Adrian Bird

Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex

Cytosine residues in the sequence 5′CpG (cytosine–guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development, and in repression of viral genomes,. The methyl-CpG-binding proteins MeCP1 (ref. 5) and MeCP2 (ref. 6) interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner,. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

DNA methylation landscapes: provocative insights from epigenomics

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.

Methylation-induced repression--belts, braces, and chromatin.

Methyl-CpG-binding repressors presumably go where methylation takes them, but what determines where in the genome methyl-CpGs lie? Our profound ignorance of the mechanisms involved may be short-lived. Expectations have been raised by the discovery of two new mammalian cytosine DNA methyltransferases, DNMT3a and 3b, that are important in establishing embryonic methylation patterns (Okano et al. 1999xOkano, M, Bell, D.W, Haber, D.A, and Li, E. Cell. 1999; 99: 247–257Abstract | Full Text | Full Text PDF | PubMed | Scopus (2612)See all ReferencesOkano et al. 1999). The gene for each protein is essential for normal mouse development and analysis of DNAs from double mutant (Dnmt3a−/−, Dnmt3b−/−) embryos and embryonal stem cells show that de novo methylation of certain sequences is defective. DNMT1 is often considered as a maintenance methyltransferase that can complete hemimethylated sites following DNA replication, but can not transfer methyl groups to nonmethylated DNA. In vitro, however, DNMT1 can methylate nonmethylated DNA. Whether there are two or three mammalian de novo methyltransferases remains to be determined. The big question, however, is this: how are de novo methyltransferases recruited to, or excluded from, particular regions of the genome in normal cells? What dictates whether CpG islands (e.g., those on the X chromosome, at imprinted genes and other loci), repetitive DNA, viral DNA sequences, or mobile elements should either evade methylation, or succumb to it? Although the signals that cause susceptibility or resistance to methylation are still unknown, genetic approaches in plants are leading the way forward. Short inverted repeats of DNA sequence and double-stranded RNA can promote methylation of homologous sequences (Selker 1999xSelker, E.U. Cell. 1999; 97: 157–160Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesSelker 1999). Intriguingly, a SWI/SNF family member has genetic connections to DNA methylation patterns in Arabidopsis (Jeddeloh et al. 1999xJeddeloh, J.A, Stokes, T.L, and Richards, E.J. Nat. Genet. 1999; 22: 94–97CrossRef | PubMed | Scopus (444)See all ReferencesJeddeloh et al. 1999); could this mean that chromatin disruption is a prerequisite for de novo methylation in this plant? An alternative possibility is that DNA methylation is the default state of parts of the genome and that demethylation, either active or passive, is the key determinant of methylation patterns. We can look forward to exciting revelations as the knowledge vacuum in this area is filled over the next few years.

CpG islands and the regulation of transcription

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.

A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.

Rett syndrome (RTT) is an inherited neurodevelopmental disorder of females that occurs once in 10,000–15,000 births. 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 X-linked gene MECP2 (refs. 3–12), encoding a protein that binds to methylated sites in genomic DNA and facilitates gene silencing. Previous work with Mecp2-null embryonic stem cells indicated that MeCP2 is essential for mouse embryogenesis. 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.

Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins

ABSTRACT Methylation at the DNA sequence 5′-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal.

MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin

MeCP2 is an abundant mammalian protein that binds to methylated CpG. We have found that native and recombinant MeCP2 repress transcription in vitro from methylated promoters but do not repress nonmethylated promoters. Repression is nonlinearly dependent on the local density of methylation, becoming significant at the density found in bulk vertebrate genomic DNA. Transient transfection using fusions with the GAL4 DNA binding domain identified a region of MeCP2 that is capable of long-range repression in vivo. Moreover, MeCP2 is able to displace histone H1 from preassembled chromatin that contains methyl-CpG. These properties, together with the abundance of MeCP2 and the high frequency of its 2 bp binding site, suggest a role as a global transcriptional repressor in vertebrate genomes.

Purification, sequence, and cellular localization of a novel chromosomal protein that binds to Methylated DNA

Methylation of mammalian DNA can lead to repression of transcription and alteration of chromatin structure. Recent evidence suggests that both effects are the result of an interaction between the methylated sites and methyl-CpG-binding proteins (MeCPs). MeCP1 has previously been detected in crude nuclear extracts. Here we report the identification, purification, and cDNA cloning of a novel MeCP called MeCP2. Unlike MeCP1, the new protein is able to bind to DNA that contains a single methyl-CpG pair. By staining with an antibody, we show that the distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. In mouse, for example, MeCP2 is concentrated in pericentromeric heterochromatin, which contains a large fraction (about 40%) of all genomic 5-methylcytosine.

Reversal of Neurological Defects in a Mouse Model of Rett Syndrome

Rett syndrome is an autism spectrum disorder caused by mosaic expression of mutant copies of the X-linked MECP2 gene in neurons. However, neurons do not die, which suggests that this is not a neurodegenerative disorder. An important question for future therapeutic approaches to this and related disorders concerns phenotypic reversibility. Can viable but defective neurons be repaired, or is the damage done during development without normal MeCP2 irrevocable? Using a mouse model, we demonstrate robust phenotypic reversal, as activation of MeCP2 expression leads to striking loss of advanced neurological symptoms in both immature and mature adult animals.

The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation.

DNA methylation plays an important role in mammalian development and correlates with chromatin-associated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys9 of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys9 H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys9methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.