What is Epigenetics?

“Epigenetics” refers to covalent modification of DNA, protein, or RNA, resulting in changes to the function and/or regulation of these molecules, without altering their primary sequences. In some cases, epigenetic modifications are stable and passed on to future generations, but in other instances they are dynamic and change in response to environmental stimuli. Nearly every aspect of biology is influenced by epigenetics, making it one of the most important fields in science.

Epigenetics and Me

Why do some foods cause health problems and others make us healthy? How does stress impact our long-term well-being? Why is it that the older we get, the more likely it is that age-related illness will strike us? Unlocking the secrets behind these and other questions has the potential to revolutionize life as we know it. The emerging field of epigenetics is aiming to do just that.

The importance of nature versus nurture has long been disputed. It cannot be denied that environment greatly influences how a child grows and develops, nor can it be denied that our DNA is the blueprint that makes us who we are. Epigenetics merges these two seemingly contradictory lines of thought to explain how environmental factors cause physical modifications to DNA and its associated structures, which result in altered functions.

The most commonly known epigenetic modification is DNA methylation. Although many technologies have been developed in the past to characterize genomic DNA methylation, none of them has been able to efficiently determine DNA methylation patterns on a genomic scale. Until now.

More on Epigenetics

Many cellular processes, including gene expression and DNA replication, are often regulated by mechanisms that fall into the category of “classical genetics”. This generally means that they are controlled by elements such as promoters, enhancers, or binding sites for repressor proteins, that are present or absent in the DNA sequence. An example of this type of regulation is the control of expression of a cellular oncogene. In normal (non-cancer) cells, this gene would not be expressed. However, in a cancer cell, this gene could have aquired a mutation, which is a change to the DNA sequence, that allows the oncogene to be expressed, and thus can contribute to the progression of cancer.

In addition to the regulatory mechanisms of classical genetics, nearly all cellular processes can also be regulated by epigenetic mechanisms. Epigenetic mechanisms can be just as important to biological events as genetic mechanisms, and can also result in stable and heritable changes. However, the big difference between genetic and epigenetic regulation is that epigenetic mechanisms do not involve a change to the DNA sequence, whereas genetic mechanisms involve the primary DNA sequence and changes or mutations to this sequence. Epigenetic regulation involves the modification of DNA and the proteins associated with DNA, which results in changes to the conformation of DNA and accessibility of other factors to DNA, without a change to the sequence of the DNA.

The Greek prefix “epi” means “on top of” or “over”, so the term “Epigenetics” literally describes regulation at a level above, or in addition to, those of genetic mechanisms. Common types of epigenetic regulation are DNA methylation and hydroxymethylation, histone modification, chromatin remodeling, and regulation by small and large non-coding RNAs. The field of epigenetics was given its name and a vague definition only ~50 years ago, but is now a dynamic and rapidly expanding discipline, challenging and revising traditional paradigms of inheritance.

Through epigenetics, the classic works of Charles Darwin, Gregor Mendel, and Jean-Baptiste Lamarck and others 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 cellular differentiation, embryology, the regulation of gene expression, aging, cancer, and other diseases.

Epigenetic Regulation

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To understand epigenetics requires an understanding of chromatin structure. Chromatin, which is organized into repeating units called nucleosomes, is the complex of DNA, proteins, 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. Histones and DNA can be chemically modified with epigenetic marks that influence chromatin structure by altering the electrostatic nature of the chromatin and/or by altering the affinity of interactions with chromatin-binding proteins. In mammalian cells, most of the chromatin exists in a condensed, transcriptionally silent state called heterochromatin. Heterochromatin generally has high levels of DNA methylation and the nucleosomes in heterochromatin contain histones with post-translational modifications that are conducive for gene silencing. Euchromatin is less condensed, and contains most actively transcribed genes. Euchromatin exhibits lower levels of DNA methylation, relative to heterochromatin, and the nucleosomes in euchromatin contain histones with modifications that promote gene expression.

