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PubMedDate not supplied

The critical DNA flanking sequences of a CpG oligodeoxynucleotide, but not the 6 base CpG motif, can be replaced with RNA without quantitative or qualitative changes in Toll-like receptor 9-mediated activity.

Double- and single-stranded oligodeoxynucleotides containing unmethylated cytosine-guanosine (CpG) dinucleotides (CpG-ODN) activate immune cells via TLR9. In this report we synthesized hybrid DNA-RNA molecules (HDR) in order to further explore the structure-immune function relationship of CpG-ODN in TLR9 signaling and the potential immunomodulatory properties of RNA. We demonstrate that replacement of the deoxyadenosine flanking sequences, critical for the immune activating properties of CpG-ODN, with a similar number of adenosines, although not guanosines, cytosines, or uracils, maintains complete immunostimulatory activity of the hybrid oligonucleotide in vitro, whereas a similar RNA replacement of even 1 base of the required unmethylated 6 base DNA motif (purine-purine-CpG-pyrimidine-pyrimidine) results in a complete loss of activity. Regardless of whether the critical flanking sequence was RNA or DNA there was no significant change in the quantitative or qualitative immune-stimulating activity, or TLR-specificity of the resulting sequences, thus underscoring the relatively permissive functional role of the flanking sequence, and the more specific role of the motif in mediating TLR9 signaling. These data further support a potential role for RNA in immunomodulation.

Animals↗
PubMed1992

An archipelago of CpG islands in Xq28: identification and fine mapping of 20 new CpG islands of the human X chromosome.

19 probes for CpG islands, mapping to Xq28, have been used as probes to construct a physical map of genes of this band of the human X chromosome. A total of 22 CpG islands have been precisely mapped in respect to known loci along the 9-10 Mb of Xq28. The fine mapping of such a large number of CpG islands has demonstrated that also in gene rich Giemsa light bands, like Xq28, gene distribution is non uniform: the CpG islands are clustered in the distal portion of the band in a 2 Mb region between the G6PD gene and the DXS15 locus. Moreover, 16 CpG islands were found between the G6PD and the RCP/GCP genes, a region of DNA of only about 300 kb. If this structural organization has a biological function it has yet to be determined. However, the isolation of large genomic regions enriched in gene sequences and the availability of cosmid or YAC contigs will provide the means to test the significance of such gene organization, as well as the material for large sequencing projects and gene search, for the identification of candidate genes for inherited disorders mapped to Xq28 and for comparative mapping.

Animals↗
PubMed2003

Sequence context at human single nucleotide polymorphisms: overrepresentation of CpG dinucleotide at polymorphic sites and suppression of variation in CpG islands.

Human polymorphisms originate as mutations, and the influence of context on mutagenesis should be reflected in the distribution of sequences surrounding single nucleotide polymorphisms (SNPs). We have performed a computational survey of nearly two million human SNPs to determine if sequence-dependent hotspots for polymorphism exist in the human genome. Here we show that sequences containing CpG dinucleotides, which occur at low frequencies in the human genome, are 6.7-fold more abundant at polymorphic sites than expected. In contrast, polymorphisms in CpG sequences located within CpG islands, important regulatory regions that modulate gene expression, are 6.8-fold less prevalent than expected. The distribution of polymorphic alleles at CpGs in CpG islands is also significantly different from that in non-island regions. These data strongly support a role for 5-methylcytosine deamination in the generation of human variation, and suggest that variation at CpGs in islands is suppressed.

5-Methylcytosine↗
PubMed2002

De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation.

