by early DNA replication intermediates at dual function promoters of this kind.

DNA methylation and chromatin modification are two global mechanisms that regulate gene expression. Recent studies provide insight into the mechanism of transcriptional silencing by a methyl-CpG binding protein, MeCP2. MeCP2 is shown to interact with the Sin3/histone deacetylase co-repressor complex. Thus, this interaction can provide a mechanistic explanation for the long-known relationship between DNA methylation and chromatin structure. Moreover, several studies have shown that inhibition of histone deacetylases by specific inhibitors can reactivate endogenous genes or reporter constructs previously silenced by DNA methylation. Taken together, the data strongly suggest that DNA methylation can pattern chromatin modification.

Methylation of cytosines within the sequence CpG is essential for mouse development and has been linked to transcriptional suppression in vertebrate systems. Methyl-CpG binding proteins (MeCPs) 1 and 2 bind preferentially to methylated DNA and can inhibit transcription. The gene for MeCP2 has been cloned and a methyl-CpG binding domain (MBD) within it has been defined. A search of DNA sequence databases with the MBD sequence identified a human cDNA with potential to encode an MBD-like region. Sequencing of the complete cDNA revealed that the open reading frame also encodes two cysteine-rich domains that are found in animal DNA methyltransferases (DNMTs) and in the mammalian HRX protein (also known as MLL and All-1). HRX is related to Drosophila trithorax. The protein, known as Protein Containing MBD (PCM1), was expressed in bacteria and shown to bind specifically to methylated DNA. PCM1 also repressed transcription in vitro in a methylation-dependent manner. A polyclonal antibody raised against the protein was able to 'supershift' the native MeCP11 complex from HeLa cells, indicating that PCM1 is a component of mammalian MeCP1.

The pattern of methylation in human genes for 18S and 28S ribosomal RNA has been investigated using methylation-sensitive restriction enzymes. We find that the transcribed region of the repeat unit is predominantly unmethylated, in agreement with previous studies. In contrast the non-transcribed spacer, which makes up the majority of the 43 kb repeat unit, is highly methylated in blood cell DNA. The boundaries between methylated and non-methylated domains appear to be relatively sharp, and occur approximately 1.5 kb upstream of the 5' edge of the proximal promoter and approximately 1.0 kb downstream of the 3' end of the transcribed region. A small proportion of all repeat units are methylated throughout the transcribed region, and may represent silent genes. The coincidence between the methylation pattern, the transcription pattern and other features of the repeat unit has implications for our understanding of the mechanism by which patterns of DNA methylation are generated.

Of the estimated 45,000 CpG islands in the human genome, the overwhelming majority are found at the 5' ends of genes and their identification and cloning are proving very useful for finding and isolating genes. Recent work has shed light on the chromosomal distribution and origin of CpG islands. It has been shown unequivocally that CpG islands are concentrated in the R band chromosomal regions and that intact transcription factor binding sites and required for their maintenance. Cases of methylation of CpG islands and inactivation of the associated genes have been reported which may be important in ageing, tumorigenesis and imprinting.

Mutations in the gene encoding the methyl-CpG-binding protein MECP2 are the major cause of Rett syndrome, an autism spectrum disorder mainly affecting young females. MeCP2 is an abundant chromatin-associated protein, but how and when its absence begins to alter brain function is still far from clear. Using a stem cell-based system allowing the synchronous differentiation of neuronal progenitors, we found that in the absence of MeCP2, the size of neuronal nuclei fails to increase at normal rates during differentiation. This is accompanied by a marked decrease in the rate of ribonucleotide incorporation, indicating an early role of MeCP2 in regulating total gene transcription, not restricted to selected mRNAs. We also found that the levels of brain-derived neurotrophic factor (BDNF) were decreased in mutant neurons, while those of the presynaptic protein synaptophysin increased at similar rates in wild-type and mutant neurons. By contrast, nuclear size, transcription rates, and BDNF levels remained unchanged in astrocytes lacking MeCP2. Re-expressing MeCP2 in mutant neurons rescued the nuclear size phenotype as well as BDNF levels. These results reveal a new role of MeCP2 in regulating overall RNA synthesis in neurons during the course of their maturation, in line with recent findings indicating a reduced nucleolar size in neurons of the developing brain of mice lacking Mecp2.