DNA can be modified by methylation of cytosine bases5. The enzymes that methylate DNA are called DNA methyltransferases. In humans, the DNA methyltransferases DNMT3A and DNMT3B methylate the genome during embryonic development, whereas the maintenance DNA methyltransferase, DNMT1, methylates hemimethylated DNA (methylated on only one strand) following mitosis. Methylated DNA generally represses 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 relative to other dinucleotide combinations and are widely dispersed throughout the human genome. The majority of CpGs are located in non-coding regions and are typically methylated. However, many of the remaining CpG dinucleotides are found in clusters upstream of a gene’s coding sequence, in domains referred to as CpG islands. These CpG islands are typically unmethylated, or hypomethylated, to allow for the expression of downstream genes. DNA regions near CpG islands, referred to as island “shores” are often methylated and may serve to fine-tune the expression levels of nearby genes.

Histones are also subject to several different covalent modifications, including methylation, acetylation, phosphorylation, ubiquitination, and sumoylation1, 4. 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 based on 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. Acetylated histone residues can also serve as binding sites for other histone-modifying enzymes or chromatin-remodeling factors that promote gene expression. Histone methylation can be either repressive or activating, depending on the location of the methylated residue. For example, methylation of the lysine at the fourth residue of histone H3 (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 specific subunits of the histone octamer, replacing them with histone variants, resulting in a non-canonical structure. Not all nucleosome-remodeling proteins possess the same functions. The SWI/SNF family can slide nucleosomes, eject histones, and displace H2A-H2B dimers. The ISWI family 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.

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 specific subunits of the histone octamer, replacing them with histone variants, resulting in a non-canonical structure. Not all nucleosome-remodeling proteins possess the same functions. The SWI/SNF family can slide nucleosomes, eject histones, and displace H2A-H2B dimers. The ISWI family 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.

References

1. Allis, CD et al. In CD Allis, T Jenuwein, & D Reinberg (Eds.), Epigenetics (p. 23-62). New York: Cold Spring Harbor Laboratory Press (2007).
2. Cairns, BR. Nat Struct Mol Biol 14: 989-996 (2007).
3. Henderson, IR & Jacobsen, SE. Nature 447: 418-424 (2007).
4. Kouzarides, T & Berger, SL. In CD Allis, T Jenuwein, & D Reinberg (Eds.), Epigenetics (p. 191-210). New York: Cold Spring Harbor Press (2007).
5. Miranda, TB & Jones, PA. J Cell Physiol 213: 384-390 (2007).

Epigenetics Research Techniques

The epigenetic modifications of DNA, histones, and RNA can often be distinguished from their unmodified counterparts using both standard molecular biology techniques and Next-Gen whole-genome approaches. Methods such as bisulfite treatment for investigating DNA methylation and chromatin immunoprecipitation for investigating histone modifications and protein-chromatin interactions are extremely powerful for performing experiments to determine the mechanisms of epigenetic regulation and the importance of epigenetics to different aspects of gene expression and cell biology.

The methylation of cytosine in DNA introduces biochemical properties that can be used to distinguish methylated DNA from non-methylated DNA. Most importantly, sodium bisulfite deaminates cytosine into uracil, but 5-methylcytosine is resistant to this conversion, meaning a base change following bisulfite treatment can be used to experimentally determine DNA methylation status. Additionally, there are several methylation-sensitive restriction endonucleases whose ability to cut DNA at specific sequences is dependent upon the DNA methylation state of the sequence. These features, when coupled with other common molecular biological methods, such as DNA sequencing, PCR, real-time PCR, Southern blotting, primer extension, HPLC, and MALDI-TOF MS, provide the epigenetic scientist with a variety of tools to investigate epigenetic processes1, 2. Additionally, methylated DNA immunoprecipitation (MeDIP) assays utilize an antibody that specifically recognizes methylated DNA, and the immunoprecipitated methylated DNA can be identified by PCR, Next-Gen sequencing, or microarray hybridization approaches. As DNA sequencing becomes more affordable and capable of high throughput approaches, it is becoming feasible for routine whole-genome sequencing of bisulfite-converted or MeDIP-precipitated DNA to detect genome-wide DNA methylation profiles.

Histone modifications can also be studied using antibodies specific for particular histone modifications, and the chromatin associated with those modifications can be detected by chromatin immunoprecipitation (ChIP) assays. ChIP assays are also very useful to determine whether other non-histone proteins, such as chromatin-remodeling and histone-modifying factors are associated with chromatin. The chromatin immunoprecipitated in ChIP assays can be identified by performing PCR for specific genes of interest, or genome-wide by hybridization to a microarray (ChIP-chip), or by direct Next-Gen sequencing of the immunoprecipitated chromatin (ChIP-Seq).

