Epigenetics involves cellular processes that alter the expression of genes and change the phenotype of an individual; however, these processes do not alter the nucleotide sequence within the DNA. As a result, epigenetic changes are not considered to be mutations. Instead, epigenetic mechanisms modify the structure of the DNA or the chromatin (i.e., the histones within nucleosomes) surrounding a gene or, in one case, alters the structure of an entire chromosome. These structural modifications either activate or silence transcription.
The epigenetic factors that modify the DNA or alter chromatin structure can be established during the formation of gamete cells, embryonic development, or in adult organism in the response to environmental agents. Processes that promote epigenetic changes during gamete formation include genomic imprinting. In genomic imprinting, the epigenetic changes established during gamete formation in one of the two parents are passed to their offspring. These epigenetic changes that are inherited are said to display epigenetic inheritance. Epigenetic changes established during embryonic development include X chromosome inactivation (XCI), and the processes that govern the differentiation of embryonic cells into adult cell types, including muscle cells, neurons, or epithelial cells. Environmental factors that influence epigenetic changes in an adult organism include diet, stress, the unique environment of space, and the toxins found in cigarette smoke.
Epigenetic changes established during embryonic development or by environmental factors are often permanent in the individual. For example, when an epigenetic change is established in a cell, this affected cell divides by mitosis, and the epigenetic changes are preserved in the daughter cells. This allows daughter cells to "remember" the epigenetic changes of the parental cell. Even though the epigenetic changes may be permanent in the individual, most epigenetic changes are erased during gamete formation and as a result, are not passed on to offspring. An exception to this general rule is genomic imprinting (see below).
Three major epigenetic mechanisms influence the transcription of genes (see figure 15.1). These epigenetic mechanisms include:
Genomic imprinting involves inheriting a silenced gene from one parent. Since the active copy of the gene is inherited from the other parent, genomic imprinting causes the offspring to only express one of the two possible alleles that control a trait (monoallelic expression). The genomic imprint (in other words, the DNA methylation pattern) is established on the allele during gamete formation in one of the parents, is passed on to the offspring, and is retained throughout the lifetime of the offspring.
A well known example of genomic imprinting involves the regulation of the insulin-like growth factor 2 (Igf2) gene that controls body size in mice (see figure 15.2). There are two Igf2 alleles: the Igf2 allele produces normal body size, while the Igf2 - allele produces dwarf body size. In the case of the Igf2 gene, the maternally-inherited allele is silenced, resulting in the offspring expressing the paternally-inherited allele only. For example, suppose a homozygous dwarf female (Igf2 - Igf2 -) mouse is mated to a homozygous normal male (Igf2 Igf2) mouse. All the offspring are normal body size because the offspring inherited the active Igf2 allele from the father (the Igf2 - allele from the mother has been silenced). Note that the offspring are heterozygous (Igf2 Igf2 -). If a homozygous normal female (Igf2 Igf2) mouse is mated to a homozygous dwarf male (Igf2 - Igf2 -) mouse, all the offspring are dwarf because the offspring inherited the active Igf2 - allele from their father (the Igf2 allele from the mother has been silenced). Note that the offspring are also heterozygous (Igf2 Igf2 -). The results of these two crosses violate Mendel’s laws of inheritance; the two crosses produce offspring with the same genotype (Igf2 Igf2 -), yet have different phenotypes.
Genomic imprinting has three stages (see figure 15.3):
Genomic imprinting involves DNA methylation patterns established during gamete formation. Genomic imprinting also involves several DNA sequences located near the Igf2 gene (see figure 15.4). The Igf2 gene in mice is located near another gene called H19. The function of the H19 gene is unknown; however, an enhancer sequence that functions to regulate the transcription of the Igf2 gene is located next to the H19 gene. DNA methylation occurs at two DNA sequences on each side of the Igf2 gene. The first DNA sequence is the imprinting control region (ICR) and is located between the H19 and Igf2 genes. A second DNA sequence called the differentially methylated region (DMR) is located downstream of Igf2.
During the formation of egg cells, both the ICR and the DMR sequences are unmethylated. The absence of methylation allows CTC-binding factor (CTCF) proteins to bind to cytosine-thymine-cytosine trinucleotide sequences (CTC sequences) within both ICR and DMR. The CTCF proteins bound to the ICR and DMR sequences also bind to each other, forcing a loop to form in the DNA. This loop containing the Igf2 is considered a heterochromatin structure. When the Igf2 gene is found within heterochromatin, an activator protein fails to bind to the enhancer DNA sequence adjacent to H19. As a result, the Igf2 gene is silenced in egg cells.
