Biomedical subjects

Richard R Meehan

Publications and source records attributed to Richard R Meehan.

10 recordsLinked to original sources

PubMed2006

Methylation-sensitive polymerase chain reaction.

Here, we describe a robust and reproducible methylation-sensitive polymerase chain reaction (MS-PCR) method to detect the percentage methylation in repeat sequences of individual pre-implantation ovine embryos produced by different embryo technologies. This method allows the comparison of embryos produced by nuclear transfer with other production and embryo culture methods, accounting for the heterogeneity between embryos within a single treatment. DNA extracted from single embryos is digested with a methylation-sensitive restriction enzyme to determine the percentage methylation after PCR amplification in comparison with an undigested control. The undigested control represents 100% methylation because methylation-sensitive enzymes do not cut methylated DNA, allowing the entire sample to be amplified by PCR. Image analysis quantification of the digested subsample PCR product on an ethidium bromide-stained agarose gel is proportional to the amount of methylated DNA in each embryo. By comparing quadruplicate values obtained for each embryo against a standard curve, we are able to ensure the validity of our results for each individual embryo. Compared with bisulphite sequencing methods, the method described is rapid, inexpensive, and relatively high-throughput.

Animals↗

PubMed2005

Epigenetic silencing in embryogenesis.

Transcription profiling of early embryos emphasizes that differential gene expression is a fundamental control mechanism of development. Precise regulatory mechanisms operate on a background of epigenetic changes in chromatin composition, modification, and architecture that are integral for the development of a pluripotent embryo into an adult.

Animals↗

PubMed2004

Kaiso is a genome-wide repressor of transcription that is essential for amphibian development.

DNA methylation in animals is thought to repress transcription via methyl-CpG specific binding proteins, which recruit enzymatic machinery promoting the formation of inactive chromatin at targeted loci. Loss of DNA methylation can result in the activation of normally silent genes during mouse and amphibian development. Paradoxically, global changes in gene expression have not been observed in mice that are null for the methyl-CpG specific repressors MeCP2, MBD1 or MBD2. Here, we demonstrate that xKaiso, a novel methyl-CpG specific repressor protein, is required to maintain transcription silencing during early Xenopus laevis development. In the absence of xKaiso function, premature zygotic gene expression occurs before the mid-blastula transition (MBT). Subsequent phenotypes (developmental arrest and apoptosis) strongly resemble those observed for hypomethylated embryos. Injection of wild-type human kaiso mRNA can rescue the phenotype and associated gene expression changes of xKaiso-depleted embryos. Our results, including gene expression profiling, are consistent with an essential role for xKaiso as a global repressor of methylated genes during early vertebrate development.

Animals↗

PubMed2004

A determining influence for CpG dinucleotides on nucleosome positioning in vitro.

DNA sequence information that directs the translational positioning of nucleosomes can be attenuated by cytosine methylation when a short run of CpG dinucleotides is located close to the dyad axis of the nucleosome. Here, we show that point mutations introduced to re-pattern methylation at the (CpG)3 element in the chicken betaA-globin promoter sequence themselves strongly influenced nucleosome formation in reconstituted chromatin. The disruptive effect of cytosine methylation on nucleosome formation was found to be determined by the sequence context of CpG dinucleotides, not just their location in the positioning sequence. Additional mutations indicated that methylation can also promote the occupation of certain nucleosome positions. DNase I analysis demonstrated that these genetic and epigenetic modifications altered the structural characteristics of the (CpG)3 element. Our findings support a proposal that the intrinsic structural properties of the DNA at the -1.5 site, as occupied by (CpG)3 in the nucleosome studied, can be decisive for nucleosome formation and stability, and that changes in anisotropic DNA bending or flexibility at this site explain why nucleosome positioning can be exquisitely sensitive to genetic and epigenetic modification of the DNA sequence.

Animals↗

PubMed2004

The effect of interspecific oocytes on demethylation of sperm DNA.