References

1. Esteller, M. N Engl J Med 358: 1148-1159 (2008). 2. Suzuki, M. Nat Rev Genet 9: 465-476 (2008).

Model Systems for Epigenetics Research

The current concept of epigenetics is derived from observations of several model species ranging from unicellular fungi to mammals1. Each model organism offers a different advantage and all are important for learning about the processes and mechanisms involved in epigenetic regulation.

Multiple species of 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. One of the best known examples 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 repeat induced 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 partitioning 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 fruit 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 coined2. 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 flies 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 a mutant white allele. Instead, some cells in these flies have undergone a chromosomal inversion that moved the wild type white gene in close proximity to pericentromeric heterochromatin, which spreads to suppresses the expression of the of the white gene. 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, and are now the subject of intense research in species ranging from yeast to humans4. PcG proteins repress transcription in Drosophila by binding to Polycomb response elements (PRE). Different PcG protein complex components have been shown to methylate histones or bind to modified histones, making transcriptionally repressive 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 complexes3. The worm Caenorhabditis elegans, long used to study development and neurobiology, has been used to study epigenetic-mediated 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 on epigenetic states.

References

1. Allis, CD et al. In CD Allis, T Jenuwein, & D Reinberg (Eds.), Epigenetics (p. 23-62). New York: Cold Spring Harbor Laboratory Press (2007). 2. Elgin, SC & Reuter, G. In CD Allis, T Jenuwein, & D Reinberg (Eds.), Epigenetics (p. 81-100). New York: Cold Spring Harbor Laboratory Press (2007). 3. Henderson, IR & Jacobsen, SE. Nature 447: 418-424 (2007). 4. Scheuttengruber, B et al. Cell 128: 735-745 (2007).

Epigenetics in Development & Disease

Epigenetics is a prominent theme in the study of human development from fertilization through aging and to death3, 4. Epigenetic marks control the expression of genes that function in embryonic development, and other epigenetic programming events occur concurrently. These include the erasure and re-establishment of DNA methylation marks, 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 initial 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. The epigenomic state is dynamic and tightly regulated, and misregulation of epigenetic patterns are observed in many human diseases and multiple types of cancers.

Changes in the epigenome are also correlated with 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 biomarkers are also associated with disease.

In the last several years, scientists have discovered numerous DNA methylation biomarkers 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 expression1. Conversely, other genes such as MAGE and uPA, are usually methylated and repressed, but are hypomethylated and expressed in some cancers. Furthermore, a host of other diseases have epigenetic etiologies2, 5. 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.

References

1. Esteller, M. Nat Rev Genet 8: 286-297 (2007). 2. Feinberg, AP. Nature 447: 433-440 (2007). 3. Reik, W. Nature 447: 425-432 (2007). 4. Sedivy, JM et al. Exp Cell Res 314: 1909-1917 (2008). 5. Zoghbi, HY & Beaudet AL. In CD Allis, T Jenuwein, & D Reinberg (Eds.), Epigenetics (p. 435-456). New York: Cold Spring Harbor Laboratory Press (2007).

Epigenetic Biomarkers & Treatments

The wealth of new data and knowledge relating to epigenetics obtained in recent years highlights a vibrant future for epigenetics research. Of great importance for the future are the integration of high-throughput sequencing technologies and the means to maintain and manipulate the large amount of data produced by sequencing epigenomes. The influx of epigenomic data will augment the growing database of known epigenetic marks and encourage more research to describe the functions of these marks in various tissues, stages of development, and disease states. As more epigenetic marks are associated with specific diseases, tools can be developed to diagnose patients and gauge the severity of disease. There is also great interest in therapeutic epigenetics. Several drugs, such as DNA methyltransferase inhibitors and histone deacetylase inhibitors, are already used in cancer treatment1.

There are issues with specificity and efficacy of these drugs, so further research into their mechanisms is needed to develop better therapeutic agents. Likewise, better understanding of the various epigenetic diseases and syndromes may lead to effective drugs designed to overcome epigenetic defects. Recent advances in embryology have posed more questions related to epigenetics, particularly to the mechanics of genome demethylation and the re-establishment of methylation in early embryonic development3. The epigenetic marks associated with the production of pluripotent embryonic stem cells is also of high interest for its relevance in reprogramming differentiated cells to make induced pluripotent stem cells2. Beyond embryonic development, phenomena relating to the acquisition of epigenetic marks during an organism’s life span and their passage to offspring is a tantalizing area of research with many questions to be answered regarding mechanisms, and environmental influences. It is a very exciting time to be studying epigenetics, and this field of research has the potential to completely transform medicine and greatly improve human lives.