Rett syndrome is a neurological disorder caused by mutation of the X-linked MECP2 gene. Mice lacking functional Mecp2 display a spectrum of Rett syndrome-like signs, including disturbances in motor function and abnormal patterns of breathing, accompanied by structural defects in central motor areas and the brainstem. Although routinely classified as a neurodevelopmental disorder, many aspects of the mouse phenotype can be effectively reversed by activation of a quiescent Mecp2 gene in adults. This suggests that absence of Mecp2 during brain development does not irreversibly compromise brain function. It is conceivable, however, that deep-seated neurological defects persist in mice rescued by late activation of Mecp2. To test this possibility, we have quantitatively analysed structural and functional plasticity of the rescued adult male mouse brain. Activation of Mecp2 in ∼70% of neurons reversed many morphological defects in the motor cortex, including neuronal size and dendritic complexity. Restoration of Mecp2 expression was also accompanied by a significant improvement in respiratory and sensory-motor functions, including breathing pattern, grip strength, balance beam and rotarod performance. Our findings sustain the view that MeCP2 does not play a pivotal role in brain development, but may instead be required to maintain full neurological function once development is complete.

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CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are “orphans” that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development., Author Summary In the decade since the sequence of the human genome was announced, efforts have been made to annotate all genes with their regulatory sequences. CpG islands are short regions containing the sequence CG at high density that map to regions controlling the expression of most human genes (known as promoters). Using a biochemical method, we have identified and mapped all CpG islands in the human and mouse genomes and find that over half are remote from known gene promoters—so-called “orphans.” Mice, which were thought to possess far fewer CpG islands than humans, turn out to have a very similar number. Surprisingly, orphan CpG islands in both species often mark hitherto unknown promoters. The activity of these novel promoters is particularly dynamic during normal development, as they are often silenced by DNA methylation. In colorectal cancers, however, aberrant DNA methylation affects all CpG islands equally.

In the vertebrate genomes studied to date the 5' end of many genes are associated with distinctive sequences known as CpG islands. CpG islands have three properties: they are non-methylated; the dinucleotide CpG occurs at the frequency predicted by base composition; and they are GC-rich. Unexpectedly we have found that CpG islands in certain fish only have the first two properties; that is, their GC-content is not elevated compared to bulk genomic DNA. Based on this finding, we speculate that the GC-richness of CpG islands in vertebrates other than fish is a passive consequence of a higher mutation rate in regions of open chromatin under conditions where the nucleotide precursor pools are biased.

The genomes of many animals, plants and fungi are tagged by methylation of DNA cytosine. To understand the biological significance of this epigenetic mark it is essential to know where in the genome it is located. New techniques are making it easier to map DNA methylation patterns on a large scale and the results have already provided surprises. In particular, the conventional view that DNA methylation functions predominantly to irreversibly silence transcription is being challenged. Not only is promoter methylation often highly dynamic during development, but many organisms also seem to target DNA methylation specifically to the bodies of active genes.

The p14(ARF) and p16(INK4A) tumor suppressor genes are commonly inactivated by aberrant methylation of their promoter regions in human colon cancer. The methyl-CpG-binding domain protein MBD2 is physically associated with the methylated promoters of the p14(ARF) and p16(INK4A) genes in specific tumor cell lines. Moreover, deficiency of MBD2 strongly inhibits intestinal tumorigenesis in the Min mouse, raising the possibility that the protein might be involved in transcriptional repression of methylated tumor suppressor genes. The aim of this study was to evaluate the role of MBD2 in the silencing of p14(ARF) and p16(INK4A) in cancer.The MBD2 protein was stably knocked down by RNA interference in RKO, a colon cancer cell line in which both p14(ARF) and p16(INK4A) are silenced by methylation.We demonstrate here that MBD2 associates with the methylated promoter of the p14(ARF) gene in the RKO colon cancer cell line. Depletion of MBD2 by RNAi leads to selective upregulation of the p14(ARF) but not the p16(INK4A) gene transcript. In addition, p14(ARF) repression can be restored by expressing mouse MBD2 protein in MBD2-deficient RKO cells.These findings implicate MBD2 in transcriptional repression of the methylated p14(ARF) tumor suppressor gene and suggest that repression by MBD2 selectively affects a subset of methylated promoters.

