Showing papers on "Epigenetics published in 1995"

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

Abstract: A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms.

DNA methylation in early development

Abstract: Several lines of evidence strongly suggest that DNA methylation is involved in embryo development. Perhaps the most direct evidence comes from experiments with methyltransferase deficient mice. Embryos that express low levels of the maintenance methyltransferase do not develop to term and die at the 5 to 20 somite stage corresponding to the level of the enzyme. In the mouse, dramatic methylation changes have been observed during the early steps of embryo development. Most genes are subject to a process of active demethylation starting promptly after fertilization. Except for a small number of methylated CpG sites in imprinted genes the vast majority of the sites are unmethylated by the stage of cavitation (16 cell). Such genome-wide demethylation may signify an erasure of epigenetic information originating in the highly differentiated gametes. This erasure may be essential for the establishment of a pluripotent state, before specific cell lineages can be determined. The process of laying down a new developmental program involves, initially, global de novo methylation at the stage of pregastrulation followed by gene specific demethylations associated with the onset of activity of these genes. CpG islands characteristic of housekeeping genes, appear to be protected from the global de novo methylation. An exception to this rule is the hypermethylation of CpG islands in X-linked housekeeping genes on the inactive X chromosome and of specific differentially methylated CpG sites in imprinted genes. Primordial germ cells escape the global de novo methylation which takes place at the pregastrula stage and undergo a very similar de novo methylation process in the differentiated gonads (15.5-18.5 days post coitum), forming the methylation patterns which are specific to the gametes. Specific demethylations then form a terminal methylation pattern which is then clonaly inherited in the soma and erased after fertilization.

Hypomethylation of the amyloid precursor protein gene in the brain of an Alzheimer's disease patient.

Abstract: Aberrant expression of the amyloid precursor protein (APP) gene may contribute to the beta-amyloid deposition seen in Alzheimer's disease and Down syndrome patients. Genomic DNA was isolated from human brain tissue and digested with HpaII, an enzyme sensitive to CpG methylation. Southern-blot analysis revealed the absence of methylation at a site in the APP gene of an Alzheimer's disease subject. This site was methylated in a nondemented subject and a subject with a non-Alzheimer's type of dementia (Pick's disease). This is the first report of an epigenetic defect in an Alzheimer's disease patient and the observation suggests that hypomethylation of the APP gene may be one of several factors contributing to aberrant gene expression in Alzheimer's disease.

Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms.

Abstract: Genomic imprinting determines the monoallelic expression of a small number of genes during at least later stages of development. To obtain information necessary for the elucidation of imprinting mechanisms, we assessed the allele-specific expression and total expression level of four imprinted genes during early stages of development of normal F1 hybrid mice utilizing quantitative allele-specific reverse transcription-PCR (RT-PCR) single-nucleotide primer extension assays. The Igf2r and Snrpn genes were activated by the early 4-cell stage and exhibited biallelic and monoallelic expression, respectively, throughout preimplantation development. Thus, with respect to different imprinted genes, epigenetic systems determining monoallelic expression are not uniform in their time of establishment. Biallelic expression of Igf2r was observed in single blastomeres, discounting the possibility of random allelic inactivation at this stage. The closely linked H19 and Igf2 genes were activated after the blastocyst stage and often exhibited biallelic and monoallelic expression respectively in tissues of pregastrulation postimplantation-stage embryos, rather than reciprocal monoallelic modes as observed at later stages. This raises the possibility that imprinting of H19 is involved only in the maintenance and not in the initiation of monoallelic expression of Igf2. Monoallelic expression of Snrpn was observed in each blastomere at the 4-cell stage, demonstrating that the germ line, which exhibits biallelic expression of imprinted genes, must be derived from cells in which imprinting was once manifest.