During the formation of sperm cells, the ICR and DMR sequences are methylated by de novo methylation. CTCF proteins fail to bind to methylated ICR and DMR sequences, preventing the formation of a DNA loop containing the Igf2 gene. Without the DNA loop, the Igf2 gene is essentially located within euchromatin. In the absence of loop formation, an activator protein binds to the enhancer next to the H19 gene, and the Igf2 gene is transcribed. Note that even though DNA methylation usually silences genes by preventing activator proteins from binding to enhancer DNA sequences (see Part 14); in the case of the Igf2 gene, DNA methylation prevents the binding of proteins that form heterochromatin. As a result, in mice, the methylation of sequences near the Igf2 gene activates transcription.
Genomic imprinting plays an important role in two genetic diseases in humans: Angelman syndrome (AS) and Prader-Willi syndrome (PWS). AS patients are thin, hyperactive, display mental deficiencies, have involuntary muscle contractions, and seizures. PWS patients have an uncontrollable appetite, obesity, diabetes, small hands/feet, and like AS patients, have mental deficiencies.
In addition to genomic imprinting, both AS and PWS involve an identical deletion in the long arm (q arm) of chromosome 15 (see figure 15.5). This region of chromosome 15 contains a small group of genes that are either maternally or paternally imprinted. For example, in AS, a gene on chromosome 15 called UBE3A is imprinted (silenced) during sperm formation, meaning that a sperm cell contains a silenced UBE3A allele. If an offspring inherits this silenced UBE3A allele from the father and inherits a deletion copy of chromosome 15 (missing the UBE3A gene) from the mother, the offspring has no fuctional UBE3A alleles. The absence of a functional UBE3A allele produces the AS disease phenotype.
The genes involved in PWS have not been determined; however, candidate genes on chromosome 15 include SNRPN (encodes a splicing factor protein) and NDN. In PWS, the SNRPN and NDN genes are imprinted (silenced) during egg formation. If the offspring inherits silenced SNRPN and NDN alleles from the mother and inherits a deletion copy of chromosome 15 (missing the SNRPN and NDN genes) from the father, the offspring lack functional SNRPN and NDN alleles. The absence of active SNRPN and NDN alleles is thought to produce the PWS disease phenotype.
We learned previously (see Part 2) that during embryogenesis, one of the two X chromosomes in female mammals is randomly chosen for inactivation. This random inactivation process is called X chromosome inactivation (XCI). After XCI occurs, the inactive X chromosome is maintained in a transcriptionally silent state with each cell division.
XCI is an epigenetic process that involves RNA-associated silencing. Recall that each X chromosome contains a region near the centromere called the X inactivation center (Xic) that plays an important role in XCI. Within the Xic are two genes, the Xist and Tsix genes. The Xist gene is expressed preferentially from the X chromosome that will be inactivated, while the Tsix gene is expressed from the X chromosome that will remain active. The XCI process involves the Xist and Tsix genes as follows (see figure 15.6):
Methylation of CpG sites contributes to fragile X syndrome. Fragile X syndrome is the most common form of inherited mental retardation, affecting 1 in 4000 males and 1 in 8000 females. Fragile X syndrome is named because of a site on the X chromosome that looks like a gap (see figure 15.7). This gap region tends to break and is therefore called a fragile site.
A trinucleotide repeat expansion (TNRE) mutation contributes to fragile X syndrome. In the TNRE that causes fragile X syndrome, the number of copies of a 5’-CGG-3’ sequence increases from generation to generation due to errors in DNA replication. When the number of 5’-CGG-3’ trinucleotides exceeds 230 copies, disease symptoms are produced. Recall that a TNRE mutation is also responsible for Huntington’s disease (see Part 7). In fragile X syndrome, the 5’-CGG-3’ trinucleotide repeats on the X chromosome are found in the first exon of the FMR1 gene. This beginning region of FMR1 is transcribed to produce the 5’-UTR in the mRNA. The expansion of the trinucleotide repeat is thought to form a CpG island within exon 1 of the FMR1 gene. These numerous CpG sites near the beginning of the FMR1 gene can become hypermethylated, silencing the transcription of the FMR1 gene. Since the protein product of the FMR1 gene is known to be expressed in the brain, the silencing of the FMR1 gene is thought to prevent protein production, leading to disease symptoms.
