Biomedical subjects

Adrian P Bird

Publications and source records attributed to Adrian P Bird.

11 recordsLinked to original sources

PubMed2006

Engineering a high-affinity methyl-CpG-binding protein.

Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.

Animals↗

PubMed2006

Genomic DNA methylation: the mark and its mediators.

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.

Animals↗

PubMed2005

Cutting edge: a critical role for gene silencing in preventing excessive type 1 immunity.

Immunity often depends on proper cell fate choice by helper T lymphocytes. A naive cell, with minimal expression of IFN-gamma and IL-4, must give rise to progeny expressing high levels of either one, but not both, of those cytokines to defend against protozoan and helminthic pathogens, respectively. In the present study, we show that inactivation of the Mbd2 gene, which links DNA methylation and repressed chromatin, results in enhanced resistance to the protozoan parasite Leishmania major but impaired immunity to the intestinal helminth Trichuris muris. Helper T cells from methyl CpG-binding domain protein-2-deficient mice exhibit exuberant patterns of cytokine expression despite appropriate silencing of genes encoding the lineage-specifying factors T-bet and GATA-3. These results suggest that gene silencing can facilitate the ability of a progenitor cell to give rise to appropriately differentiated daughter cells in vivo. These findings also point to novel pathways that could participate in genetic control of resistance to infection and autoimmunity.

Animals↗

PubMed2005

DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG.

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.

Adenine↗

PubMed2005

Female-specific hyperacetylation of histone H4 in the chicken Z chromosome.

Birds undergo genetic sex determination using a ZW sex chromosome system. Although the avian mechanisms of neither sex determination nor dosage compensation are understood, a female-specific non-coding RNA (MHM) is expressed soon after fertilisation from the single Z chicken chromosome and is likely to have a role in one or both processes. We have now discovered a prominent female-specific modification to the Z chromatin in the region of the MHM locus. We find that chicken chromatin at Zp21, including the MHM locus, is strongly enriched for acetylation of histone H4 at lysine residue 16 in female but not male chromosomes. Interestingly, this specific histone modification is also enriched along the length of the up-regulated Drosophila melanogaster male X chromosome where it plays a vital role in the dosage compensation process.

Acetylation↗

PubMed2004

MeCP2 behaves as an elongated monomer that does not stably associate with the Sin3a chromatin remodeling complex.

MeCP2 is a transcription factor that recognizes and binds symmetrically methylated CpG dinucleotides to repress transcription. MeCP2 can associate with the Sin3a/histone deacetylase corepressor complex and mediate repression in a histone deacetylase-dependent manner. In extracts from rodent tissues, cultured cells, and Xenopus laevis oocytes, we find that only a small amount of mammalian MeCP2 interacts with Sin3a and that this interaction is not stable. Purification of rat brain MeCP2 (53 kDa) indicates no associated proteins despite an apparent molecular mass by size exclusion chromatography of 400-500 kDa. Biophysical analysis demonstrated that the large apparent size was not because of homo-multimerization, as MeCP2 consistently behaves as a monomeric protein that has an elongated shape. Our findings indicate the MeCP2 is not an obligate component of the Sin3a corepressor complex and may therefore engage a more diverse range of cofactors for repressive function.

Animals↗

PubMed2004

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains.

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.

Alternative Splicing↗

PubMed2003

MBD1, MBD2 and CGBP genes at chromosome 18q21 are infrequently mutated in human colon and lung cancers.

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.

Chromosomes, Human, Pair 18↗

PubMed2003

The methylated component of the Neurospora crassa genome.

Cytosine methylation is common, but not ubiquitous, in eukaryotes. Mammals and the fungus Neurospora crassa have about 2-3% of cytosines methylated. In mammals, methylation is almost exclusively in the under-represented CpG dinucleotides, and most CpGs are methylated whereas in Neurospora, methylation is not preferentially in CpG dinucleotides and the bulk of the genome is unmethylated. DNA methylation is essential in mammals but is dispensable in Neurospora, making this simple eukaryote a favoured organism in which to study methylation. Recent studies indicate that DNA methylation in Neurospora depends on one DNA methyltransferase, DIM-2 (ref. 6), directed by a histone H3 methyltransferase, DIM-5 (ref. 7), but little is known about its cellular and evolutionary functions. As only four methylated sequences have been reported previously in N. crassa, we used methyl-binding-domain agarose chromatography to isolate the methylated component of the genome. DNA sequence analysis shows that the methylated component of the genome consists almost exclusively of relics of transposons that were subject to repeat-induced point mutation--a genome defence system that mutates duplicated sequences.

Chromatography, Affinity↗

PubMed2002

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 Lys(9) of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys(9) 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 Lys(9) methylation. 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.

Animals↗

PubMed2002

Gene silencing quantitatively controls the function of a developmental trans-activator.

How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.

Animals↗