CpG islands are normally methylation free in cells of the animal, even when the associated gene is transcriptionally silent. In mouse NIH 3T3 and L cells, however, over half of the islands are heavily methylated. Near identity of the methylated subset in the two cell lines suggested that methylation is confined to genes that are nonessential in culture. In agreement with this, islands at several tissue-specific genes, but not at housekeeping genes, have become methylated in many human and mouse cell lines. At the chromatin level, methylated islands are Mspl resistant compared with their nonmethylated counterparts. We suggest that mutation-like gene inactivation due to CpG island methylation is widespread in many cell lines and could explain the loss of cell type-specific functions in culture.

Actively transcribed chromatin is structurally different from bulk inactive chromatin. It has been difficult to define the molecular basis of the difference, however, because purified fractions of active chromatin were not available. We have overcome this problem by releasing oligonucleosomes from the nonmethylated CpG-rich islands (CpG islands) of HeLa cell nuclei using restriction endonucleases. Since CpG islands very often include the promoters and 5' transcribed regions of genes, they represent a model for the "active" chromatin structure. CpG island chromatin differs in three respects from bulk chromatin prepared in the same way: histone H1 is present in very low amounts; histones H3 and H4 are highly acetylated; and nucleosome-free regions are present. Except for the latter regions, the average nucleosomal spacing is similar to that of bulk chromatin.

Gene expression in the gut is segmentally regulated, but little is known of the molecular origin of patterning. Analysis of gene expression in colons from mice lacking the methyl-CpG binding repressor MBD2 revealed frequent activation of genes that are normally only expressed in the exocrine pancreas and duodenum. Reduced DNA methylation activated the same gene set in the colon. No significant differences in DNA methylation between the colon and duodenum were detected, but MBD2 was significantly more abundant in the colon. The relevance of MBD2 concentration was tested in a human colon cancer cell line. Depletion of MBD2 was again found to activate exocrine pancreatic genes. Gene activation in this cell culture model was accompanied by loss of promoter-bound MBD2 and increased histone acetylation. The results suggest that modulation of MBD2 during gut development establishes a region-specific gene expression pattern that is essential for establishing correct segmental character.

Geneticists study the gene; however, for epigeneticists, there is no obvious 'epigene'. Nevertheless, during the past year, more than 2,500 articles, numerous scientific meetings and a new journal were devoted to the subject of epigenetics. It encompasses some of the most exciting contemporary biology and is portrayed by the popular press as a revolutionary new science--an antidote to the idea that we are hard-wired by our genes. So what is epigenetics?

DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T] > or = 4) adjacent to methyl-CpG. [A/T] > or = 4 was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome.

The invertebrate chordate Ciona intestinalis is a widely used model organism in biological research. Individuals from waters ranging from arctic to temperate are morphologically almost indistinguishable. However, we found significant differences in whole genomic DNA sequence between northern European and Pacific C. intestinalis. Intronic and transposon sequences often appear unrelated between these geographic origins and amino acid substitutions in protein coding sequences indicate a divergence time in excess of 20 MYA. This finding suggests the existence of two cryptic species within the present C. intestinalis species. We found five marker loci which distinguish the two genetic forms by PCR. This analysis revealed that specimens from Naples, Italy, have the Pacific-type genome, perhaps due to human-mediated marine transport of species. Despite major genomic divergence, the two forms could be hybridized in the laboratory.

One could be forgiven for thinking that biology is in turmoil following recent discoveries that seem to undermine conventional wisdom surrounding the role of genomes in evolution (Ball, 2013). Is the vast excess of genomic DNA—hitherto dismissed as junk—indispensable after all? Are the effects of the environment often transmitted between generations regardless of the information in genes? What is a gene these days anyway? Such speculations are exhilarating, but I suggest that most biologists are not more confused than ever about the role of genes in evolution but, rather, less.