Epigenetic processes are important in the embryonic development of multicellular organisms. Embryonic development, starting with a fertilized egg and eventually producing an entire adult organism, initiates with the activation of genes that produce the overall body plan. For example, a group of genes called Hox genes specify the structures that form along the anteroposterior axis in animals. The Hox genes are actively transcribed during embryonic development when body parts are forming; the Hox genes are not transcribed in the cells of an adult organism. Epigenetic factors permanently silence Hox genes after the Hox protein products have been used to help form the body plan. Further, epigenetic processes that occur in development ensure that the different cell types in the body have specific phenotypes. For instance, muscle-specific genes are actively transcribed in muscles, whereas genes that specify another fate (neuron, epithelial cell) are permenantly silenced in muscle cells.
Two protein complexes, called the trithorax group (TrxG) and the polycomb group (PcG), are thought to regulate the epigenetic changes that occur during embryonic development and the differentiation of cell types. The TrxG protein complex is involved in gene activation processes, while the PcG protein complex is involved in gene silencing processes. The TrxG and PcG complexes are both histone methyltransferases, which accomplish epigenetic changes by adding methyl groups to the tail domains of histone H3. The TrxG complex recognizes histone H3 and adds three methyl groups (trimethylation) to a lysine amino acid at position 4 within the histone tail. Trimethylation of lysine 4 within histone H3 is an activating epigenetic mark. PcG also recognizes histone H3 and adds three methyl groups to a lysine at position 27; however, this modification to lysine 27 is a silencing epigenetic mark. Note that inactive X chromosomes (i.e., Barr bodies) have abundant trimethylation of histone H3 at lysine 27 (see above).
We will now consider how a PcG complex silences transcription, by describing how the Hox genes are inactivated after the Hox proteins have been used to help determine the body plan during embryogenesis (see figure 15.8). The silencing of the Hox genes occurs by:
It is important to note that the epigenetic silencing of the Hox gene is maintained during subsequent cell divisions, ensuring that the Hox genes remain silent in the adult organism.
One of the best examples of how environmental changes can influence transcription involves the Agouti gene in mice. The protein product of the Agouti gene catalyzes yellow pigment formation in the hairs of developing mouse pups. The Agouti gene has three alleles: A, a, and Avy. If a mouse has the AA or Aa genotype, the mouse coat color is agouti (brown). However, if the hairs from an agouti mouse are examined closely, each hair contains a stripe of yellow pigment sandwiched between layers of black pigment. In mice that have the aa genotype, the mouse is black due to the absence of yellow pigment production.
The Avy allele results in the overexpression of the Agouti gene. Homozygous Avy mice do not survive; however, if mice have AAvy or Avya genotypes, a variety of phenotypes are possible; some mice are yellow, some are mottled (black and yellow fur patches), and some are pseudoagouti (mostly black with a little yellow). The extent of the yellow fur color reflects the degree of Avy allele expression. If the Avy allele is highly overexpressed, then a yellow coat is produced. Intermediate levels of Avy allele overexpression produces the mottled phenotype. If the Avy allele displays low levels of overexpression, then the pseudo-agouti coat is produced. Interestingly, mice that have high levels of Avy allele overexpression are also prone to develop obesity, diabetes, and cancer (see figure 15.9). Mice with the AA, Aa, and aa genotypes are lean in appearance and are less susceptible to diabetes and cancer. Moreover, mice with lower Avy allele overexpression (i.e., pseudoagouti) are also leaner and more resistant to diabetes and cancer.
The variation in coat color phenotypes among Avy heterozygotes can be partially explained by their mother’s diet during pregnancy. When pregnant female mice are fed a diet supplemented with the vitamins folic acid and vitamin B12, the offspring that are heterozygous for the Avy allele tend to have darker coats and are leaner compared to the heterozygous offspring of mice fed a non-supplemented diet. Moreover, the offspring of the pregnant mice fed the diet rich in folic acid and vitamin B12 had higher levels of methylation of the CpG islands adjacent to the Avy allele than the heterozygous offspring of mice fed a non-supplemented diet. These results suggest that supplementing the diets of pregnant mice with folic acid and vitamin B12 increases DNA methylation near the Avy allele in the offspring, leading to decreased Avy allele overexpression and decreased risk of obesity and cancer. Importantly, the behavior of a mother mouse (eating a diet supplemented with folic acid and vitamin B12) was shown to influence the expression of a gene in her pups.