Epigenetic programming of differential gene expression in development and evolution

Abstract: This review covers data on changing patterns of DNA methylation and the regulation of gene expression in mouse embryonic development. Global demethylation occurs from the eight-cell stage to the blastocyst stage in preimplantation embryos, and global de novo methylation begins at implantation. We have used X-chromosome inactivation in female embryos as a model system to study specific CpG sites in the X-linked Pgk-1 and G6pd housekeeping genes and in the imprinted regulatory Xist gene to elucidate the role of methylation in the initiation and maintenance of differential gene activity. Methylation of the X-linked housekeeping genes occurs very close in time to their inactivation, thus raising the question as to whether methylation could be causal to inactivation, as well as being involved in its maintenance. A methylation difference between sperm and eggs in the promoter region of the Xist gene, located at the X-chromosome inactivation centre, is correlated with imprinted preferential inactivation of the paternal X chromosome in extra-embryonic tissues. Based on our data, a picture of the inheritance of methylation imprints and speculation on the significance of the Xist imprint in development is presented. On a more general level, an hypothesis of evolution by "adaptive epigenetic/genetic inheritance" is considered. This proposes modification of germ line DNA in response to a change in environment and mutation at the site of modification (e.g., of methylated cytosine to thymine). Epigenetic inheritance could function to shift patterns of gene expression to buffer the evolving system against changes in environment. If the altered patterns of gene activity and inactivity persist, the modifications may become "fixed" as mutations; alternatively, previously silenced gene networks might be recruited into function, thus appearing as if they are "acquired characteristics." An extension of this hypothesis is "foreign gene acquisition and sorting" (selection or silencing of gene function according to use). "Kidnapping" and sorting of foreign genes in this way could explain the observation that increased complexity in evolution is associated with more "junk" DNA. Adaptive epigenetic/genetic inheritance challenges the "central dogma" that information is unidirectional from the DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution.

Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor.

Abstract: In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation

Abstract: Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Mechanisms involved in the immortalization of mammalian cells by ionizing radiation and chemical carcinogens.

Abstract: Immortalization is a prerequisite for the clonal evolution and malignant transformation of normal mammalian cells in culture. In order to gain a mechanistic insight into the genetics of carcinogen-induced cellular immortality, a cell culture assay has been developed based on the use of freshly explanted Syrian hamster dermal (SHD) fibroblasts. The relative efficacies of a variety of chemical and physical carcinogens at immortalizing SHD cells (against a zero background of spontaneous immortalization) were compared. Ionizing radiation and nickel chloride appeared to be more effective as immortalizing agents than powerful point mutagens, suggesting (but not proving) that clastogenic damage may be more significant in the immortalization process than point mutation. Frequencies of induced immortality (10(-6)-10(-7)/treated cell) were arguably consistent with a direct mutational mechanism involving a single genetic target. However, detailed cytogenetic characterization of a panel of newly immortalized cell lines revealed no non-random chromosomal alterations in the cells at the level of G-banding. Furthermore, additional experiments with the SHD system have provided confirmatory evidence that immortalization can occur as an indirect consequence of carcinogen exposure following an induced high frequency change in the treated population, rather than through direct targeted mutagenesis. Previous somatic cell genetic studies have suggested the possibility that a target gene for immortalization exists on the human and Chinese hamster X chromosomes. Here we provide strong evidence that the normal SHD X chromosome displays powerful senescence-inducing properties when introduced, by microcell transfer, into newly immortalized SHD recipients. These results suggest that induction of the immortal phenotype in SHD cells by carcinogens results primarily from functional inactivation of a senescence gene which may be X-linked. One possible mechanism for senescence gene inactivation consistent with our observations is through a sub-microscopic interstitial genetic deletion. However, the considerable efficacy of nickel (a human carcinogen) as an immortalizing agent at nonmutagenic doses raises the alternative possibility that immortalization may occur through an epigenetic mechanism.

Epigenetic regulation of gene expression: the effect of altered chromatin structure from yeast to mammals

Abstract: Epigenetic gene regulation refers to different states of phenotypic expression caused by differential effects of chromosome or chromatin packaging rather than by differences in DNA sequence. Examples of epigenetic regulation can be found in organisms as diverse as the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and mammals. Three major types of epigenetic regulation are considered in this review : dosage compensation, imprinting and position effect variegation. While the specific details and mechanisms of each is quite different, they all involve either local or extensive alterations in chromatin structure. A number of genes implicated in epigenetic regulation have been isolated and their products identified as proteins or RNA molecules involved at various levels in DNA, chromatin or chromosome binding. While in general our understanding of mammalian epigenetic phenomena is not as advanced as that in model systems, the detailed molecular and genetic understanding of processes responsible for conditional gene silencing in invertebrate systems provides strong models for consideration of such effects in human and mouse genetics.