References

1. Esteller, M. Nat Rev Genet 8: 286-297 (2007). 2. Mikkelsen, TS et al. Nature 454: 49-55 (2008). 3. Reik, W. Nature 447: 425-432 (2007).

Epigenetics Glossary of Terms

5-hydroxymethylcytosine (5hmc): an epigenetic modification of DNA; can result from the oxidation of 5-methylcytosine modification by the Tet family of enzymes.

5-methylcytosine (5mC): an epigenetic modification of DNA that usually occurs at CpG dinucleotides; the 5mC modification usually correlates with repressed gene expression.


Adult stem cell: multipotent stem cells present in differentiated tissue; also known as tissue-specific stem cells.

Bisulfite conversion: the deamination of non-methylated cytosine bases to uracil by treatment with sodium bisulfite (NaHSO3); 5mC bases are resistant to bisulfite conversion.

Bisulfite sequencing: determining the sequence of bisulfite-converted DNA; considered the “gold standard” of DNA methylation analysis.

Blastocyst: an early embryonic structure consisting of distinct outer trophectoderm cells, which develop into the placenta, and the inner cell mass, which develops into the fetus.

Body methylation: methylation of DNA bases within coding sequences of actively transcribed genes found within euchromatin.

Bromodomain: a protein motif that binds acetylated lysine residues; commonly present in proteins that recognize acetylated histones, such as chromatin-remodeling factors.

ChIP-on-chip: a combination of chromatin immunoprecipitation and DNA hybridization to genomic microarrays (also known as ChIP-chip).

ChIP-Seq: a method combining chromatin immunoprecipitation and DNA sequencing to analyze specific DNA-protein interactions. Next-gen DNA sequencing is often performed, resulting in a genome-wide analysis of protein-chromatin interactions.

Chromatin: the complex of DNA, histones, RNA, and other proteins that comprise the structural basis of chromosomes.

Chromatin immunoprecipitation (ChIP): a method used to identify proteins bound to DNA and the sequence to which they bind using an antibody to specifically immunoprecipitate the protein of interest; the DNA sequence that co-precipitates with the protein can be identified by PCR, hybridization, or sequencing.

Chromodomain: a motif of 40-50 amino acids common to proteins that function in chromatin remodeling; may function in binding DNA, RNA, and protein; often binds methylated histones.

Combined Bisulfite Restriction Analysis (COBRA): a quantitative technique for the detection of methylated DNA in which DNA is subjected to bisulfite conversion and digestion with restriction endonucleases that are specific for sequences containing CpG sites (and thus are subject to methylation); the digestion products are a direct reflection of DNA methylation at the restriction sites.

Constitutive heterochromatin: heterochromatin, often located near centromeres (also known as pericentric heterochromatin), that is irreversibly silenced; DNA within constitutive heterochromatin is typically AT-rich.

CpG islands: regions of DNA enriched for CG dinucleotides; CpG islands are typically 300-3000 bp long, located upstream of gene coding regions, and usually protected from DNA methylation.

De novo methylation: the establishment of genomic DNA methylation during embryonic development; in mammals, after genomic DNA is demethylated in the zygote, the methyltransferases Dnmt3a and Dnmt3b methylate DNA between embryonic implantation and gastrulation.

Differentially DNA-Methylated Region (DMR): a region of DNA that is methylated differentially in the two chromosomes of a diploid cell; often associated with genomic imprinting.

DNA methylation: a heritable, reversible epigenetic modification in which a methyl group is covalently added to a DNA sequence, usually the 5th carbon of the cytosine pyrimidine ring in a CpG dinucleotide, although CpHpG and CpHpH sequences can be methylated in plants.

DNA methyltransferase: an enzyme that catalyzes the addition of a methyl group to a DNA nitrogenous base; the 5mC class adds a methyl group to the 5-carbon position of cytosine bases; humans produce DNMT1, the maintenance methyltransferase, which is active at hemimethylated sites, and the methyltransferases DNMT3a and DNMT3b, which function during embryonic development and shortly after birth.