Another environmental agent that affects the Agouti phenotype is the chemical bisphenol A (BPA), a chemical found in some plastics, including plastics that were at one time commonplace in water bottles. The exposure of pregnant female mice to BPA produces more Avy heterozygote offspring that have yellow coats and are obese compared to the heterozygous offspring of mice not exposed to BPA. BPA is thought to inhibit the DNA methylation process, resulting in low levels of CpG island methylation near the Avy allele. As a result, the Avy allele is overexpressed in these mice, producing yellow coats and obesity. Incidentally, the addition of folic acid and vitamin B12 to the diet of these pregnant mice counteracted the effect of BPA. Again, the environment of the mother mouse influenced the expression of a gene in her pups.
Cancer is a condition characterized by uncontrolled cell division. Multiple mutations are typically required to convert a normal cell into a cancerous cell. If some of these mutations occur in the genes that produce proteins involved in DNA methylation, histones acetylation, or histone methylation, the epigenetic markings of many genes are altered. As a result, these mutations have epigenetic consequences, ultimately producing genes that are either overactive or not expressed sufficiently. For example, higher than normal expression of oncogenes can result in higher rates of cell division, promoting cancer. Alternatively, lower than normal expression of cancer-preventing tumor-suppressor genes can also promote the formation of cancer.
Mutations in the genes involved in DNA methylation have been associated with certain cancers. For example, mutations in the gene that produces DNA methyltransferase have been associated with acute myeloid leukemia. Note that the result of these mutations would be decreased methylation of the CpG islands adjacent to many genes, including oncogenes. As a result, these mutations lead to higher oncogene transcription and higher rates of cell division.
Mutations in the genes involved in histone modifications have also been linked to certain cancers. For example, mutations in the genes that produce histone acetyltransferases (HATs) have been associated with colorectal, breast, and pancreatic cancer. In this case, the defective HAT would result in lower expression of many genes, including tumor-suppressor genes. Since tumor suppressor genes encode repressor proteins that silence cancer genes, the overall effect is a higher rate of cancer formation.
Finally, certain chemicals are known to produce the epigenetic changes associated with cancer. For example, the polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke are associated with lung, breast, stomach, and skin cancer. These PAHs are thought to contribute to cancer by altering the DNA methylation patterns adjacent to many genes, including oncogenes and tumor-suppressor genes.
As we have seen, epigenetic processes are associated with several human diseases (AS, PWS, fragile X syndrome, acute myeloid leukemia, colorectal cancer). Scientists and physicians are interested in the possibility of treating diseases by converting the abnormal methylation or acetylation patterns in diseased cells back to the normal state. These types of restorative changes in epigenetic patterns are called epigenetic therapy. Inhibiting the DNA methylation process could reactivate silenced tumor suppressor genes in some cancers. One way to do this is to use DNA methyltransferase inhibitors, such as 5-azacytidine. Similarly, histone deacetylases (HDACs) remove acetyl groups from histone tails, potentially silencing tumor suppressor genes. HDAC inhibitors, such as phenylbutyric acid, could reverse this effect, activating the silenced genes.
Identical twins have the same DNA sequences. They also have the same epigenetic markings in their genome (epigenome) when they are born. However, beginning at birth, the epigenetic processes in the twins behave independently of each other, so that later in life, the twins have very different epigenomes. Twin studies suggest that environmental factors influence the epigenetic patterns within the genome and may explain why individuals with the same DNA sequences do not necessarily have the same overall phenotype. For example, if one twin has been diagnosed with schizophrenia, the identical twin has only a 40-50% chance of having schizophrenia, despite the twins having the same genome. This difference in phenotype may be explained by the different environmental factors encountered by each twin during their lifetime, resulting in distinctive epigenetic patterns in each genome.
Another example of how the environment can influence the epigenomes of identical twins involves Scott and Mark Kelly (see figure 15.11). Scott spent a year on the International Space Station, while his twin brother Mark stayed home. A recent NASA study showed that time in space altered the expression of many of Scott’s genes, including those genes involved in response to hypoxia (oxygen depletion) and inflammation. Moreover, the expression of approximately 7% of Scott’s genes has not returned to baseline levels even after spending several years on Earth since his time on the space station. This alteration in Scott’s gene expression is thought to be the result of epigenetic changes resulting from the unique environment of outer space.
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