Dynamics of Nucleosome Assembly and Effects of DNA Methylation

The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)₂ tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)₂ tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA.     

Understanding the Connection between Epigenetic DNA Methylation and Nucleosome Positioning from Computer Simulations

Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.     

Study of Tissue-Specific CpG Methylation of DNA in Extended Genomic Loci

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.     

Effect of CpG Island Methylation on MicroRNA Expression in the k-562 Cell Line

To test the hypothesis that methylation of a CpG island is associated with regulation of microRNA expression, we investigated CpG islands in the upstream sequences of microRNA precursors (pre-miRNAs) through bioinformatic analysis and determined whether the CpG islands were methylated by methylation-specific PCR in the k-562 cell line. We used 5-azacytidine for DNA demethylation, and changes in microRNA expression were detected by microarray assay, RT-PCR, and real-time PCR after 5-azacytidine induction. We showed that the CpG islands in the upstream regions of 18 pre-miRNAs were methylated, including miR-663, miR-369, miR-615, and miR-410, and promoter activity was detected in the upstream region of pre-miR-663. We found that a decrease in methylation of a CpG island could up-regulate the expression of miR-663, suggesting that miR-663 could be regulated by DNA methylation. Expression levels of miR-369, miR-615, and miR-410 were not regulated by DNA methylation in this cell line.     

The loss of CpG dinucleotides from DNA. III. Methylation and evolution of histone genes

From nucleotide sequences of more than 70 histones genes in 15 species of eucaryotes the probable frequency was determined for CpG----TpG + CpA substitutions, occurring as a result of deamination of 5-methylcytosine residues in DNA. It was found that histone genes differ in the character of CpG methylation with respect to the species studied and may be divided into three groups differing in the value of CpG suppression. In one of them, M-, CpG dinucleotides must have not been methylated throughout the existence of these genes; in another, M+, nearly every other CpG has undergone transition. In the third group, M +/-, no more than 20% of CpG have steadily undergone methylation (and mutation). The CpG deficiency in M+ and M +/- histone genes is in general proportional to the level of methylation of total DNA in different species. It has been noted that the genes of different core histones in the same organism are characterized, as a rule, by the same type of CpG methylation and belong to the same group. Genes H1 and H5 show a higher level of CpG suppression and thus have a higher degree of methylation than the genes of core histones from the same organism. The most conserved among the histone genes, those for H3 and H4 in particular, must have not been methylated in the majority of the species studied. The distribution of methylated and non-methylated spacers and coding sequences of histone genes of man, mouse, hen and yeast reveals a mosaic pattern. It has been found that 5'-flanked regions in most cases are methylated more than respective genes, while the G + C content in them is significantly lower, compared with the coding gene sequences. The absence of methylation in the 5'-regulatory regions does not appear to be mandatory for histone genes. It has been established that the genes of the same histones may differ in the level of methylation even in more or less closely related species. Group M- comprises genes of core histones of man, hen, sea urchin, Drosophila, Neurospora and wheat; group M +/- includes analogous genes of mouse, Xenopus, trout and sea urchins. The results obtained testify against the possible universal involvement of methylation in the regulation of histone gene expression.     

Salt-Induced Conformation and Interaction Changes of Nucleosome Core Particles

Small angle x-ray scattering was used to follow changes in the conformation and interactions of nucleosome core particles (NCP) as a function of the monovalent salt concentration C_s. The maximal extension (D_max) of the NCP (145 ± 3-bp DNA) increases from 137 ± 5 Å to 165 ± 5 Å when C_s rises from 10 to 50 mM and remains constant with further increases of C_s up to 200 mM. In view of the very weak increase of the R_g value in the same C_s range, we attribute this D_max variation to tail extension, a proposal confirmed by simulations of the entire I(q) curves, considering an ideal solution of particles with tails either condensed or extended. This tail extension is observed at higher salt values when particles contain longer DNA fragments (165 ± 10 bp). The maximal extension of the tails always coincides with the screening of repulsive interactions between particles. The second virial coefficient becomes smaller than the hard sphere virial coefficient and eventually becomes negative (net attractive interactions) for NCP₁₄₅. Addition of salt simultaneously screens Coulombic repulsive interactions between NCP and Coulombic attractive interactions between tails and DNA inside the NCP. We discuss how the coupling of these two phenomena may be of biological relevance.     

DNA甲基化与组蛋白修饰对克隆胚发育的影响

在正常的受精、发育过程中,基因组DNA不对称的去甲基化、重新甲基化以及组蛋白修饰作用发生在整个受精和胚胎发育过程中。本文将从DNA甲基化、DNA不对称的去甲基化和组蛋白修饰作用就其原理,相互之间的关系及其对胚胎发育情况的影响作以综述,并对近年来,DNA甲基化与组蛋白修饰作用在胚胎发育过程中的研究作以总结。     

Regionally Specific and Genome-Wide Analyses Conclusively Demonstrate the Absence of CpG Methylation in Human Mitochondrial DNA

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.     

Positioning of a Nucleosome on Mouse Satellite DNA Inserted into a Yeast Plasmid Is Determined by Its DNA Sequence and an Adjacent Nucleosome

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.     

Effect of DNA Local Conformation on the Affinity and Binding Specificity of bis-Netropsins to DNA

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked tail-to-tail via cis-diammineplatinum group (Nt–Pt(NH ₃ )–Nt) or aliphatic pentamethylene chain (Nt–(CH ₂ ) ₅ –Nt), has been studied. Both bis-netropsins have been shown to bind to DNA oligomer 5"-CCTATATCC-3" (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5"-CCXCC-3" [where X = TTATT (II), TTAT (III), TTTTT (IV), and AATTT (V)] along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of Nt–(CH ₂ ) ₅ –Nt with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin ( ₂) to monodentate binding constant ( ₁) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for Nt–Pt(NH ₃ )–Nt and Nt–(CH ₂ ) ₅ –Nt binding as a hairpin has been found to be DNA duplex 5"-CGTATACG-3".     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.