Dosage compensation: mechanisms involved in equalizing the expression of genes encoded on the X-chromosome between the two sexes; some examples are X chromosome inactivation in female mammals, X chromosome upregulation in male Drosophila flies, and partial repression of both X chromosomes in hermaphrodite C. elegans worms.

Embryo: an individual organism between the onset of multicellularity through birth; alternatively defined as beginning with implantation of the blastocyst in the uterus; in human development the term is usually used until the 8th week of pregnancy, from which point the term fetus is used.

Embryonic stem cell (ES cell): pluripotent stem cells found in the blastocyst, inner cell mass, and embryo.

Epiallele: variations in the epigenetic status of a gene or locus; often associated with differential methylation.

Epigenetic mark: a modifying moiety that carries an epigenetic signal; examples include methylation of DNA, methylation, acetylation, phosphorylation, ubiquitination, and sumoylation of histones.

Epigenetic silencing: the suppression of gene transcription or expression because of epigenetic factors such as RNAi, DNA methylation, histone modification, or chromatin remodeling.

Epigenetic therapy: application of chemical compounds, such as DNA methyltransferase inhibitors (e.g. 5-azacytidine, 5-aza-2’-deoxycytidine), to target epigenetically regulated mechanisms in patients.

Epigenetics: heritable traits that can be maintained through cell division and sexual reproduction that are not the result of a change in DNA sequence; epigenetic factors include chromatin conformation, DNA methylation, histone modification, and RNAi.

Epigenome: all of the epigenetic marks present throughout the genome of a cell.

Euchromatin: decondensed chromatin that is conformationally favorable for transcription; euchromatin typically has less DNA methylation than heterochromatin, and its associated histones have modifications that favor gene transcription.

Facultative heterochromatin: heterochromatin that may become transcriptionally active in specific cell development fates.

Hemimethylated: the status of a symmetrical DNA sequence (such as CG or CHG) that is methylated on only one strand.

Heterochromatin: condensed chromatin that is conformationally unfavorable for transcription; heterochromatin typically has more DNA methylation than euchromatin, is associated with histones containing repressive modifications, and can be associated with repressive non-coding RNAs.

Histone acetyltransferase (HAT): enzyme that acetylates histones at specific lysine residues.

Histone deacetylase (HDAC): enzyme that remove acetyl groups from N(6)-acetyl-lysine residues on a histone.

Histone: chromosomal architectural proteins that bind DNA within nucleosomes; in eukaryotes there are 4 core histones, H2A, H2B, H3, and H4, the non-nucleosomal linker histone H1, and variant histones.

Histone code: the hypothesis that the locations and types of histone modifications, through chromatin remodeling and/or recruitment of transcription factors, predicts the effects of those modifications on gene expression.

Histone methyltransferase: a class of enzymes that add methyl groups to specific histone residues; members include histone-lysine N-methyltransferase and histone-arginine N-methyltransferase.

Histone modification: posttranslational addition or removal of epigenetic marks from histones; includes methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and the removal of these marks.

Histone variants: paralogous histones that can replace the major core histone proteins and may have distinct gene regulatory functions; also known as replacement histones.

Hypermethylation: increase in the level of DNA methylation in a population of cells relative to a reference or normal sample; may be used to describe a specific nucleotide or a group of nucleotides.

Hypomethylation: decrease in the level of DNA methylation in a population of cells relative to a reference or normal sample; may be used to describe a specific nucleotide or a group of nucleotides.

Imprinting: epigenetic regulation in which maternally and paternally inherited alleles are differentially expressed owing to cis-acting modifications of DNA or histones inherited from parental chromosomes.

Inner cell mass (ICM): pluripotent cells located in the interior of the blastocyst that develop into the fetus.

Induced Pluripotent Stem (iPS) Cells: differentiated cells reprogrammed to pluripotency by ectopic expression of reprogramming factors such as Oct3/4, Sox2, Klf4, and c-Myc.

Large non-coding RNA: non-coding RNA larger than 200 nucleotides; can have roles in epigenetic regulation of gene expression

Loss of imprinting (LOI): activation of an allele normally silenced by genomic imprinting; LOI causes excess gene product to be produced and is often associated with tumorigenesis.

Methylation sensitive PCR (MSP): a technique used to determine the methylation status of specific DNA sequences by PCR amplification of a bisulfite-converted template with different primer sets that distinguish methylated DNA and non-methylated (C?T converted) DNA.

