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Epigenetics Overview

What is epigenetics?

The term 'epigenetics' addresses all heritable and potentially reversible changes in genome function that do not alter the nucleotide sequence within the DNA.

The packaging of DNA affects its functionality.

All higher organisms face the same dilemma: they need to store an enormous number of individual genes within a tiny nucleus, and they need to ensure that defined groups of genes are expressed at the right time and in the right tissue. This conflict between efficient storage and functionality of genetic information requires a control system that defines how individual genetic regions are packaged and where they are located within the nucleus. This will have direct consequences for the competence of such regions to be expressed, as highly condensed regions will be less accessible to transcription complexes than 'open' regions. Condensation can even affect a complete chromosome as illustrated by the Barr bodies in mammalian females. One of the two X-chromosomes in female cells undergoes extensive chromatin condensation, which switches of most of the genes. In the interphase nuclei of female mammals, the condensed X-chromosome is visible as dense, stainable structure, called a Barr body.

Chromatin modification - a tool for epigenetic systems

In eukaryotes, genetic information is assembled into chromatin. The DNA strand loops in two circles around a core complex of eight histone molecules, forming a histone monomer as the first level of genome organisation. The histone complex consists of a central 'fold' unit that associates with the DNA strands, and of unstructured 'tails' that protrude to the outside. These tails represent the communication relays for factors and signals that influence the degree of condensation of the local chromatin. Certain aminoacids within the tail region can be modified, for example via acetylation or methylation of lysine residues, generating new 'epigenetic states'. These changes can affect the local chromatin structure, either directly, when, for example, the acetylation of a particular lysine residue alters its previously positive charge, which weakens its association with a negatively charged region in an adjacent nucleosome, or indirectly, when, for example, a methylated lysine residue within a defined sequence context becomes a signal for a protein that establishes a repressive multi-protein complex. In some species, the reversible modification of chromatin is accompanied by a reversible modification at the DNA level, the methylation of cytosine residues, which also can act as a target for repressive chromatin factors.

Reversibility and heritability: two key features of epigenetic systems

The DNA code defines how a combination of four nucleotides generates information that can be copied into RNA, which, in case of a protein-encoding gene, translates the information into an amino acid chain. The DNA sequence therefore determines the potential of a gene to generate a phenotype via RNA or protein synthesis. If the DNA sequence is damaged or altered, this will frequently result in the loss of the gene function. These genetics mutations are usually irreversible as they are linked to changes that cannot be corrected. A modification at the histone tails can have the same effect as a mutation of the DNA, for example, if it leads to the formation of a chromatin state that is no longer accessible to the transcription machinery. The consequence will be that that genetic information can not be converted into RNA or protein information, and the phenotype of the gene will therefore be lost. In contrast to irreversible changes made to the DNA, however, any epigenetic modification to an aminoacid of one of the histone tails can be reversed, and the active state can be restored. Epigenetic modifications can be introduced at an early stage and can be faithfully transmitted to daughter cells following cell division. They can also be reset at specific stages, for example during meiosis, to restore the original epigenetic pattern. Again, we can look at the Barr body to illustrate this principle: Male mammals contain only one X-chromosome, which they obtain from their mother. This X-chromosome needs to be functional and must therefore not be condensed. One of the two X-chromosomes of the mother, however, is condensed and inactive. Before it can be passed on to the next generation via the female germ line, the condensation of this X-chromosome must be reversed. Throughout these processes, the genes on the X-chromosome do not change, they simply switch between an 'off' and 'on' state, which is determined by their chromatin structure.

Epigenetic systems can work at the posttranscriptional level

Changes in chromatin state are only one aspect of epigenetic phenomena. Another are changes in RNA stability that prevent the expression of a phenotype although a gene has been faithfully transcribed. Small RNA molecules play a key role in this process, as they regulate the degradation or translational inhibition of homologous RNA molecules. Small RNAs also provide a link to chromatin modification systems as they direct chromatin modification functions to homologous DNA regions.