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants.

Abstract: The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.

The reduced expression of endogenous duplications (REED) in the maize R gene family is mediated by DNA methylation

Abstract: The duplicated R and Sn genes regulate the maize anthocyanin biosynthetic pathway and encode tissue-specific products that are homologous to helix-loop-helix transcriptional activators As a consequence of their coupling in the genome, Sn is partially silenced Genomic restriction analysis failed to reveal gross structural DNA alterations between the strong original phenotype and the weak derivatives However, the differences in pigmentation were inversely correlated with differences in the methylation of the Sn promoter Accordingly, treatment with 5-azacytidine (AZA), a demethylating agent, restored a strong pigmentation pattern that was transmitted to the progeny and that was correlated with differential expression of the Sn transcript Genomic sequencing confirmed that methylation of the Sn promoter was more apparent in the less pigmented seedlings and was greatly reduced in the AZA revertants In addition, some methylcytosines were located in non-symmetrical C sequences These findings provide an insight into Sn and R interaction, a process that we have termed Reduced Expression of Endogenous Duplications (REED) We propose that increasing the copy number of regulatory genes by endogenous duplication leads to such epigenetic mechanisms of silencing Further understanding of the REED process may have broader implications for gene regulation and may identify new levels of regulation within eukaryotic genomes

DNA methylation and polyamines in embryonic development and cancer.

Abstract: Mammalian DNA contains relatively large amounts of a modified base, 5-methyl-cytosine (m5C). Methylation of cytosine is catalyzed by DNA(cytosine-5)methyltransferase (DNA MTase). DNA methylation seems to play an important role in the regulation of gene expression during development. Thus, m5C may inhibit transcription by preventing the binding of transcription factors and/or by altering chromatin structure. The DNA methylation patterns of the male and female pronuclei are erased in the morula and early blastula, and when the blastocyst forms, most of the DNA has become demethylated. Following implantation, however, there is a surge of de novo methylation affecting the entire genome, and already by gastrulation DNA is methylated to an extent characteristic of that of the adult animal. During subsequent development, tissue-specific genes undergo programmed demethylation, which may cause their activation. Site-directed mutagenesis of the DNA MTase gene, has recently shown that DNA methylation is absolutely required for normal development of the early mouse embryo. DNA methylation and polyamine synthesis depend on a common substrate, S-adenosylmethionine (AdoMet). As a consequence, changes in cellular polyamine levels may affect the degree of DNA methylation. When the first step in the polyamine biosynthetic pathway is blocked, F9 teratocarcinoma stem cells accumulate large amounts of decarboxylated AdoMet, the aminopropyl group donor in polyamine synthesis, and go through terminal differentiation into parietal endoderm cells. The accumulation of decarboxylated AdoMet is a direct consequence of the polyamine-depleted state of the cell. Although the decarboxylated AdoMet molecule contains a methyl group, it does not act as a methyl group donor in DNA methylation. Instead it acts as a competitive inhibitor of DNA MTase. A consequence of polyamine depletion is therefore genome-wide loss of DNA methylation due to insufficient maintenance methylation during successive rounds of DNA replication. Our recent finding that prevention of the accumulation of decarboxylated AdoMet counteracts the differentiative effect lends further support to the hypothesis proposed.

Genomic imprinting: causes and consequences.

