The field of epigenetics, transcending genetics, genomics, and molecular biology, is now poised to be the vanguard of biological science. The rise of epigenetics marks a maturation of the field, which only 50 years ago was given its name and a vague definition, but is now a dynamic discipline, challenging and revising traditional paradigms of inheritance. Through epigenetics the classic works of Charles Darwin, Gregor Mendel, and Jean-Baptiste Lamarck are now seen in different ways. As more factors influencing heredity are discovered, today’s scientists are using epigenetics to decipher the roles of DNA, RNA, proteins, and environment in inheritance. The future of epigenetics will reveal the complexities of genetic regulation, cellular differentiation, embryology, aging, cancer, and other diseases. In this review fundamental concepts of epigenetics will be described, including chromatin structure, epigenetic markers, model systems, research methods, and future directions.
Note: If you’d like to print out a hard copy of these reviews, you can find it at Zymo Research’s website in their Publications.
Darwin, Mendel, and Lamarck looking sharp portrait style.
Chromatin Structure and Epigenetic Markers
To understand epigenetics requires an understanding of chromatin structure (Figure 2). Chromatin, which is organized into repeating units called nucleosomes, is the complex of DNA, protein, and RNAs that comprises chromosomes1. A nucleosome consists of 147 bp of double-stranded DNA wrapped around an octamer of histone proteins, usually two copies each of the core histones H2A, H2B, H3, and H4. In mammalian cells, most of the chromatin exists in a condensed, transcriptionally silent form called heterochromatin. Euchromatin is less condensed, and contains most actively transcribed genes. Histones and DNA are chemically modified with epigenetic markers that influence chromatin structure by altering the electrostatic nature of the chromatin or by altering the affinity of chromatin-binding proteins. DNA can be modified by methylation of cytosine bases10. The enzymes that methylate DNA are called DNA methyltransferases.
In humans the de novo DNA methyltransferases DNMT3A and DNMT3B methylate the genome during embryonic development, whereas the maintenance DNA methyltransferase DNMT1 methylates hemimethylated DNA following mitosis. Methylated DNA is suppressive of gene expression, as it attracts methylcytosine binding proteins that promote chromatin condensation into transcriptionally repressive conformations. In mammals, only cytosines preceding guanines (CpG dinucleotides) are known to be highly methylated. CpG dinucleotides are underrepresented and widely dispersed in the human genome. Although the majority of CpGs are located in non-coding regions and typically methylated, most remaining CpG dinucleotides are found in clusters upstream of gene coding sequences. These clusters, called CpG islands, are typically non-methylated so as to allow gene expression.
Histones are subject to several different covalent modifications, including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation1,8. The hypothesis of the histone code was developed to suggest that combinations of histone modifications ultimately control gene expression. While it is not clear that this hypothesis is universal, several supporting examples have been reported. Histone modifications can have varying effects owing to the type of modification and the location of the modification on the histone.
The best-characterized histone modifications are acetylation and methylation. Acetylation of histone lysine residues is associated with euchromatin because it weakens the charge attraction between histone and DNA, serving to decondense chromatin and facilitate transcription. Histone methylation can be either repressive or activating, depending upon location. For example, methylation of the lysine at the fourth residue of histone 3 (H3K4Me) promotes a transcriptionally active conformation, whereas H3K9Me promotes a transcriptionally repressive conformation. H3K36Me can be activating or repressive, depending upon proximity to a gene promoter region.
Histones are not the only proteins that interact with DNA in chromatin. Nucleosome remodeling complexes manipulate chromatin structure, thereby affecting gene silencing and expression. Chromatin remodeling proteins affect chromatin structure in various ways. They can expose DNA wrapped in nucleosomes by sliding histones along the DNA, or detach the histone octamer completely from a DNA sequence. They can also remove only the H2A-H2B subunits of the histone octamer, leaving the H3-H4 subunits, and resulting in a non-canonical structure. In recent studies, the reverse reaction has also been observed for the insertion of variant histone proteins. Not all nucleosome remodeling proteins possess the same functions.
The SWI/SNF family, which is found in yeast, fly, plants, and humans, can slide nucleosomes, eject histones, and displace H2A-H2B dimers. The ISWI family, which is only found in mammals, is capable of sliding, but not histone ejection. Some ISWI family proteins can displace H2A-H2B dimers, while others cannot. The Mi-2/NuRD complex has DNA sliding activity, and, unique among chromatin remodeling complexes, also carries histone deacetylase activity2.
Non-Coding RNAs in Epigenetics
RNAs are known to play several interesting roles in the control of chromatin structure. In plants a process called RNA-directed DNA methylation uses siRNAs generated by RNA Polymerase IV and the DICER LIKE 3 protein to localize the DNA methyltransferase DRM2 to its specific target sequence7. Another epigenetic trait dependent upon RNA is X chromosome inactivation1. This process occurs in female mammals to control expression dosage of the genes encoded on the X chromosome. In females, one of the two X chromosomes is inactivated in a process featuring the expression of the RNA Xist, which binds to the entire length of the chromosome from which it is transcribed. Xist recruits chromatin-remodeling proteins and blocks transcription machinery from binding to the inactivated chromosome.