In contrast to mice, in sheep no genome-wide demethylation of the paternal genome occurs within the first postfertilization cell cycle. This difference could be due either to an absence of a sheep demethylase activity that is present in mouse ooplasm or to an increased protection of methylated cytosine residues in sheep sperm. Here, we use interspecies intracytoplasmic sperm injection to demonstrate that sheep sperm DNA can be demethylated in mouse oocytes. Surprisingly, mouse sperm can also be demethylated to a limited extent in sheep oocytes. Our results suggest that the murine demethylation process is facilitated either by a sperm-derived factor or by male pronuclear chromatin composition.

Animals↗

PubMed2003

Molecular cloning and developmental expression of two Chloride Intracellular Channel (CLIC) genes in Xenopus laevis.

CLIC proteins are components or regulators of novel intracellular anion channels in mammalian cells, and previous studies have suggested that human nuclear membrane-associated CLIC1 and mouse inner mitochondrial membrane CLIC4 are involved in cell division and apoptosis. We have isolated Xenopus homologues of CLIC1 and CLIC4 and shown them to be well conserved during chordate evolution, but poorly conserved in invertebrates. Consistent with fundamental cellular roles, Xenopus CLIC genes are expressed at every stage of embryonic development. Expression is localised to mesodermal and ectodermal tissues, with particularly marked expression of xCLIC4 in the developing nervous system. This is the first description of non-mammalian CLIC expression, and use of Xenopus laevis as a model organism may provide insights into the role of CLIC-associated ion channels in animal development.

Amino Acid Sequence↗

PubMed2003

HP1 binding to native chromatin in vitro is determined by the hinge region and not by the chromodomain.

We have isolated the complete coding sequences for two Xenopus laevis isoforms of heterochromatin protein 1, corresponding to HP1alpha and HP1gamma. The sequence of xHP1alpha shows considerable divergence from its mammalian homologues, whereas xHP1gamma is highly conserved. Functionally, xHP1alpha behaves identically to human HP1alpha. We observe unexpected differences between the two HP1 variants in binding native soluble chromatin, which seem to correlate with their distinct nuclear distributions in vivo. A surprising finding is that the characteristic interaction of HP1 chromodomains with histone H3 at methylated lysine 9 is not detected in preformed chromatin due to its inaccessibility. Instead, we localize a strong chromatin-binding activity to the short hinge region between the chromodomain and the chromoshadow domain of xHP1alpha but not xHP1gamma. This novel chromatin-binding activity has a non-specific DNA-binding component in addition to a linker histone-dependent preference for an altered chromatin structure with a likely heterochromatin organization.

Amino Acid Sequence↗

PubMed2003

DNA methylation in animal development.

Nuclear transfer experiments have demonstrated that epigenetic mechanisms operate to limit gene expression during animal development. In somatic cells, silenced genes are associated with defined chromatin states which are characterised by hypermethylation of DNA, hypoacetylation of histones and specific patterns of methylation at distinct residues of the N-terminal tails of histone H3 and H4. This review describes the role of the DNA methylation-mediated repression system (Dnmt1's, MeCPs and MBDs and associated chromatin remodelling activities) in animal development. DNA methylation is essential for normal vertebrate development but has distinct regulatory roles in non-mammalian and mammalian vertebrates. In mammals, DNA methylation has an additional role in regulating imprinting. This suggests that epigenetic regulation is plastic in its application and should be considered in a developmental context that may be species specific.

Animals↗

PubMed2003

A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos.

MeCP2 is a DNA binding protein that represses transcription of methylated genes in vitro, but the endogenous function of MeCP2 in vivo is unclear. Here, we demonstrate that in Xenopus laevis embryos MeCP2 is a partner of the SMRT corepressor complex that regulates the expression of a neuronal repressor xHairy2a in differentiating neuroectoderm. The MeCP2/SMRT complex is bound to the promoter of the silenced xHairy2a gene and is displaced upon activation by the Notch intracellular domain (NICD). A truncated form of MeCP2 (R168X) found in patients with Rett syndrome cannot interact with the SMRT complex or fully activate xHairy2a during primary neurogenesis. This disruption of MeCP2 activity results in abnormal patterning of primary neurons during neuronal differentiation. Our results support a model whereby the dynamic association of MeCP2 with methylated DNA and the SMRT complex regulates a gene involved in cell fate decisions during primary neurogenesis in Xenopus.

Animals↗

PubMed2002

DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos.

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.

Animals↗