Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases

Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.     

Variability in allelic DNA methylation in spermatozoa

In certain segments of human DNA, the methylation of deoxycytidine residues has been found to be highly specific and interindividually conserved. Imprinted DNA sequences in diploid primary cells show allele-specific differences in DNA methylation, usually with the active chromosomal regions being unmethylated and the inactive regions being methylated. We show here that DNA from spermatozoa exhibits variations in allelic methylation patterns. Since germ cells are haploid, individual spermatozoa can differ in DNA methylation patterns not only in the maternally or paternally derived allele, but also within each allele.     

The biochemical and cytological characterization of Vicia faba DNA by means of MboI,AluI and Bam HI restriction endonucleases

Summary Restriction endonucleases were employed to characterize both cytologically and electrophoretically the DNA of Vicia faba. The electrophoretic pattern of total DNA digested with AluI and MboI shows a continuous smear. Bam HI also shows a continuous smear for the bigger polynucleotide fragments and several bands in the lower part of the lane. Digestion of fixed chromosomal DNA produces metaphase longitudinal differentiation when MboI and AluI are used, while no appreciable banding pattern is present when Bam HI is employed. These results are discussed in relation to the organization of chromosomal DNA, to other data in the literature on chromosome banding and on the digestion of total DNA of other species.     

No relationship between genetic instability in Bloom's syndrome and DNA hypomethylation of some major repetitive sequences

Bloom's syndrome (BS) is an autosomal recessive disorder, characterized by a high incidence of cancer at a young age. Cytogenetically, BS cells exhibit a high frequency of chromosomal damage and sister chromatid exchange (SCE). Thus, BS provides a human model of a genetic disorder exhibiting both chromosomal instability and a high incidence of cancer. In addition to its involvement in gene regulation, CpG methylation has recently been suggested to play an important role in the evolution and stability of chromosome structure. We have examined DNA methylation profiles of total DNA and some selected repeated sequences in normal and BS cells. No specific DNA hypomethylation in either total blood or lymphoblastoid cell lines from BS patients has been detected, suggesting that the genomic instability observed in BS is not directly related to a major DNA demethylation of the total CCGG sites, or of Alu or chromosome 1 satellite 2 repeated sequences.     

Transformation of Hprt gene with sperm DNA

Inactivation of the X chromosome during mammalian spermatogenesis has been postulated to occur by the same mechanism that controls female somatic X chromosome inactivation. We have used DNA-mediated transformation of HPRT- cells to test this idea, because it has been shown previously that inactive X chromosome DNA from somatic cells will not transform HPRT- cells. Isolated DNA from the mature sperm of five mammals (human, mouse, horse, bull, rabbit) were all capable of HPRT transformation, and transformants were confirmed electrophoretically. Measures were taken to ensure that the transformation frequencies observed could not be due to somatic contamination. The positive HPRT transformation result indicates that mature sperm X chromosomal DNA is not modified in the same manner as that of female inactive X chromosomal DNA. Since there is evidence for methylation of the somatic inactive X chromosome, it is possible that methylation, at least for the genes studied, is not involved in sperm X chromosome inactivation.     

DNA methylation and chromatin structure: a view from below

An understanding of the function and control of DNA methylation in eukaryotes has been elusive. Studies of Neurospora crassa have led to a model that accounts for the chromosomal distribution of methylation and suggests a basic function for DNA methylation in eukaryotes.     

Altered LINE-1 Methylation in Mothers of Children with Down Syndrome

Down syndrome (DS, also known as trisomy 21) most often results from chromosomal nondisjunction during oogenesis. Numerous studies sustain a causal link between global DNA hypomethylation and genetic instability. It has been suggested that DNA hypomethylation might affect the structure and dynamics of chromatin regions that are critical for chromosome stability and segregation, thus favouring chromosomal nondisjunction during meiosis. Maternal global DNA hypomethylation has not yet been analyzed as a potential risk factor for chromosome 21 nondisjunction. This study aimed to asses the risk for DS in association with maternal global DNA methylation and the impact of endogenous and exogenous factors that reportedly influence DNA methylation status. Global DNA methylation was analyzed in peripheral blood lymphocytes by quantifying LINE-1 methylation using the MethyLight method. Levels of global DNA methylation were significantly lower among mothers of children with maternally derived trisomy 21 than among control mothers (P = 0.000). The combination of MTHFR C677T genotype and diet significantly influenced global DNA methylation (R² = 4.5%, P = 0.046). The lowest values of global DNA methylation were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate. Although our findings revealed an association between maternal global DNA hypomethylation and trisomy 21 of maternal origin, further progress and final conclusions regarding the role of global DNA methylation and the occurrence of trisomy 21 are facing major challenges.     