Methylated DNA Immunoprecipitation (Methyl-DIP or MeDIP): a technique used to identify methylated DNA by precipitation with an antibody specific for 5mC and followed by detection of precipitated DNA by PCR, hybridization to a genomic microarray, or sequencing.

Methylation-sensitive Single-Nucleotide Primer Extension (Ms-SNuPE): a technique used to query methylation status of a targeted base bisulfite conversion followed by primer extension with labeled dCTP or dTTP to distinguish methylated and non-methylated DNA.

microRNA (miRNA): Small RNA molecules (usually 21-23 nulceotides) that play a role in regulating gene expression by transiently suppressing translation of an mRNA molecule or by directing its cleavage.

Multipotency: the property of stem cells describing their ability to differentiate into cells of a specific lineage, but not other lineages; example: hematopoietic stem cells can differentiate into multiple types of blood cells, but not into muscle cells, skin cells, or cells of any other lineage.

Non-coding RNA (ncRNA): RNA molecules that do not contain protein-coding potential; ncRNAs can be highly abundant and functionally important RNA molecules that can epigenetically regulated gene expression or other cellular processes, such as nuclear organization and splicing; the majority of most genomes are transcribed as non-coding RNA; examples include microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), large non-coding RNAs (lncRNAs), natural antisense transcripts (NATs) and large intergenic RNAs (lincRNAs).

Nucleosome: the repeating unit of chromatin structure; one nucleosome is comprised of 147 bp of DNA wrapped around a protein octamer including two molecules each of the core histones H2A,histone H2B, histone H3, and histone H4.

Piwi-interacting RNA (piRNA): the largest class of small non-coding RNA molecules expressed in animal cells; piRNAs are 26-34 nucleotides in length and form RNA-protein complexes through interactions with piwi proteins; piRNAs differ from miRNAs and siRNA in both methods of biogenesis and function, but are known to play a role in silencing retrotransposons in germ cells.

Pluripotency: the property of embryonic stem cells to differentiate into cells of any three germ layers (endoderm, mesoderm, ectoderm); pluripotent cells are more differentiated than totipotent cells and less differentiated than multipotent cells.

Polycomb-group (PcG): a group of proteins functioning in histone modification, histone binding, or DNA binding that facilitate gene repression; named for the Drosophila melanogaster Polycomb gene.

Position effect variegation (PEV): the variable silencing of a gene because of its proximity to heterochromatin.

RNA interference (RNAi ): posttranscriptional gene silencing mediated by small RNA sequences that are capable of hybridizing to a target mRNA sequences.

Small activating RNA (saRNA): miRNAs that can activate gene expression by binding to promoter sequences.

Small interfering RNAs (siRNA): small RNAs (21-24 nt) that function in gene silencing, heterochromatin assembly, and RNA directed DNA methylation.

Somatic cell nuclear transfer (SCNT): transplantation of a diploid nucleus from a somatic cell to an enucleated egg cell, artificially mimicking fertilization and potentiating development; SCNT is used for reproductive cloning.

Stem cell: an undifferentiated cell that is capable of producing daughter stem cells by mitosis or differentiating into specialized cell types.

Totipotency: the property of fertilized egg cells and early zygotic cells to differentiate into embryonic and extraembryonic cells.

Trithorax-group (trxG): a group of proteins functioning in transcriptional regulation, chromatin remodeling, and histone lysine methyltransferase activity that facilitate gene expression; named for the Drosophila melanogaster trithorax gene.

Tumor suppressor gene: a gene that functions in regulation of cell cycle and/or promotes apoptosis, protecting the individual from the development of cancer; tumor suppressor genes are often mutated in cancer.

Uniparental disomy: the condition in which an offspring inherits both copies of a chromosome (or a segment thereof) from the same parent. Genomic imprinting under such conditions can cause loss of expression or aberrant expression of alleles.

X-inactivation: a dosage compensation mechanism in which one of two X-chromosomes in the cells of female mammals is epigenetically silenced.

Xist: X inactive specific transcript; the non-coding RNA transcribed from the X-inactivation center (Xic) that binds along the entire chromosome from which it is transcribed to mediate X chromosome inactivation in placental mammals.

Zygote: the totipotent cell that results from the union of the oocyte and sperm gametes.

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