Abstract: Contributors Part I. Genomic Imprinting in Mammals: 1. The role of imprinting in early mammalian development A. Gilligan, and D. Solter 2. The evolution of parental imprinting: a review of hypotheses D. Haig and R. Trivers 3. Genetic variations in parental imprinting on mouse chromosome 17 J. Forejt, S. Gregorov..., M. Landikova, J. Capkova and L. M. Silver Part II. Chromatin Structure and DNA Modifications: 4. Epigenetic inheritance: the chromatin connection A. P. Wolffe 5. Chromobox genes and the molecular mechanisms of cellular determination P. B. Singh and T. C. James 6. The biochemical basis of allele-specific gene expression in genomic imprinting and X inactivation T. H. Bestor 7. DNA methylation and mammalian development R. Jaenisch, C. Beard and E. Li Part III. Mechanisms of Imprinting: 8. X chromosome inactivation and imprinting M. F. Lyon 9. Imprinting of H19 and Xist in uniparental embryos M. A. Surani, A. C. Ferguson-Smith, H. Sasaki and S. C. Barton 10. Imprinted genes, allelic methylation, and imprinted modifiers of methylation W. Reik, R. Feil, N. D. Allen, T. F. Moore and J. Walter 11. Genomic imprinting of the H19 and Igf2 genes in the mouse S. M. Tilgham, M. S. Bartolomei, A. L. Webber, M. E. Brunkow, J. Saam, P. A. Leighton and K. Pfeifer 12. Plasticity of imprinting R. Ohlsson, T. Ekstrom, G. Franklin, S. Pfeifer-Ohlsson, H. Cui, S. Miller, R. Fisher and C. Walsh 13. Regional regulation of allele-specific gene expression I. Simon and H. Cedar Part IV. Genomic Imprinting in Embryonal Tumors and Overgrowth Disorders: 14. Genomic imprinting in embryonal tumors and overgrowth disorders A. E. Reeve 15. Tracking imprinting: the Beckwith-Wiedemann syndrome M. Mannens 16. Genomic imprinting in Beckwith-Wiedemann syndrome R. Weksberg and J. Squire 17. Mitotic crossing over and the disruption of genomic imprinting G. B. Cote 18. Evaluating H19 as an imprinted tumor suppressor gene B. Tycko 19. A domain of abnormal imprinting in human cancer A. P. Feinberg Part V. Genomic Imprinting and the Prader-Willi Syndrome: 20. Parent-of-origin-specific DNA methylation and imprinting mutations on human chromosome 15 B. Horsthemke, B. Dittrich and K. Buiting 21. The SNRPN gene and Prader-Willi syndrome U. Francke, J. A. Kerns and J. Giacalone Part VI. Imprinting: A Search for New Genes and Unifying Principles: 22. Use of chromosome rearrangements for investigations into imprinting in the mouse B. M. Cattanach, J. Barr and J. Jones 23. A new imprinted gene, U2af-related sequence, isolated by a methylation-sensitive genome scanning method T. Mukai, I. Hatada, T. Yamaoka, K. Kitagawa, X.-D. Wang, T. Sugama, J. Masuda and J. Ogata 24. The mouse Igf2/MPR gene: a model for all gametic imprinted genes? D. P. Barlow Index.

Genetic and epigenetic inactivation of repetitive sequences in Neurospora crassa: RIP, DNA methylation, and quelling.

Abstract: The genome of the filamentous fungus Neurospora crassa is approximately 100-fold smaller than that of vertebrates, and contains little repetitive DNA. The existence of repeat-induced point mutation (RIP; reviewed in Selker 1990b) and quelling (Pandit and Russo 1992; Romano and Macino 1992) may account for the rarity of repetitive sequences. RIP is a dramatic example of gene inactivation: duplicated gene-sized DNA sequences are efficiently detected and peppered with transition mutations in the sexual phase of the Neurospora life cycle. Asexual inactivation of repeated genes, termed quelling, has also been observed in some Neurospora transformants. DNA methylation is often associated with quelling and RIP, but methylation has not been demonstrated to be a requirement for these processes. Many eukaryotic organisms can silence repetitive genes. The phenomena in higher plants referred to as trans-inactivation, co-suppression, and RIGS (repeat induced gene silencing), reviewed in Matzke and Matzke (1993) and in other chapters of this volume, show similarities to RIP and quelling. It is still unclear, however, whether the phenomena in plants are mechanistically related to gene inactivation processes operating in fungi.

Meiotic transmission of a hypomethylated repetitive DNA family in tobacco.