Satellite 2 demethylation induced by 5-azacytidine is associated with missegregation of chromosomes 1 and 16 in human somatic cells

Satellite sequences are an important part of the pericentromeric regions in mammalian genomes; they play a relevant role in chromosome stability and DNA hypomethylation of these sequences has been reported in ICF syndrome and in some cancers that are closely associated with chromosomal abnormalities. Epigenetic modifications of satellite sequences and their consequences have not been extensively studied in human cells. In the present work, we evaluated satellite 2 methylation patterns in human lymphocytes exposed to 5-azacytidine (5-azaC) and assessed the relationship between these patterns and chromosome missegregation. Human lymphocytes were exposed to 10μM 5-azaC for 24, 48, and 72h. Segregation errors were evaluated in binucleate cells using FISH against pericentromeric regions of chromosomes 1, 9, and 16. DNA methylation patterns were evaluated by immunodetection, and by bisulfite plus urea conversion and sequencing. We have identified that 5-azaC induced missegregation of chromosomes 1 and 16, which have highly methylated satellite 2, after 72h of exposure. Chromosome methylation patterns showed a notable decrease in pericentromeric methylation. Bisulfite conversion and sequencing analysis demonstrated demethylation of satellite 2 associated to 5-azaC exposure, principally after 72h of treatment. This change occurred in a non-specific pattern. Our study demonstrates an association between loss of satellite 2 DNA methylation and chromosome loss in human lymphocytes.     

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin,and the effect of DNA replication inhibition

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-³H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.     

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

An analysis of the action of nonspecific and restriction nucleases on metaphase chromosomes in situ

Patterns of differential staining of Drosophila, mouse, rat, cattle and pig chromosomes were examined after the treatment with nucleases (DNAase I, DNAase II) and restriction enzymes (AluI, HpaII, MspI, BpE, EcoRI). The above effects depend on the species used, on the enzymes and substitution of thymine for bromodeoxyuridine in the chromosomal DNA. It is supposed that such a phenomenon may not only result from the irregular distribution of specific restriction sites along chromosomes but also depend on the specificity of supramolecular organization of the chromosomal DNA.     

High-resolution mapping of DNA methylation in human genome using oligonucleotide tiling array

DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region (∼1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5′ regulatory regions and the 3′ regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

DNA modifications in the mammalian brain

DNA methylation is a crucial epigenetic mark in mammalian development, genomic imprinting, X-inactivation, chromosomal stability and suppressing parasitic DNA elements. DNA methylation in neurons has also been suggested to play important roles for mammalian neuronal functions, and learning and memory. In this review, we first summarize recent discoveries and fundamental principles of DNA modifications in the general epigenetics field. We then describe the profiles of different DNA modifications in the mammalian brain genome. Finally, we discuss roles of DNA modifications in mammalian brain development and function.     

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2-(13)C]cytosine and [2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.     

The 5'' flanking region of human epsilon-globin gene.

The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat.     

Unmethylated DNA in mouse L cells. I. DNA fibre autoradiography.

Enzymatic methylation of DNA in mouse L cells has been studied using DNA fibre autoradiography to analyse the distribution of 5-methylcytosine in chromosomal DNA. The autoradiographic pattern of DNA labelled in the 5-methylcytosine is in several respects similar to the pattern of DNA replication. Two mean features are apparent: (1) the silver grains appear in well defined sections, and (2) the labelled sections are arranged in tandem along each DNA double helix. After a short pulse of radioactivity in the rate of growth of labeled sections in the pattern of DNA replication and the enzymatic methylation of DNA are identical. Unlike the replication pattern, DNA labeled during the S phase with L-[Me-3H] methionine is not completely labeled. There are distinct, 8-20 mum intervals in the autoradiographic pattern of this DNA. The length of these intervals may correspond to unmethylated sections of chromosomal DNA of about 23 to 58 kilo base pairs. These unmethylated sections of chromosomal DNA represent about 10% of the total genome.