Model Systems and the Diversity of Epigenetics Research
The current concept of epigenetics is derived from observations of several model species ranging from unicellular fungi to mammals1. Yeast have been used as model systems to study chromatin structure. Work on the budding yeast Saccharomyces cerevisiae has helped elucidate chromosome structure and telomere silencing. The Sir family of proteins, all but one of which are unique to budding yeast, maintains chromatin silencing in S. cerevisiae. Perhaps the best known example of epigenetic gene silencing in S. cerevisiae is mating-type switching, which features the translocation of alleles between transcriptionally active and silent regions of a chromosome. The fission yeast, Schizosaccharomyces pombe, is also a model for chromatin structure, but differs significantly from budding yeast.
Instead of using the Sir proteins to control chromatin structure, S. pombe is more similar to higher eukaryotes, using histone modification and siRNA. Not surprisingly, SWI/SNF chromatin remodeling proteins of S. pombe are also more similar to those of higher eukaryotes than the divergent S. cerevisiae.
Although DNA methylation is not observed in yeast, it is present in the fungus Neurospora crassa, making it a model organism for DNA methylation studies1. A phenomenon called repeatinduced point mutation (RIP) was discovered in N. crassa. RIP is a genome defense mechanism in which repeated sequences are prone to cytosine methylation and deamination to induce G:C to A:T mutations. Another model organism that has contributed to epigenetics is the protozoan Tetrahymena thermophila. Epigenetic mechanisms are used to regulate gene expression on the two nuclei of ciliates. Ciliates have a micronucleus that is dormant during most of the life cycle, but active during reproduction. The partition of active and suppressed nuclei in T. thermophila enabled the identification of histone variants, the first histone acetyltransferase, histone lysine methylation, and histone phosphorylation. T. thermophila is also an interesting organism for the study of RNAi. It has one RNAi pathway that functions in gene silencing and a second pathway that functions in DNA rearrangement and deletion during sexual reproduction1.
The fly Drosophila melanogaster is a classic genetic model organism that is also a model for epigenetic research. Observations of epigenetic phenomena in Drosophila were made decades before the term epigenetics was devised3. Position effect variegation – the change in phenotype due to the change of a gene’s position in the genome- was first described in the 1930s through observations of eye color (Figure 3). Drosophila normally have red eyes, but mutations in the white gene cause white eyes. However, some flies have patchy red and white eyes, but not because of the white mutant allele. Instead, some cells have undergone a chromosomal inversion that moved the wild type white gene in close proximity to pericentromeric heterochromatin, suppressing expression. Further research of suppressors and enhancers of position effect variegation have lead to the discovery of more epigenetic factors such as chromatin remodeling proteins and histone modifying proteins. The Polycomb group (PcG) and Trithorax group (TrxG) proteins were originally discovered in Drosophila, but are the subject of research in species ranging from yeast to human12. PcG proteins repress transcription by binding to Polycomb response elements (PRE). Different PcG protein complexes have been shown to methylate histones or bind to modified histones, making transcriptionally suppressive architectural changes. Conversely, the TrxG proteins bind to PREs, but serve to activate transcription via histone modification and chromatin remodeling.
Three other model organisms well-established in genetic research are also important for epigenetic research. Plants, such as Arabidopsis thaliana, have epigenetic mechanisms as sophisticated as mammals, including RNAi pathways, DNA methylation, histone modification, and chromosome remodeling complexes7. The worm, Caenorhabditis elegans, long used to study development, has been used to study cellular differentiation, X-linked dosage compensation, and RNAi1. Finally, mice, as mammals, are more similar to humans than any of these model systems, and are used as models for epigenetic research, particularly in embryology and stem cell research, but also in environmental studies, including effects of behavior and nutrition. Epigenetics is a prominent theme in the study of human development from fertilization through aging and to death11,13.
Epigenetic markers control the expression of genes that function in embryonic development, but other epigenetic programming events occur concurrently. These include the erasure and re-establishment of DNA methylation markers, genetic imprinting, X-chromosome inactivation, the development of pluripotent stem cells, and the differentiation of somatic cells. Although the most dramatic epigenetic events, such as the establishment of the epigenome, take place during embryonic development, the maintenance of the epigenetic state is important throughout life for the production of differentiated cells from adult stem cells and proper gene expression in specific cell types. A review of epigenetics and embryonic development can be found in a separate article in this Newsletter (see Epigenetic Regulation in Mammalian Genomes). However, the epigenome is dynamic, as it exhibits changes during the aging process. For example, gene promoters become hypermethylated as an individual ages, whereas the CpG sequences of non-coding centromeric repeat regions become hypomethylated. Interestingly, many of the known age related DNA methylation markers are also associated with disease.
In the last several years scientists have discovered numerous DNA methylation markers that are correlated with cancer. In a variety of cancers tumor suppressor genes such as p16, p14, and MGMT exhibit hypermethylation in the CpG islands upstream of the coding regions, repressing their expression4. Conversely, other genes such as MAGE and uPA, are usually methylated and repressed, but are hypomethylated and expressed in some cancers. The role of DNA methylation in cancer is reviewed in greater detail in another article in this Newsletter (page 9). Furthermore, a host of other diseases have epigenetic etiologies6,15. Prader-Willi syndrome, Angelman syndrome and pseudohypoparathyroidism, are all the result of uniparental disomy (UDP), a condition in which a person inherits both homologous chromosomes (or segments of chromosomes) from the same parent. UDP can be the result of gene deletion, translocation, or a defect in imprinting. Other epigenetic diseases are caused by mutations in genes necessary for chromatin structure. Rett Syndrome, for example, is caused by a genetic defect in MECP2, a methyl-CpG-binding protein that functions in gene repression.
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