Abstract: We have recently shown that hypomethylation of cytosine residues in the HRS60 family of repetitive DNA sequences can be induced with 5-azacytidine (5-azaC) in tobacco tissue cultures. We have also proven that such a DNA methylation status is maintained during the recovery of protoplasts, plant regeneration, and vegetative development. In the present paper we follow meiotic transmission of hypomethylated HRS60 DNA. Plants obtained from seeds treated with 5-azaC were either self pollinated or crossed with a non-treated control in a reciprocal way. Analysis of the methylation status of the HRS60 DNA revealed that these sequences were hypomethylated in the progenies up to the extent found in the parental 5-azaC-treated plant. Since no parent-of-origin effect was observed, we presume that both male and female gametes transmit an artificial methylation imprint to a similar extent. This result is supported by methylcytosine evaluation in the total genomic DNA samples. A temporal analysis of 5-azaC effects on germinating seeds and a phenotypic evaluation of 5-azaC-treated tobacco plants are also presented.

A novel L23-related gene 40 kb downstream of the imprinted H19 gene is biallelically expressed in mid-fetal and adult human tissues

Abstract: The closely linked IGF2 and H19 genes on human chromosome 11p15.5 are monoallelically expressed as a result of genomic imprinting and show altered expression in Wilms' tumors (WTs). To map regional imprinting we have sought to isolate additional human genes close to IGF2/H19 and to characterize their allelic expression patterns. Here we report a novel gene, provisionally named L23MRP [L23 (mitochondrial)-related protein], which is oriented 'tail-to-tail' with H19 and is transcribed to within 40 kb of the last H19 exon. L23MRP is expressed biallelically in many mid-fetal and adult human tissues. This gene is also expressed at normal levels in WTs which have lost expression of H19 either via loss of the maternal chromosome 11p15.5 or via an epigenetic pathway involving site-specific DNA hypermethylation. These data indicate that, at least in post-embryonic stages, L23MRP is functionally insulated from the IGF2/H19 imprinted domain.

Genetic and epigenetic mechanisms in the pathogenesis of neurofibromatosis type I.

Abstract: Neurofibromatosis type I (NF1) is a common genetic disease which leads to a variety of clinical features affecting cells of neural crest origin. In the period since the NF1 gene was isolated in 1991, our understanding of the genetics of NF1 has increased remarkably. One of the most striking aspects of NF1 genetics is its complexity, both in terms of gene organization and expression. The gene is large and, when mutated, gives rise to diverse manifestations. A growing body of data suggests that mutations in the NF1 gene alone may not be responsible for all of the features of this disease. Epigenetic mechanisms, those which affect the NF1 transcript, play a role in the normal expression of the NF1 gene. Therefore, aberrations in those epigenetic processes are most likely pathogenic. Herein we summarize salient aspects of the vast body of NF1 literature and provide some insights into the myriad of regulatory mechanisms that may go awry in the genesis of this common but complex disease.

Hypomethylation of DNA: a possible epigenetic mechanism involved in tumor promotion.