DNA methyltransferases, Dnmt3a and Dnmt3b, are required for de novo methylation in embryonic stem (ES) cells and postimplantation embryos. However, the mechanism of de novo methylation is largely unknown. In this study, we have analyzed the sequence specificity of Dnmt3a and Dnmt3b during de novo methylation of murine Moloney leukemia virus provirus DNA in virus-infected ES cells. Provirus DNA from infected wild-type (J1), Dnmt1-/- (c/c), and Dnmt3a3b-/- (3a3b-/-) ES cells were analyzed using the bisulfite sequencing method. We demonstrate that Dnmt3 enzymes methylate predominantly CpG sites in vivo and confirm that Dnmt3 enzymes, but not Dnmt1, are responsible for de novo methylation. However, the sequence context and CpG density do not appear to influence de novo methylation, though strand bias is detectable. Interestingly, non-CpG methylation is detected as a component of de novo methylation. CpA methylation was detected at approximately 1.4% of all sites in J1 and approximately 1.0% in c/c, but only approximately 0.2% in 3a3b-/-. Few methylated CpT or CpC sites were detected. Similar results from nearest neighbor analysis of global endogenous methylation levels indicated a correlation between Dnmt3a and Dnmt3b presence and CpA methylation. These results demonstrate that the Dnmt3 enzymes methylate predominantly CpG sites and at a low frequency CpA sites with no apparent sequence preferences.

5-Methylcytosine↗
PubMed2004

CpG oligodeoxynucleotides induce expression of proinflammatory cytokines and chemokines in astrocytes: the role of c-Jun N-terminal kinase in CpG ODN-mediated NF-kappaB activation.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate the cells of the innate immune system through a specific receptor called Toll-like receptor-9 (TLR9). It was reported that CpG ODN stimulation induces activation of astrocytes and microglia. However, the precise intracellular signaling pathways that lead to this glial cell activation have not been clearly elucidated. In this study, we found that CpG ODN induce mRNA expression of adhesion molecules and matrix metalloproteinase-9 (MMP-9), as well as proinflammatory cytokines and chemokines, in mouse astrocytes. CpG ODN stimulation in astrocytes induces the activation of IkappaB kinase (IKK) and c-Jun N-terminal kinase (JNK), whereas it inhibits the constitutive ERK1/2 activation. The abrogation of JNK activity using a pharmacological inhibitor showed that JNK activation is essential for the induction of cytokine and chemokine gene expression. This effect of JNK does not require the phosphorylation of c-Jun; rather, it works via the potentiation of NF-kappaB signaling.

Animals↗
PubMed2006

Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2.

Vertebrates produce multiple chondroitin sulfate proteoglycans that play important roles in development and tissue mechanics. In the nematode Caenorhabditis elegans, the chondroitin chains lack sulfate but nevertheless play essential roles in embryonic development and vulval morphogenesis. However, assignment of these functions to specific proteoglycans has been limited by the lack of identified core proteins. We used a combination of biochemical purification, Western blotting, and mass spectrometry to identify nine C. elegans chondroitin proteoglycan core proteins, none of which have homologues in vertebrates or other invertebrates such as Drosophila melanogaster or Hydra vulgaris. CPG-1/CEJ-1 and CPG-2 are expressed during embryonic development and bind chitin, suggesting a structural role in the egg. RNA interference (RNAi) depletion of individual CPGs had no effect on embryonic viability, but simultaneous depletion of CPG-1/CEJ-1 and CPG-2 resulted in multinucleated single-cell embryos. This embryonic lethality phenocopies RNAi depletion of the SQV-5 chondroitin synthase, suggesting that chondroitin chains on these two proteoglycans are required for cytokinesis.

Amino Acid Sequence↗
PubMed2006

A profile of methyl-CpG binding domain protein occupancy of hypermethylated promoter CpG islands of tumor suppressor genes in human cancer.

Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.

Binding Sites↗
PubMed2001

Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis.

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs). The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues, and was even higher in HCCs. Significant overexpression of DNMT3b and reduced expression of DNMT2 were observed in HCCs compared with the corresponding noncancerous liver tissues. DNA hypermethylation on CpG islands of the p16 (8% and 66%) and hMLH1 (0% and 0%) genes and methylated in tumor (MINT) 1 (6% and 34%), 2 (24% and 58%), 12 (21% and 33%), 25 (0% and 5%), and 31 (0% and 23%) clones, and DNA hypomethylation on satellites 2 and 3 (18% and 67%), were detected in noncancerous liver tissues and HCCs, respectively. There was no significant correlation between the expression level of any DNA methyltransferase and DNA methylation status. Reduced expression of DNA repair protein, MBD4, was significantly correlated with poorer tumor differentiation and involvement of portal vein. Slightly reduced expression of MBD2 was detected in HCCs, and the expression of MeCP2 was particularly reduced in HCCs with portal vein involvement. These data suggest that overexpression of DNMT1 and DNMT3a, DNA hypermethylation on CpG islands, and DNA hypomethylation on pericentromeric satellite regions are early events during hepatocarcinogenesis, and that reduced expression of MBD4 may play a role in malignant progression of HCC.

Adult↗
PubMed1996

PCR induction of a TaqI restriction site at any CpG dinucleotide using two mismatched primers (CpG-PCR).

A final common pathway has been devised for analysis of de novo mutation at any CpG site. Artificial restriction sites can be introduced in known DNA sequences by using either or both sense and antisense mismatched PCR primers. Sitting of the primers directly adjacent 5' and 3' to the CpG site yields a 52-bp PCR product, resulting from the sum of the two 25-mer oligonucleotides plus the two intervening bases (C and G), and also yields consistent digestion fragments. Three out of four possible four-base palindromes (TaqI, HHaI, and MspI) were investigated for mutations R329X and E80K in the human LDL receptor gene, and for mutations R395W and R612C, and TaqI site was forced using PCR in which both primers had 3' mismatched T. Both empirically and on theoretical grounds, Taq1 is the forced restriction site of choice. The approach has been adapted to the high-throughput microplate diagonal gel electrophoresis (MADGE) system for selective analysis of mutations at CpG sites, which may account for 20% of all single base variation in the human genome.

Base Composition↗
PubMed2003

Treatment of tumor cells with histone deacetylase inhibitors results in altered recruitment of methyl-CpG binding proteins to a methylated CpG island.

When human cancer cells with silencing of the CDH1 gene associated with CpG island methylation and histone deacetylation were treated with histone deacetylase inhibitors, alteration in recruitment of methyl-CpG binding proteins (MBPs) to the methylated CDH1-CpG island was observed, as well as altered histone acetylation status. This change was independent of the histone deacetylase inhibitor used. These results suggest that histone hyperacetylation provides a more open chromatin structure conformation for the recruitment of additional MBPs.

Acetylation↗
PubMed2002

Differential patterns of methylation of the IFN-gamma promoter at CpG and non-CpG sites underlie differences in IFN-gamma gene expression between human neonatal and adult CD45RO- T cells.

IFN-gamma is a potent pleiotropic Th1 cytokine, the production of which is tightly regulated during fetal development. Negative control of fetal/neonatal IFN-gamma production is generally attributed to the Th1-antagonistic effect of mediators produced by the placenta, but evidence exists of additional and more direct transcriptional regulation. We report that neonatal (cord blood) CD3(+)/CD45RO(-) T cells, in particular the CD4(+)/CD45RO(-) subset, are hypermethylated at CpG and non-CpG (CpA and CpT) sites within and adjacent to the IFN-gamma promoter. In contrast, CpG methylation patterns in cord blood IFN-gamma-producing CD8(+)/CD45RO(-) T cells and CD56(+)/CD16(+)/CD3(-) NK cells did not differ significantly from those in their adult counterparts. Consistent with this finding, IFN-gamma production by stimulated naive cord blood CD4(+) T cells is reduced 5- to 10-fold relative to adult CD4(+) T cells, whereas production levels in neonatal and adult CD8(+) T cells are of a similar order. Evidence of significant CpA and CpT methylation was not discovered in promoter sequence from other cytokines (IL-4, TNF-alpha, or IFN-gammaR alpha-chain). We additionally demonstrate that overexpression of DNA methyltransferase 3a in embryonic kidney carcinoma cells is accompanied by CpA methylation of the IFN-gamma promoter.

5-Methylcytosine↗
PubMed2005

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome.

An effective tool for the global analysis of both DNA methylation status and protein-chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20,736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein-chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

Base Sequence↗
PubMed1998

Methylation status of CpG sites and methyl-CpG binding proteins are involved in the promoter regulation of the mouse Xist gene.

The mouse Xist gene is expressed exclusively from the inactive X chromosome and is involved in the initiation of X inactivation. We previously reported that the -1157/+917 region of the Xist promoter was ubiquitously functional in mammalian cells and that experiments in a transient expression system revealed no trans-acting element responsible for the inactive X specific expression of Xist. In somatic tissues, the 5' end of the silent Xist allele on the active X is known to be fully methylated whereas the expressed allele on the inactive X is unmethylated. In the present study we have used a bisulphite genomic sequencing method to evaluate DNA methylation at all cytosines including CpG dinucleotides within the Xist promoter. We report and confirm that methylation of specific sites plays a key role in Xist gene expression. In vitro DNA methylation of the 5'-region drastically reduced transcriptional activity in transiently transfected fibroblasts. Mobility shift assays showed that methylation does not inhibit Xist promoter activity by preventing the binding of transcription factors and that two distinct nuclear proteins bind in a sequence methyl-CpG-specific manner. Therefore, we suggest that Xist repression involves its promoter methylation and two distinct methylated DNA binding proteins.

Animals↗
PubMed1992

Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein.

Repression of transcription from densely methylated genes can be mediated by the methyl-CpG binding protein MeCP-1 (Boyes and Bird, 1991). Here we have investigated the effect of methylation on genes with a low density of methyl-CpG. We found that sparse methylation could repress transfected genes completely, but the inhibition was fully overcome by the presence in cis of an SV40 enhancer. Densely methylated genes, however, could not be reactivated by the enhancer. In vitro studies showed that the sparsely methylated genes bound weakly to MeCP-1 and that binding interfered with transcription. In the absence of available MeCP-1, methylation had minimal effects on transcription. From these and other results we propose that sparsely methylated genes form an unstable complex with MeCP-1 which prevents transcription when the promoter is weak. This complex can be disrupted by a strong promoter, thereby allowing the methylated gene to be transcribed.

Animals↗
PubMed1990

Establishment of de novo DNA methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter.

The establishment of de novo patterns of DNA methylation in mammalian genomes is characterized by the gradual spreading of methylation, which has been documented to occur across an entire integrated adenovirus genome as well as at the nucleotide level in the integrated late E2A promoter of adenovirus type 2. By applying the techniques of genomic sequencing and dimethylsulfate or DNase I genomic footprinting in vivo, we have now demonstrated that the spreading of methylation in cell lines that carry the late E2A promoter with three in vitro pre-methylated 5'-CCGG-3' sequences initially involves a DNA domain of this promoter that is devoid of bound proteins. Subsequently, methylation further spreads to neighboring regions, and the patterns of complexed transcription factors are altered. Evidence has been adduced that DNA methylation at sequences homologous to the AP-1 and octamer binding factor sites interferes with protein binding. In contrast, the methylation of sequences in the vicinity of but not involving sequences homologous to an AP-2 site still permits the binding of proteins to these sites. It is significant that during the spreading of methylation a few 5'-CG-3' sequences can remain hemimethylated for several cell generations, before they also become methylated in both complements. Moreover, in cell line HE2, the integrated, heavily methylated late E2A promoter has been shown by the genomic sequencing technique to contain 5-methyldeoxycytidine residues, not only in all 5'-CG-3' dinucleotides but also in a 5'CA-3' and a 5'-CT-3' dinucleotide sequence. Hence, 5-methyldeoxycytidine occurs in a silenced mammalian DNA sequence also in dinucleotides other than 5'-CG-3'. This finding raises the question of whether 5-methyldeoxycytidine in non-5'-CG-3' dinucleotides can be maintained in the methylated state during continuous cell propagation.

Adenoviridae↗