Cells of a multicellular organism are genetically homogeneous but structurally and functionally heterogeneous owing to the differential expression of genes. Many of these differences in gene expression arise during development and are subsequently retained through mitosis. Stable alterations of this kind are said to be 'epigenetic', because they are heritable in the short term but do not involve mutations of the DNA itself. Research over the past few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. Here, we review advances in the understanding of the mechanism and role of DNA methylation in biological processes. Epigenetic effects by means of DNA methylation have an important role in development but can also arise stochastically as animals age. Identification of proteins that mediate these effects has provided insight into this complex process and diseases that occur when it is perturbed. External influences on epigenetic processes are seen in the effects of diet on long-term diseases such as cancer. Thus, epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression. The extent to which environmental effects can provoke epigenetic responses represents an exciting area of future research.

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Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals

Gene transcription can be activated or inhibited by a reversible modification of the gene; this modification is termed an epigenetic change. This account of epigenetics in cancer reviews the mechan...

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Epigenetics in Cancer

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods-in germ cells and in preimplantation embryos-in which methylation patterns are reprogrammed genome wide, generating cells with a broad developmental potential. Epigenetic reprogramming in germ cells is critical for imprinting; reprogramming in early embryos also affects imprinting. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. A role of reprogramming in stem cell differentiation is also envisaged. DNA methylation is one of the best-studied epigenetic modifications of DNA in all unicellular and multicellular organisms. In mammals and other vertebrates, methylation occurs predominantly at the symmetrical dinucleotide CpG (1-4). Symmetrical methylation and the discovery of a DNA methyltransferase that prefers a hemimethylated substrate, Dnmt1 (4), suggested a mechanism by which specific patterns of methylation in the genome could be maintained. Patterns imposed on the genome at defined developmental time points in precursor cells could be maintained by Dnmt1, and would lead to predetermined programs of gene expression during development in descendants of the precursor cells (5, 6). This provided a means to explain how patterns of differentiation could be maintained by populations of cells. In addition, specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. Neat and convincing as this model is, it is still largely unsubstantiated. While effects of methylation on expression of specific genes, particularly imprinted ones (7) and some retrotransposons (8), have been demonstrated in vivo, it is still unclear whether or not methylation is involved in the control of gene expression during normal development (9-13). Although enzymes have been identified that can methylate DNA de novo (Dnmt3a and Dnmt3b) (14), it is unknown how specific patterns of methylation are established in the genome. Mechanisms for active demethylation have been suggested, but no enzymes have been identified that carry out this function in vivo (15-17). Genomewide alterations in methylation-brought about, for example, by knockouts of the methylase genes-result in embryo lethality or developmental defects, but the basis for abnormal development still remains to be discovered (7, 14). What is clear, however, is that in mammals there are developmental periods of genomewide reprogramming of methylation patterns in vivo. Typically, a substantial part of the genome is demethylated, and after some time remethylated, in a cell- or tissue-specific pattern. The developmental dynamics of these reprogramming events, as well as some of the enzymatic mechanisms involved and the biological purposes, are beginning to be understood. Here we look at what is known about reprogramming in mammals and discuss how it might relate to developmental potency and imprinting.

Epigenetic Reprogramming in Mammalian Development

Ke yW ords 5-methylcytosine, DNA methyltransferase, transposons, genomic imprinting ■ Abstract Large-genome eukaryotes use heritable cytosine methylation to silence promoters, especially those associated with transposons and imprinted genes. Cytosine methylation does not reinforce or replace ancestral gene regulation pathways but instead endows methylated genomes with the ability to repress specific promoters in a manner that is buffered against changes in the internal and external environment. Recent studies have shown that the targeting of de novo methylation depends on multiple inputs; these include the interaction of repeated sequences, local states of histone lysine methylation, small RNAs and components of the RNAi pathway, and divergent and catalytically inert cytosine methyltransferase homologues that have acquired regulatory roles. There are multiple families of DNA (cytosine-5) methyltransferases in eukaryotes, and each family appears to be controlled by different regulatory inputs. Sequence-specific DNA- binding proteins, which regulate most aspects of gene expression, do not appear to be involved in the establishment or maintenance of genomic methylation patterns.

Eukaryotic cytosine methyltransferases.

Epigenetic phenomena in animals and plants are mediated by DNA methylation and stable chromatin modifications. There has been considerable interest in whether environmental factors modulate the establishment and maintenance of epigenetic modifications, and could thereby influence gene expression and phenotype. Chemical pollutants, dietary components, temperature changes and other external stresses can indeed have long-lasting effects on development, metabolism and health, sometimes even in subsequent generations. Although the underlying mechanisms remain largely unknown, particularly in humans, mechanistic insights are emerging from experimental model systems. These have implications for structuring future research and understanding disease and development.

Epigenetics and the environment: emerging patterns and implications.

Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed in most cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression.

https://science.sciencemag.org/content/sci/293/5527/95.full.pdf

Epigenetic Instability in ES Cells and Cloned Mice

To assess the extent of abnormal gene expression in clones, we assessed global gene expression by microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived by nuclear transfer (NT) from both cultured embryonic stem cells and freshly isolated cumulus cells. Direct comparison of gene expression profiles of more than 10,000 genes showed that for both donor cell types ≈4% of the expressed genes in the NT placentas differed dramatically in expression levels from those in controls and that the majority of abnormally expressed genes were common to both types of clones. Importantly, however, the expression of a smaller set of genes differed between the embryonic stem cell- and cumulus cell-derived clones. The livers of the cloned mice also showed abnormal gene expression, although to a lesser extent, and with a different set of affected genes, than seen in the placentas. Our results demonstrate frequent abnormal gene expression in clones, in which most expression abnormalities appear common to the NT procedure whereas others appear to reflect the particular donor nucleus.

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Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei

Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.

Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality

The generation of adult animals by nuclear cloning from adult donor cells is extremely inefficient, with most clones dying soon after implantation. In contrast, cloning from embryonic stem cell donor nuclei is significanty more efficient than from adult donor cells. However, regardless of donor cell type, all clones that survive to birth and beyond suffer serious phenotypic and gene expression abnormalities. All available evidence is consistent with the notion that the anomalous phenotypes of cloned animals are caused by faulty epigenetic reprogramming of the donor nucleus. Faulty reprogramming appears to be caused by the cloning process itself as well as by the epigenetic state of the donor nucleus. In contrast to reproductive cloning, faulty reprogramming of the donor nucleus does not tend to interfere with the application of nuclear transfer technology for therapeutic purposes (therapeutic cloning).

David Humpherys

Papers

Epigenetic Instability in ES Cells and Cloned Mice

Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei

Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality

Nuclear cloning, stem cells, and genomic reprogramming.