Abstract: The overall objective of our research is to discern mechanisms that can facilitate the aberrant expression of oncogenes involved in carcinogenesis. We are testing the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism underlying the aberrant expression of oncogenes involved in carcinogenesis. Hypomethylation may be a mechanism underlying the role of cell proliferation in carcinogenesis, and hypomethylation could possibly result from an enzymatic replacement of 5-methylcytosine (5MeC) with cytosine that is not linked to DNA replication. The testing of this hypothesis can serve as a focal point for a mechanism of action-oriented approach for considering key aspects of carcinogenesis: aberrant gene expression, heritable epigenetic events, species-to-species extrapolation/unique species sensitivity, tumor promotion, and thresholds. DNA methylation plays a role in the regulation of gene activity. There is a persuasive body of evidence indicating that differential methylation of DNA (i.e., 5-methylcytosine v. cytosine) is a determinant of chromatin structure and that the methyl group provides a chemical signal which is recognized by trans-acting factors that regulate transcription. Hypomethylation of a gene is necessary but not sufficient for its expression and, therefore, a hypomethylated gene can be considered to possess an increased potential for expression as compared to a hypermethylated gene. Changes in the methylation status of a gene provide a mechanism by which its potential for expression can be altered in an epigenetic heritable manner, and it is expected that modifications in DNA methylation would result from threshold-exhibiting events. Our experimental model is liver tumorigenesis, we focus upon oncogenes (e.g., Ha-ras and raf) relevant to mouse liver tumorigenesis, employ the liver tumor prone B6C3F1 (C57BL/6 x C3H/He) mouse, and make relevant comparisons with the sensitive C3H/He paternal strain and the resistant C57BL/6 maternal strain. A unique aspect of this research is that it offers the potential to provide insight regarding molecular mechanisms that underlie promotion of carcinogenesis while at the same time the results can provide the type of information that is required in order to take a more rational approach towards carcinogen risk assessment. Specifically, the practical significance of our research is that it addresses the areas of dose-response relationships, e.g., the existence of threshold-exhibiting mechanisms, and species-to-species extrapolation issues. This is discussed within the context of the requirement for a rational approach to risk assessment.

Genetic response to metals.

Abstract: Part 1 Molecular mechanism of metal induced mutagenicity and carcinogenicity: regulation of nuclear calcium and zinc interference by toxic metal ions effects of antioxidants, zinc, and chelators on free radical status of children living in the chernobyl area the role of ascorbate in metabolism and geno-toxicity of chromium (VI) inactivation of critical cancer-related genes by nickel-induced DNA hypermethylation and increased chromatin condensation - a new model for epigenetic carcinogenesis oxidative mechanisms of nickel(II) and cobalt(II) genotoxicity the antimutagenic effects of metallothionein may involve free radical scavenging protection from metal-induced DNA damage by metallothionein in an in vitro system DNA strand breakage and lipid peroxidation as possible mechanisms of selenium toxicity role of metal in oxidative DNA damage by nonmutagenic carcinogen. Part 2 Metal-DNA, DNA cleavage, and zinc-finger proteins: sequence-selective cleavage of DNA by cationic metalloporphyrins lanthanide(III) complexes as synthetic nucleases - hydroxyalkyl group participation in catalysis initiation of DNA strand cleavage by iron bleomycin - key role of DNA in determining the pathway of reaction nickel complexes in modification of nucleic acids new methods for determining the structure of DNA and DNA-protein complexes based on the chemistry of iron(II) EDTA DNA recognition by steroid hormone receptor zinc fingers - effects of metal replacement and protein-protein dimerization interface HIV-1 tat protein forms a zinc-finger-like structure. Part 3 Metal-related genetic diseases - Menkes and Wilson diseases: Menkes disease - from patients to gene variability in clinical expression of an X-linked copper disturbance, Menkes disease development of copper-histidine treatment for Menkes disease copper-histidine therapy in Menkes disease - clinical, biochemical, and molecular aspects biochemical and clinical benefits of copper-histidine therapy in Menkes disease the Wilson disease gene - a copper-binding ATPase homologous to the Menkes disease gene zinc therapy - an advance in the treatment of Wilson's disease. Part 4 Metals and gene regulation: transcriptional regulation and function of yeast metallothionein genes transcriptional regulation of the metallothionein gene - metal-responsive element and zinc regulatory factor.

Chromatin structure and imprinting : developmental control of DNase-I sensitivity in the mouse insulin-like growth factor 2 gene

Abstract: The insulin-like growth factor 2 (Igf2) gene on distal mouse chromosome 7 is expressed predominantly from the paternal allele. In previous studies we identified two regions of paternal allele-specific methylation; one at ˜ 3 kb upstream of promoter 1, and a second in the 3′, coding portion of the gene. The 3′ region is methylated in an expressing tissue (fetal liver), whereas in a non-expressing tissue (fetal brain), it is not methylated. By contrast, in the 5′ region, the paternal allele is highly methylated in all tissues. Here, we have studied another characteristic of chromatin, namely, sensitivity to DNase-1 and have focused our developmental analysis on the two differentially methylated regions of Igf2. In the upstream region, four clustered DNase-I hypersensitive sites (HSS) were detected in embryonic stem (ES) cells and in midgestation embryos, but not in neonatal liver or brain. In promoter 1 (P1), at β 0.3 kb upstream of exon 1, we detected a tissue-specific HSS that was present in neonatal liver, in which P1 is active, but was absent in ES cells, the embryo, and in neonatal brain. No DNase-I HSS were detected in the 3′ differentially methylated region of Igf2. In all these regions, we did not detect differences in DNase-I sensitivity between the parental chromosomes. These results establish major developmental and tissue-specific control of chromatin in the Igf2 locus. The presence of the HSS upstream of Igf2 precedes transcriptional activation of the Igf2 gene and may be indicative of a promoter for another transcript that is transcribed in the opposite direction. The HSS in P1 is largely liver-specific; this promoter therefore is differently regulated than the more general fetal promoters P2 and P3. Whereas methylation can be allele-specific, presumably reflecting the gene imprint, the nuclease sensitivity, as detected by our assay, is not. These results, taken together with previous observations, reveal developmental and tissue-specific complexity in the expression of the parental imprint at the level of chromatin and transcription. We propose that epigenetic features of tissue-specific control and of the control of allelic expression are intricately linked. © 1995 Wiley-Liss, Inc.

Uniparental disomy and genomic imprinting as causes of human genetic disease.

Abstract: The existence of parent-of-origin differences in the expression of some genes, a process known as genomic imprinting, has been recognized and documented over the past several years. This epigenetic marking process results in the differential expression of normal genes depending upon whether they were inherited from the mother or the father. A number of human disorders have been identified as resulting from alterations in genomic imprinting. One process which can unmask genomic imprinting is uniparental disomy, in which both members of a chromosome pair are contributed by one sex parent. When uniparental disomy is present, genetic abnormality can result either from homozygosity of a single mutant allele which is present in two doses, or from the presence of two copies of an imprinted unexpressed gene or genes, rather than the usual one expressed and one unexpressed. Examples of human genetic disorders that are the consequence of genomic imprinting, and a discussion of current knowledge about the mechanisms of imprinting and the causes of uniparental disomy, are reviewed.

Paramutation in maize and related allelic interactions

Abstract: Paramutation has been defined as an interaction between alleles that leads to a mitotically and meiotically heritable change in one of the alleles at high frequency (Brink 1973). In the cases in which the gene function is known, it is clear that paramutation leads to reduction but not loss of phenotypic expression of the affected allele. We will discuss the genetic properties of paramutation of the maize genes b, r, and pl and review recent characterization of the effect of paramutation on gene expression and structure. As we review paramutation, we will compare apparently related phenomena, including: (1) recently described interactions between transgenes and endogenous genes in plants that lead to gene silencing, (2) interactions between transposable elements of plants that lead to heritable changes in gene expression, and (3) transvection in Drosophila and heritable epigenetic effects on gene expression in animals and fungi. For a thorough review of earlier literature on paramutation of b, r, and the handful of other examples of paramutation in plants, we refer the reader to previous reviews (Brink 1964, 1973; Brink etal. 1968; Coe 1966).

Ceratopteris: a model system for studying sex-determining mechanisms in plants

Abstract: The homosporous fern Ceratopteris richardii is a model system for studying the genetic and epigenetic factors that are involved in sex-determining processes in plants. The sex of the Ceratopteris gametophyte is determined by antheridiogen, a gibberellin-like pheromone that is secreted by the hermaphroditic gametophyte and promotes male development of sexually immature gametophytes. Abscisic acid blocks the antheridiogen response in Ceratopteris. Several mutations that affect sex expression have been isolated and characterized; these mutations define at least some of the major regulatory genes that are involved in determining the sex of the Ceratopteris gametophyte. By studying the epistatic interactions among these mutants, it is possible to determine how these genes interact with one another to form a regulatory genetic hierarchy that controls sex determination and expression in Ceratopteris. While a genetic approach is useful in identifying genes that are involved in sex determination, other molecular a...

Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

Abstract: Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.