DNA methylation regulates tissue-specific expression of Shank3

Tissue-specific gene expression can be controlled by epigenetic modifications such as DNA methylation. SHANK3, together with its homologues SHANK1 and SHANK2, has a central functional and structural role in excitatory synapses and is involved in the human chromosome 22q13 deletion syndrome. In this report, we show by DNA methylation analysis in lymphocytes, brain cortex, cerebellum and heart that the three SHANK genes possess several methylated CpG boxes, but only SHANK3 CpG islands are highly methylated in tissues where protein expression is low or absent and unmethylated where expression is present. SHANK3 protein expression is significantly reduced in hippocampal neurons after treatment with methionine, while HeLa cells become able to express SHANK3 after treatment with 5-Aza-2'-deoxycytidine. Altogether, these data suggest the existence of a specific epigenetic control mechanism regulating SHANK3, but not SHANK1 and SHANK2, expression.     

Estimation of DNA sequence diversity in bovine cytokine genes

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available. Received: 16 June 2000 / Accepted: 23 August 2000     

Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome

Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele-specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 23 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence-dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.     

Analysis of tissue-specific methylation patterns of mouse mammary tumor virus DNA by two-dimensional Southern blotting.

We used a two-dimensional Southern blotting procedure to analyze the tissue-specific methylation patterns of the five endogenous mouse mammary tumor viruses in the GR/A mouse strain. Our findings suggest that in certain tissues (brain, kidney, and liver) all proviruses are extensively methylated. In other tissues (spleen, placenta, and testes) all proviruses are hypomethylated to some degree. In these tissues individual proviruses display both quantitative and qualitative differences in methylation. We interpret the general patterns of tissue-specific hypomethylation in terms of a "hitch-hiker" model: mouse mammary tumor virus proviral methylation patterns reflect the tissue-specific activity of neighboring sequences. The observation that certain sites on particular proviruses are differentially methylated in a tissue-specific fashion may reflect tissue-specific differences in the makeup or conformation, or both, of proviral-containing chromatin.     

The chromosomal integration site determines the tissue-specific methylation of mouse mammary tumour virus proviral genes.

Multiple endogenous mouse mammary tumour virus (MMTV) proviral genes are present at different chromosomal locations in inbred mouse strains. Proviral DNA methylation is location and tissue specific. The methylation patterns are stably inherited and appear to be conferred upon the viral DNA by the flanking mouse genomic DNA. In transformed cells, either mammary carcinoma cells, or cells immortalized by SV40 in vitro, the stable pattern of methylation is lost. Although hypomethylation of proviral genes, both in normal and in transformed tissue, accompanies MMTV-specific RNA expression, it is also observed in non-expressing tissues.     

High DNA sequence diversity in pericentromeric genes of the plant Arabidopsis lyrata

Differences in neutral diversity at different loci are predicted to arise due to differences in mutation rates and from the "hitchhiking" effects of natural selection. Consistent with hitchhiking models, Drosophila melanogaster chromosome regions with very low recombination have unusually low nucleotide diversity. We compared levels of diversity from five pericentromeric regions with regions of normal recombination in Arabidopsis lyrata, an outcrossing close relative of the highly selfing A. thaliana. In contrast with the accepted theoretical prediction, and the pattern in Drosophila, we found generally high diversity in pericentromeric genes, which is consistent with the observation in A. thaliana. Our data rule out balancing selection in the pericentromeric regions, suggesting that hitchhiking is more strongly reducing diversity in the chromosome arms than the pericentromere regions.     

DNA methylation and histone acetylation of rat methionine adenosyltransferase 1A and 2A genes is tissue-specific

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet). In mammals MAT activity derives from two separate genes which display a tissue-specific pattern of expression. While MAT1A is expressed only in the adult liver, MAT2A is expressed in non-hepatic tissues. The mechanisms behind the selective expression of these two genes are not fully understood. In the present report we have evaluated MAT1A and MAT2A methylation in liver and in other tissues, such as kidney, by methylation-sensitive restriction enzyme digestion of genomic DNA. Our data indicate that MAT1A is hypomethylated in liver and hypermethylated in non-expressing tissues. The opposite situation is found for MAT2A. Additionally, histones associated to MAT1A and MAT2A genes showed enhanced levels of acetylation in expressing tissues (two-fold for MAT1A and 3.5-fold for MAT2A liver and kidney respectively). These observations support a role for chromatin structure and its modification in the tissue-specific expression of both MAT genes.     

CpG islands in genes showing tissue-specific expression

Patterns of DNA methylation at CpG dinucleotides and their relations with gene expression are complex. Methylation-free CpG clusters, so-called HTF islands, are most often associated with the promoter regions of housekeeping genes, whereas genes expressed in a single-cell type are usually deficient in these sequences. However, in the human carbonic anhydrase (CA) gene family, both the ubiquitously expressed CAII and the muscle specific CAIII appear to have such CpG islands although erythrocyte-specific CAI does not. The CAII island is quantitatively more CpG rich than that of CAIII, with a CpG:GpC ratio of 0.94 compared with 0.82 for CAIII. Estimation of CpG:GpC ratios in the proximal-promoter regions of 44 vertebrate genes suggest that 40% of genes with tissue-specific or limited tissue distribution may show methylation-free CpG clusters in their promoter regions. In many cases the CpG:GpC ratio is less than that found in housekeeping genes and this may reflect variation in the interaction of CpG clusters with regulatory factors that define different patterns of tissue expression.