Why is epigenetic research exciting?

Epigenetic research is a fast evolving field. Among the many interesting novel aspects that epigenetic research has produced are the (re)discovery of RNA molecules as essential regulators for genome structure and gene expression, and the recognition of environmental factors as modifiers of epigenetic states.

Small RNAs

For a long time, RNA has only been recognised as a carrier of information between DNA and protein components, which received the primary attention of geneticists. For a short while, there may have even been strong expectations within the genetic community that a full understanding of the DNA sequence would provide a complete understanding of an organism. It would be highly disappointing if life could be fully understood on the basis of four nucleotides, and, luckily, many steps we make in our attempt to understand life, provide us with new answers that specify interesting new questions. Epigenetic research in particular has highlighted the significance of (mainly non-coding) RNA molecules in gene regulation and genome organisation. A recent publication (Lu et al, Science (2005) 309, 1567-1569) reports the presence of more than 75,000 small RNAs in Arabidopsis seedlings and inflorescences. It is most likely that this collection will increase significantly if other tissues or material exposed to different environmental conditions will be examined. The number of transcripts appears to exceed the number of 'genes', and our previous focus on DNA transcription, promoters and their regulation by trans-acting factors needs to be extended. There are several fascinating questions that have emerged from our new knowledge about small RNAs. What determines the balance between the two contrasting but co-operative mechanisms that are based on small RNA participation: the degradation of an RNA by dicer/slicer complexes and its synthesis by RNA-dependent polymerases? What determines if and when a transcript is degraded, and into which degradation pathway it will be channelled? How and when do small RNAs affect long-distance effects within an organism? How do small RNAs select their targets for chromatin modification, and how does this influence the composition and structure of the genome? Which role do epigenetic states and mechanisms play in evolution and adaptation?

Environmental influences

The role of epigenetic patterns in adaptation received considerable attention when Marcus Pembrey and Lars Olov Bygren (Kaati et al. (2002) Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. Eur J Hum Genet 10, 682- 688; Pembrey et al. (2006) Sex-specific, male-line transgenerational responses in humans European Journal of Human Genetics 14, 159-166) published transgenerational effects in humans that reflected the nutritional conditions experienced in previous generations. Plant research on the expression stability of transgenes had already detected epigenetic changes that were induced by changing environmental conditions, and that could be faithfully transmitted to the next generation. Pembrey's and Bygren's studies of food supply and various medical conditions in a small Swedish community revealed that a famine at critical times in the lives of parent or grandparents can affect the performance of children or grandchildren. If food was not readily available during the father's slow growth period, between the ages of 9 and 12 before they reached puberty, then cardiovascular disease mortality of the children was low. If the paternal grandfather was exposed to a surplus of food during his slow growth period Diabetes mortality in grandchildren increased. Paternal grandfather's food supply was only linked to the mortality rates of grandsons, paternal grandmother's food supply was only linked to the mortality rates of granddaughters. The lifespan of the grandchildren seemed to be influenced by their paternal grandfathers' access to food during the grandfather's slow growth period. The biggest effect of food supply in grandmothers occurred when she was a fetus and infant. These timings suggest that information is being captured at key stages of egg and sperm formation, and is passed on to the offspring, possibly in form of epigenetic patterns.

If the environment was able to modify germline epigenetic imprints at specific stages in gametogenesis, we may have to reconsider Lamarck's theory about the heritability of acquired characteristics, which assumes that an organism can acquire characteristics during its lifetime and pass them on to its offspring.

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What are the advantages of working with plants to study epigenetics?

Epigenetic research offers the rare opportunity to compare related mechanisms in very different experimental systems: plants, animals and fungi. In times where biological science has produced a high level of specialisation, there is a danger that research communities loose the ability to communicate across species boundaries. Once a researcher has read the recent publications in his/her narrow field, there is often little time left to follow what is going on outside the field. The consequence is that we learn more and more about less and less, until we know everything about nothing.

