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PubMed2004

Methods of epigenetic analysis.

Epigenetics encompasses heritable changes in DNA or its associated proteins except mutations in gene sequence. Many investigators in the field of epigenetics focus on histone modifications and DNA methylation, two molecular mechanisms that are often linked and interdependent. A variety of methods are applied to the study of epigenetic processes, and the past decade has witnessed an exponential increase in novel approaches to elucidate the molecular mysteries of epigenetic inheritance. This chapter summarizes some of the most contemporary methods used to study epigenetics presented throughout the book.

Chromatin↗
PubMed1988

Unusual alkaline elution pattern induced in mammalian cell DNA by fast neutrons p(34) + Be.

Syrian hamster fibroblasts (cell line BHK 21/13) were exposed to p(34) + Be fast neutron irradiation and their DNA analysed by the alkaline elution technique. The elution profiles showed an unusual tailing off, characteristic of neutron-irradiated samples, suggesting the presence of a modification in DNA induced by the neutrons. This was not seen with 60Co gamma-irradiation. In neutron-irradiated samples the alteration of DNA appeared to persist even after 2 h of post-treatment incubation (37 degrees C) indicating the absence of repair. The modification of DNA induced by neutrons provides a possible explanation for the reduction of the shoulders in survival curves obtained with neutrons, and the high RBE of neutrons.

Animals↗
PubMed2004

Epigenetics and cancer.

Epigenetic mechanisms act to change the accessibility of chromatin to transcriptional regulation locally and globally via modifications of the DNA and by modification or rearrangement of nucleosomes. Epigenetic gene regulation collaborates with genetic alterations in cancer development. This is evident from every aspect of tumor biology including cell growth and differentiation, cell cycle control, DNA repair, angiogenesis, migration, and evasion of host immunosurveillance. In contrast to genetic cancer causes, the possibility of reversing epigenetic codes may provide new targets for therapeutic intervention.

Cell Cycle↗
PubMed2000

[Genome methylation and its role in functioning of the eukaryotic organism].

Methylation is a modification that changes the structure and functional status of DNA. Hence it is interesting to study the effect of methylation on basic processes occurring in living cells. In this review, the role of DNA methylation in recombination, replication, transcription regulation, imprinting, tumorigenesis, and tumor progression is considered.

Animals↗
PubMed1981

Synthesis of deoxythymidylate and the unusual deoxynucleotide in mature DNA of Bacillus subtilis bacteriophage SP10 occurs by postreplicational modification of 5-hydroxymethyldeoxyuridylate.

Mature DNA of Bacillus subtilis W23 phage SP10 contains a hypermodified nucleotide (YdTMP) that replaces ca. 20% of the DTMP. SP10 DNA was pulse-labeled for 1 min at 20 degrees C with 32Pi. Among the oxopyrimidine nucleotides, virtually all of the radioactivity was recovered as 5-hydroxymethyldeoxyuridylate (HMdUMP). During the subsequent chase, radioactivity was lost from HMdUMP and recovered as YdTMP. At 37 degrees C, exogenous [6-3H]5-hydroxymethyldeoxyuridine (HMdUrd) was incorporated into SP10 DNA. Label administered as HMdUrd was phosphorylated to HMdUTP in the infected cells, but all radioactivity was recovered from SP10 DNA as YdTMP and dTMP. Two heat-sensitive mutants defective in hypermodification of SP10 DNA are described. In one mutant, HMdUMP replaces YdTMP in DNA. The other mutant generates a DNA containing a novel deoxynucleotide in place of YdTMP. The novel deoxynucleotide seems to consist of PPi esterified to the 5-hydroxymethyl function of HMdUMP (PP-HMdUMP). Both mutants make normal amounts of dTMP. The data are discussed in terms of the following conclusions. (i) Both oxopyrimidine nucleotides in mature SP10 DNA are derived by postreplicative modification of HMdUMP in nascent DNA. (ii) PP-HMdUMP is an intermediate that facilitate formation of a putative exocyclic methylene intermediate which receives the hypermodification. It is also argued that PP-HMdUMP and the same exocyclic methylene intermediate could serve as intermediates in reductive modification to dTMP. (iii) YdTMP is not an intermediate in the formation of dTMP, and reductive modification proceeds independently of hypermodification.

