Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

Study of the Alzheimer's A4 precursor gene promoter region by genomic sequencing using Taq polymerase

The beta-amyloid protein has been identified as the prominent component of the fibrillary aggregates of the neuritic plaques found in Alzheimer's disease (AD). In this paper, the DNA methylation pattern of the promoter region of the Alzheimer's disease amyloid precursor gene (PAD) was assessed using the recently developed genomic sequencing technique with Taq polymerase. We analyzed seven potential methylation sites between position -460 and -275 of the PAD promoter. Three of the CpG dinucleotides we analyzed are located in the flanking regions of the AP-1 binding site and heat-shock response element consensus sequences. Of the seven CpG dinucleotides present in this region, we found none to be methylated. This finding indicates that, in healthy brain tissue, cytosine methylation of this binding motif seems not to affect protein/DNA interaction. However, it remains to be determined whether methylation of these sites is significant in AD patients.     

A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.     

DNA methylation cues in nucleosome geometry,stability and unwrapping

Cytosine methylation at the 5-carbon position is an essential DNA epigenetic mark in many eukaryotic organisms. Although countless structural and functional studies of cytosine methylation have been reported, our understanding of how it influences the nucleosome assembly, structure, and dynamics remains obscure. Here, we investigate the effects of cytosine methylation at CpG sites on nucleosome dynamics and stability. By applying long molecular dynamics simulations on several microsecond time scale, we generate extensive atomistic conformational ensembles of full nucleosomes. Our results reveal that methylation induces pronounced changes in geometry for both linker and nucleosomal DNA, leading to a more curved, under-twisted DNA, narrowing the adjacent minor grooves, and shifting the population equilibrium of sugar-phosphate backbone geometry. These DNA conformational changes are associated with a considerable enhancement of interactions between methylated DNA and the histone octamer, doubling the number of contacts at some key arginines. H2A and H3 tails play important roles in these interactions, especially for DNA methylated nucleosomes. This, in turn, prevents a spontaneous DNA unwrapping of 3–4 helical turns for the methylated nucleosome with truncated histone tails, otherwise observed in the unmethylated system on several microseconds time scale.     

Role of epigenetic DNA alterations in the pathogenesis of systemic lupus erythematosus

Epigenetic alternations in genomic DNA encompass cytosine methylation in cytosine and guanine (CpG) dinucleotide islands, which are usually extended in the promoter and first exon of genes. The DNA methylation is carried out by DNA methyltransferases (DNMT) and it serves as an epigenetic method of gene expression modulation. The epigenetic alternations in genomic DNA have been implicated in the development of malignant and autoimmune diseases. The epigenetic aberration in regulatory DNA sequences may also be responsible for the emergence of changes in the immune system in patients with systemic lupus erythematosus (SLE). The agents 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) belong to inhibitors of methyltransferase. These compounds affect the methylation level of promoter sequences and cause phenotypic changes in peripheral blood mononuclear cells (PBMC), which are similar to those observed in PBMC of SLE patients. The lack of methylcytosine in CpG dinucleotides may be responsible for the antigenic properties of microbial DNA. The presence of low-apoptotic methylated DNA fragments has been identified in plasma of SLE patients. These DNA fragments exhibit antigenic properties and may elicit the humoral response responsible for the flare of SLE. The low methylation of CpG residues in the regulatory sequences may also contribute to the elevated expression of human endogenous retroviruses (HERVs) in PBMC of SLE patients. The HERV components exhibit a profound similarity with nuclear antigens and may be responsible for the enhancement of the production of anti-antinuclear antibodies (ANA). Recent advances in the investigation of epigenetic DNA changes have formed the basis of improved understanding of etiopathogenesis of SLE, which may thereby facilitate improvement in therapeutic principles of this disease.     

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.     

The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression.

We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA.