Nanopores suggest a negligible influence of CpG methylation on nucleosome packaging and stability.
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ABSTRACT:
SUBMITTER: Langecker M
PROVIDER: S-EPMC4296928 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.
| S-EPMC3597673 | biostudies-literature
Project description: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.
| S-EPMC514372 | biostudies-literature
Project description:Cytosine methylation, predominantly of the CpG sequence in vertebrates, is one of the major epigenetic modifications crucially involved in the control of gene expression. Due to the difficulty of reconstituting site-specifically methylated nucleosomal DNA at crystallization quality, most structural analyses of CpG methylation have been performed using chemically synthesized oligonucleotides, There has been just one recent study of nucleosome core particles (NCPs) reconstituted with nonpalindromic human satellite 2-derived DNAs. Through the preparation of a 146-bp palindromic α-satellite-based nucleosomal DNA containing four CpG dinucleotide sequences and its enzymatic methylation and restriction, we reconstituted a 'symmetric' human CpG-methylated nucleosome core particle (NCP). We solved the crystal structures of the CpG-methylated and unmodified NCPs at 2.6 and 3.0 Å resolution, respectively. We observed the electron densities of two methyl groups, among the eight 5-methylcytosines introduced in the CpG-fully methylated NCP. There were no obvious structural differences between the CpG-methylated 'symmetric NCP' and the unmodified NCP. The preparation of a crystallization-grade CpG-methylated NCP provides a platform for the analysis of CpG-methyl reader and eraser proteins.
| S-EPMC4865653 | biostudies-literature
Project description: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.
| S-EPMC8881801 | biostudies-literature
Project description:DNA methylation is an important epigenetic mark that is known to induce chromatin condensation and gene silencing. We used a time-domain fluorescence lifetime measurement to quantify the effects of DNA hypermethylation on the conformation and dynamics of a nucleosome. Nucleosomes reconstituted on an unmethylated and a methylated DNA both exhibit dynamic conformations under physiological conditions. The DNA end breathing motion and the H2A-H2B dimer destabilization dominate the dynamic behavior of nucleosomes at low to medium ionic strength. Extensive DNA CpG methylation, surprisingly, does not help to restrain the DNA breathing motion, but facilitates the formation of a more open nucleosome conformation. The presence of the divalent cation, Mg(2+), essential for chromatin compaction, and the methyl donor molecule SAM, required for DNA methyltransferase reaction, facilitate the compaction of both types of nucleosomes. The difference between the unmethylated and the methylated nucleosome persists within a broad range of salt concentrations, but vanishes under high magnesium concentrations. Reduced DNA backbone rigidity due to the presence of methyl groups is believed to contribute to the observed structural and dynamic differences. The observation of this study suggests that DNA methylation alone does not compact chromatin at the nucleosomal level and provides molecular details to understand the regulatory role of DNA methylation in gene expression.
| S-EPMC3525854 | biostudies-literature
Project description:Promoter CpG island hypermethylation of tumor suppressor genes is an epigenetic hallmark of human cancer commonly associated with nucleosome occupancy and the transcriptional silencing of the neighboring gene. Nucleosomes can determine the underlying DNA methylation status. Herein, we show that the opposite is also true: DNA methylation can determine nucleosome positioning. Using a cancer model and digital nucleosome positioning techniques, we demonstrate that the induction of DNA hypomethylation events by genetic (DNMT1/DNMT3B deficient cells) or drug (a DNA demethylating agent) approaches is associated with the eviction of nucleosomes from previously hypermethylated CpG islands of tumor suppressor genes. Most importantly, the establishment of a stable cell line that restores DNMT1/DNMT3B deficiency shows that nucleosomes reoccupy their positions in de novo methylated CpG islands. Finally, we extend these results to the genomic level, combining a DNA methylation microarray and the nucleosome positioning technique. Using this global approach, we observe the dependency of nucleosome occupancy upon the DNA methylation status. Thus, our results suggest that there is a close association between hypermethylated CpG islands and the presence of nucleosomes, such that each of these epigenetic mechanisms can determine the recruitment of the other.
| S-EPMC3898323 | biostudies-other
Project description:Cytosine methylation within the 5'-C-phosphate-G-3' sequence of nucleotides (called CpG methylation) is a well-known epigenetic modification of genomic DNA that plays an important role in gene expression and development. CpG methylation is likely to be altered in the CpG islands. CpG islands are rich in cytosine, forming a structure called the i-motif via cytosine-cytosine hydrogen bonding. However, little is known about the effect of CpG methylation on the i-motif. In this study, The CpG methylation-induced structural changes on the i-motif was examined by thermal stability, circular dichroism (CD) spectroscopy, and native-polyacrylamide gel electrophoresis (Native-PAGE) evaluation of five i-motif-forming DNAs from four cancer-related genes (VEGF, C-KIT, BCL2, and HRAS). This research shows that CpG methylation increased the transitional pH of several i-motif-forming DNAs and their thermal stability. When examining the effect of CpG methylation on the i-motif in the presence of opposite G4-forming DNAs, CpG methylation influenced the proportion of G4 and i-motif formation. This study showed that CpG methylation altered the stability and structure of the i-motif in CpG islands.
| S-EPMC9223787 | biostudies-literature
Project description:Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.
| S-EPMC3196883 | biostudies-literature
Project description:Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted high-throughput nucleosome reconstitution experiments on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The DNA loci from both species contained the genes that encode the serum albumin family, the MYC protein, and the tumor suppressor p53. The results demonstrated that a small subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. We propose that these features serve to enhance the affinity of methylated DNA for the histone octamer and that this effect could be involved in gene regulatory mechanisms such as silencing. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin. For the human data, there were 3 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, and an unmethylated sonicated control. For the mouse data, there were 2 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, a methylated MNase control, and an unmethylated sonicated control.
2013-01-28 | E-GEOD-37224 | biostudies-arrayexpress
Project description:Cytosine methylation is a widespread epigenetic regulation mechanism. In healthy mature cells, methylation occurs at CpG dinucleotides within promoters, where it primarily silences gene expression by modifying the binding affinity of transcription factors to the promoters. Conversely, a recent study showed that in stem cells and cancer cell precursors, methylation also occurs at non-CpG pairs and involves introns and even gene bodies. The epigenetic role of such methylations and the molecular mechanisms by which they induce gene regulation remain elusive. The topology of both physiological and aberrant non-CpG methylation patterns still has to be detailed and could be revealed by using the differential stability of the duplexes formed between site-specific oligonucleotide probes and the corresponding methylated regions of genomic DNA. Here, we present a systematic study of the thermal stability of a DNA oligonucleotide sequence as a function of the number and position of non-CpG methylation sites. The melting temperatures were determined by monitoring the fluorescence of donor-acceptor dual-labelled oligonucleotides at various temperatures. An empirical model that estimates the methylation-induced variations in the standard values of hybridization entropy and enthalpy was developed.
| S-EPMC4678853 | biostudies-literature