Bacillus subtilis↗
PubMed2000

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells.

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 microM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Animals↗
PubMed1990

[Effective selective modification of a single-stranded fragment of DNA with alkylating derivatives of short oligodeoxyribonucleotides in the presence of reaction effectors N-(2-hydroxyethyl)phenazine derivatives of oligodeoxyribonucleotides].

Tri-, tetra-, penta- and hexanucleotides bearing a reactive 4-(N-methylamino-N-2-chloroethyl)benzylamide group can effectively and selectively modify a single-stranded DNA fragment (302 nucleotides) in the presence of effectors, N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides complementary to DNA sequences adjacent to the binding site of the reagent. The reagents investigated modify not only single-stranded but also secondary-structured DNA regions. The modification extent depends on the length of oligonucleotide parts of the reagent and effector. A gap between the two stretches associated with the target DNA prevents the effector from functioning. The substitution of an octanucleotide effector by two tetranucleotide ones only slightly reduces the modification extent with a hexanucleotide reagent. A very efficient and specific modification can be achieved by using two effectors flanking the reactive oligonucleotide derivative. The approach leads to the modification extent of up to 89% with a hexanucleotide reagent.

Alkylating Agents↗
PubMed1997

Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/dA adduct: glycosidic rotation from syn to anti at the modified deoxyadenosine.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2005

The evolution of meiosis: recruitment and modification of somatic DNA-repair proteins.

Several DNA-damage detection and repair mechanisms have evolved to repair double-strand breaks induced by mutagens. Later in evolutionary history, DNA single- and double-strand cuts made possible immune diversity by V(D)J recombination and recombination at meiosis. Such cuts are induced endogenously and are highly regulated and controlled. In meiosis, DNA cuts are essential for the initiation of homologous recombination, and for the formation of joint molecule and crossovers. Many proteins that function during somatic DNA-damage detection and repair are also active during homologous recombination. However, their meiotic functions may be altered from their somatic roles through localization, posttranslational modifications and/or interactions with meiosis-specific proteins. Presumably, somatic repair functions and meiotic recombination diverged during evolution, resulting in adaptations specific to sexual reproduction. (c) 2005 Wiley Periodicals, Inc.

Animals↗
PubMed1987

Changes of chromatin organization induced by phospholipids.

Isolated nuclei represent a suitable model for studying the influence of exogenous phospholipids, normally found as minor chromatin components, on the nuclear structure, which, in turn, could be related to the observed modifications of DNA and RNA synthesis. The morphological modifications induced on chromatin RNP granules and nuclear matrix have been analyzed both with conventional thin sectioning and with an original method based on image analysis of freeze-fractured and replicated nuclear samples. The results obtained support the hypothesis that anionic phospholipids, by removing histone H1, induce a transition of the chromatin from solenoid to nucleosome conformation and favour the RNA polymerizing activity which results in an increased release of RNP particles, while neutral phospholipids, probably affecting the matrix structure, partly impare the RNP maturation and transport, with consequent increase of chromatin condensation.

Animals↗
PubMed1996

Epigenetic modifications during oocyte growth correlates with extended parthenogenetic development in the mouse.

In mammals, the maternal and paternal genomes are required for embryonic development. This is due to genomic imprinting which leads to the expression or repression of genes solely on the basis of the parent from which they were inherited. As a result, parthenogenetic embryos die before day 10 of gestation and show limited development of extra-embryonic membranes. Maternal imprinting is established during oogenesis and is associated with allele specific modifications in DNA methylation. We have investigated epigenetic modifications during oocyte growth using nuclear transfer techniques to produce mature oocytes with maternal chromatin derived from non-growing oocytes. Parthenogenetic activation of such oocytes leads to the development of normal size fetuses with a well developed placenta on day 13.5 of gestation; three days further than previously recorded for parthenogenetic development. In contrast, after fertilization, only one embryo was recovered on 9.5 days of gestation. Further, in these embryos we investigated the well characterized methylation pattern of the maternally expressed insulin-like growth factor II receptor gene (Igf2r) and found that the pattern of methylation was indeed different to that of fertilized control embryos. Thus, the embryonic phenotypes observed here correlate with changes in epigenetic events that normally occur during oocyte growth.

Animals↗
PubMed1996

Superhelical torsion controls DNA interstrand cross-linking by antitumor cis- diamminedichloroplatinum(II).

Negatively supercoiled, relaxed and linearized forms of pSP73 DNA were modified in cell-free medium by cis-diamminedichloroplatinum(II) (cisplatin). The frequency of interstrand cross-links (ICLs) formed in these DNAs has been determined by: (i) immunochemical analysis; (ii) an assay employing NaCN as a probe of DNA ICLs of cisplatin; (iii) gel electrophoresis under denaturing conditions. At low levels of the modification of DNA (<1 Pt atom fixed per 500 bp) the number of ICLs formed by cisplatin was radically enhanced in supercoiled in comparison with linearized or relaxed DNA. At these low levels of modification, the frequency of ICLs in supercoiled DNA was enhanced with increasing level of negative supercoiling or with decreasing level of modification. In addition, the replication mapping of DNA ICLs of cisplatin was consistent with these lesions being preferentially formed in negatively supercoiled DNA between guanine residues in both the 5'-d(GC)-3' and the 5'-d(CG)-3' sites. Among the DNA adducts of cisplatin the ICL has the markedly greatest capability to unwind the double helix. We suggest that the formation of ICLs of cisplatin is thermodynamically more favored in negatively supercoiled DNA owing mainly to the relaxation of supercoils.

Antineoplastic Agents↗
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↗
PubMed2005

Cell type-specific regulation of the TGF-beta-responsive alpha2(I) collagen gene by CpG methylation.

The alpha2(I) collagen gene shows cell type-specific expression, however, the mechanism behind this specificity remains to be determined. We demonstrate here that transforming growth factor-beta (TGF-beta)-mediated induction of alpha2(I) collagen gene is regulated by DNA methylation in a cell type-specific manner. Human alpha2(I) collagen mRNA and type I collagen protein were expressed in normal human fibroblasts (NHF), and also strongly enhanced by TGF-beta; they were not detected in HaCaT, HeLa, or HepG2 cells (termed "collagen-induction resistant (CIR) cells") even following stimulation with TGF-beta. On the other hand, the transcriptional activity of exogenously transfected alpha2(I) collagen promoter was clearly up-regulated by TGF-beta in the CIR cells as well as in NHF. In the CIR cells, CpG clusters around the transcription start site of the alpha2(I) collagen gene were heavily methylated, whereas no methylation was detected in NHF. Moreover, alpha2(I) collagen gene was reactivated in the CIR cells by 5-Aza-2-deoxycytidine (5-AdC) treatment to some extent. However, demethylation by 5-AdC was limited and it was unable to recover the TGF-beta responsiveness. In NHF, the alpha2(I) collagen gene has a Smad3-accessible chromatin structure and acetylated histones in the promoter regions. By contrast, in the CIR cells, Smad3 failed to bind to the chromatin and histones were not acetylated in this area. Furthermore, in vitro methylation of the reporter gene containing the alpha2(I) collagen promoter significantly reduced both basal and TGF-beta-induced enhancement of the transcriptional activity in NHF. Thus, we propose that alpha2(I) collagen gene provides the first example of the TGF-beta responsive gene whose cell type-specificity is regulated by CpG methylation.