As epigenetic research addresses common biological mechanisms, it is more obvious than for other research fields to appreciate the specific advantages and challenges that different species and experimental systems offer. This is explicitly documented by the success of the Epigenome Network of Excellence that provides a common platform for epigenetic research labs in Europe, which work on many different model systems.

Epigenetic research is a prime example for successful 'crosstalk' among the scientific community, and plant research has made significant contributions to this research area. At the same time, there has been an extensive exchange of scientists between epigenetic labs that work on different species systems. Technically, is does not make much of a difference if you study an epigenetic system in plants, animals or fungi, if your focus is on the biochemical aspects, as you will mainly work with chromatin or RNA. Plant epigenetic research has the advantage that you can produce extensive numbers of individuals from the same line, either by seed cultivation or via 'cloning' of individual cells and regenerating them to new organisms. The analysis of such clones allows us to draw valuable conclusions about the natural variation of epigenetic states and their inheritance, a topic that has become highly relevant in view of recent progress in animal cloning. Another advantage is the large collection of mutants lines, especially in Arabidopsis, in which individual genes have been inactivated that encode proteins involved in one of the diverse epigenetic pathways. Such lines have helped significantly to identify endogenous target regions that are regulated by epigenetic pathways. A specific research area that is becoming increasingly popular for epigenetic researchers is the epigenetic response to environmental stress. More than any other organism, plants have to cope with rapidly changing environmental conditions, and especially transgenes and mobile elements have been known for a long time to respond to environmental stress via changes in chromatin structure and DNA methylation. Again, these studies provide a link to mammalian cloning and in vitro fertilisation systems, which often include the cultivation of cells under 'unusual' environmental conditions. While genetic and tissue culture aspects provide some attractive advantages for plant research, it benefits significantly from the advanced state that certain fungal and animal systems have with respect to chromatin analysis. The comparative analysis of chromatin remodelling systems and epigenetic pathways in different species helps to identify common and species-specific mechanisms.

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Which specific contributions have been made by epigenetic research on plants?

Plant research has made major contributions to epigenetics. The basic concept of epigenetic states was already discussed by Barbara McClintock in her studies of transposable elements and their regulation in maize. In her article 'Chromosome Organization and Genic Expression', published in 1951 in the Cold Spring Harbor Symposia on Quantitative Biology (16, 13-47) , she discusses the nature of a mutable locus: 'The basic mechanism responsible for a change at a mutable locus is considered to be one that is associated with a structural alteration of the chromatin materials at the locus'. At a time when neither the molecular basis of genetic information in form of DNA nor the concept of chromatin remodelling was known, she already predicted the concept of differentiation based on epigenetic gene regulation, when she stated in the same article that 'the progeny of two sister cells are not alike with respect to the type of genic action that will occur. Differential mitoses also produce alterations that allow particular genes to be reactive. Other genes, although present, may remain inactive. This inactivity or suppression is considered to occur because the genes are 'covered' by other nongenic chromatin materials. Genic activity may be possible only when a physical change in the covering material allows the reactive components of the gene to be 'exposed' and thus capable of functioning'.

Barbara McClintock's work has been inspirational to several generations of plant researcher. Among their many significant contributions made during the last two decades are:

  • the demonstration of a link between loss of DNA methylation and transposon activation (Chandler, V., Rivin, C. and Walbot, V. (1986) Stable non-mutator stocks of maize have sequences homologous to the Mu1 transposable element. Genetics 114, 1007-1021)
  • the identification of the role of DNA homology in inducing gene silencing (Matzke, M.A., Primig, M., Trnovsky, J. and Matzke, A.J.M., (1989). Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8, 643-649)
  • the identification of sense homology as a trigger for RNA degradation (Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in Trans. Plant Cell, 2, 279-289. Van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M., and Stuitje, A. R. (1990). Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291-299)
  • the observation that epigenetic states are responsive to environmental factors (Meyer, P., Linn, F., Heidmann, I., Meyer, H., Niedenhof, I. and Saedler, H. (1992) Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Molecular & General Genetics, 231, 345-352)
  • the demonstration of a link between gene silencing and virus resistance (Lindbo J.A., Silva-Rosales L., Proebsting W.M. and Dougherty W.G. (1993) Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5, 1749-1759
  • the discovery of RNA-mediated DNA methylation (Wassenegger, M., Heimes, S., Riedel, L. and Sanger, H.L. (1994) RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576)
  • the observation that ploidy levels affect epigenetic states (Mittelsten Scheid, O., Jakovleva, L., Afsar, K., Maluszynska Y. and Paszkowski, J. (1996) A change of ploidy can modify epigenetic silencing. Proc. Natl. Acad. Sci. USA 93, 7114-7119)
  • the discovery of 'aberrant RNAs' and presentation of a concept of endonucleolytic cleavage of dsRNA in post-transcriptional silencing (Metzlaff, M., O'Dell, M., Cluster, P. D. and Flavell, R. B. (1997) RNA-Mediated RNA Degradation and Chalcone Synthase A Silencing in Petunia. Cell 88, 845-854)
  • The discovery of a mobile silencing signal (Palauqui, J.C., Elmayan, T., Pollien, J. M. and Vaucheret, H. (1997) Systemic Acquired Silencing: Transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J 16, 4738-4745)
  • the discovery of small RNAs as key component in post-transcriptional silencing (Hamilton, A. J. and Baulcombe, D. C. (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952)
  • the development of the concept of promoter silencing via dsRNA constructs (Mette, M.F., Aufsatz, W., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. (2000) Transcriptional gene silencing and promoter methylation triggered by double stranded RNA EMBO J. 19, 5194-5201)
  • the identification of novel cytosine methylation functions (Cao, X. and Jacobsen, S.E. (2002) Locus specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc. Nat. Acad. Sci. U. S. A. 99, 16491-16498)
  • the demonstration of a link between DNA methylation and histone methylation (James P. Jackson, Anders M. Lindroth, Xiaofeng Cao, Steven E. Jacobsen. (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556-560)
  • the discovery of imprinting pathways in plants involving active demethylation (Grossniklaus, U., Vielle-Calzada, J-P., Hoeppner, M.A., Gagliano, W.B. (1998) Maternal control of embryogenesis by MEDEA, a Polycomb-group gene in Arabidopsis. Science 280, 446-450. Choi, Y., Gehring, M., Johnson, L., Hannon, M., Harada, J., Goldberg, R.B., Jacobsen, S.E. and Fischer, R L. (2002) DEMETER, a DNA Glycosylase Domain Protein, Is Required for Endosperm Gene Imprinting and Seed Viability in Arabidopsis. Cell 110, 33-42)
  • the development of models for the role of transposable elements in epigenetic regulation of adjacent genes (Masson, P., Surosky, R., Kingsbury, J.A., and Fedoroff N.V. (1987) Genetic and molecular analysis of the Spm-dependent a-m2 alleles of the maize a locus. Genetics 117, 117-137. Coen, E.S., Carpenter, R. and Martin, C. (1986) Transposable elements generate novel spatial paterns of gene expression in Antirrhinum majus. Cell 47, 285-296). Lippman, Z., Gendrel, A. V., Black, M., Vaughn, M. W., Dedhia, N., McCombie, W. R., Lavine, K., Mittal, V., May, B., Kasschau, K. D., Carrington, J. C., Doerge, R. W., Colot, V., and Martienssen, R. (2004). Role of transposable elements in heterochromatin and epigenetic control. Nature, 430, 471-76)

This is just a small collection of examples that illustrate the significance and wider impact of plant research for epigenetics. Very often, the significance of work that resulted from research on plants has only fully been appreciated when related effects were observed in mammals or humans. The success in animal cloning and the discovery of transgenerational effects are only two such examples.

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