Animals↗
PubMed2004

Expression of mRNAs for DNA methyltransferases and methyl-CpG-binding proteins in the human female germ line, preimplantation embryos, and embryonic stem cells.

Recent evidence indicates that mammalian gametogenesis and preimplantation development may be adversely affected by both assisted reproductive and stem cell technologies. Thus, a better understanding of the developmental regulation of the underlying epigenetic processes that include DNA methylation is required. We have, therefore, monitored the expression, by PCR, of the mRNAs of DNA methyltransferases (DNMTs), methyl-CpG-binding domain proteins (MBDs), and CpG binding protein (CGBP) in a developmental series of amplified cDNA samples derived from staged human ovarian follicles, oocytes, preimplantation embryos, human embryonic stem (hES) cells and in similar murine cDNA samples. Transcripts of these genes were detected in human ovarian follicles (DNMT3A, DNMT3b1, DNMT3b4, DNMT1, MDBs1-4, MeCP2, CGBP), germinal vesicle (GV) oocytes (DNMT3A, DNMT3b1, DNMT1, MDBs1-4, MeCP2, CGBP), mature oocytes (DNMT3A, DNMT3b1, DNMT1, CGBP), and preimplantation embryos (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD2, MDB4, CGBP). Differential expression of DNMT3B gene transcripts in undifferentiated (DNMT3b1) and in vitro differentiated human ES cells (DNMT3b3) further demonstrated an association of the DNMT3b1 transcript variant with totipotent and pluripotent human cells. Significantly, whilst the murine Dnmt3L gene is both expressed and essential for imprint establishment during murine oogenesis, transcripts of the human DNMT3L gene were only detected after fertilisation. Therefore, the mechanisms and/or the timing of imprint establishment may differ in humans.

Blastocyst↗
PubMed2001

5-Aza-2'-deoxycytidine induces histone hyperacetylation of mouse centromeric heterochromatin by a mechanism independent of DNA demethylation.

5-Aza-2'-deoxycytidine (5-azadC) is widely used as a potent inhibitor of DNA methyltransferase. Cells treated with this drug show various phenomena such as the reactivation of repressed genes, change in replication timing, and decondensation of heterochromatin. A number of studies using this drug have been reported so far but it is still controversial whether such changes are due to 5-azadC-induced demethylation itself or the side effects of the drug. Here we report that 5-azadC treatment induces histone hyperacetylation in mouse centromeric heterochromatin which normally contains methylated DNA and hypoacetylated histones. Treatment also affects the intranuclear distribution of histone deacetylase 2 (HDAC2). However, histone hyperacetylation was not observed in DNA methyltransferase 1-deficient cells with a reduced level of genomic DNA methylation. Our results suggest that 5-azadC-induced histone hyperacetylation is independent of DNA demethylation and that DNA methylation is not essential for the maintenance of the histone hypoacetylated state in centromeric heterochromatin.

Acetylation↗
PubMed2006

[Methylation and other new concepts for the origin of hepatocellular carcinoma].

Methylation takes place when a methyl group, comprised of a carbon and three hydrogen atoms (CH3), attaches to the individual DNA bases. This modification of the DNA leads to a change in gene expression. Changes in the methylation status of the DNA belong to the most common gene changes leading to malignant tumours. Recent studies have shown that three different mechanisms play a role in the effect of methylation: global hypomethylation, hypermethylation of individual gene segments and the deregulated expression of DNA methyltransferases. Such DNA methylation is also one of the most homogenous and consistent molecular changes in hepatocellular carcinoma (HCC). Moreover, the hypermethylation of specific genes has been shown to precede the development of malignant tumours. Thus, DNA methylation is a possible biomarker for the early discovery of HCC. The reversibility of methylation also offers a possible future treatment option.

Carcinoma, Hepatocellular↗