Cover1 - Chromosome Structure2 - Chromosome Compaction3 - Chromosome Variation5 - Nucleic Acid Structure6 - DNA Replication7 - Mutations and DNA Repair8 - Polymerase Chain Reaction (PCR)9 - Transcription10 - RNA Modifications11 - Translation12 - Gene Cloning13 - The lac Operon14 - Gene Regulation in Eukaryotes15 - Epigenetics16 - Genome Editing

15 - Epigenetics

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 chromatin surrounding a gene or, in one case, alters the structure of an entire chromosome. These structural modifications to the chromatin or a chromosome either activate or silence transcription.

Key Questions

  • What is meant by epigenetics?

Overview of Epigenetics

The epigenetic factors that alter chromatin can be established during gamete formation, embryonic development, or in 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 are passed from parents 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 such as muscle cells and neurons. Environmental factors that influence epigenetic changes include diet, stress, the unique environment of space, and the toxins found in cigarette smoke.

Epigenetic changes established during differentiation or by environmental factors may be permanent in an individual. When an epigenetic change is established in a cell, this affected cell will divide 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 above).

Key Questions

  • What epigenetic process occurs during gamete formation?
  • What is meant by epigenetic inheritance?
  • What epigenetic processes occur during development?
  • List some environmental factors that promote epigenetic changes. 

Epigenetic Mechanisms

Three major epigenetic mechanisms influence the transcription of genes (see figure 15.1).  These epigenetic mechanisms include:

13.1_Epigenetic_Mechanisms.jpg
Figure 15.1 Epigenetic Mechanisms --- Image created by SL

Key Question

  • Describe the three major mechanisms that promote epigenetic changes.
  • Methylation of CpG islands in the DNA typically silences transcription. What effect does methylating histone H3 have on transcription?
  • What are two examples of ncRNAs that participate in epigenetics?

Genomic Imprinting

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 for a trait (monoallelic expression). The genomic imprint (in other words, the DNA methylation pattern) is established on the allele during the formation of gametes 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 insulin-like growth factor 2 (Igf2) gene in mice (see figure 15.2).  The Igf2 gene controls body size in mice.  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 they inherited the active Igf2 allele from the father (the Igf2- allele from the mother has been inactivated). 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 they inherited the active Igf2- allele from their father (the Igf2 allele from the mother has been inactivated). Note that the offspring are also heterozygous (Igf2 Igf2-).

The two crosses described above violate Mendel’s laws of inheritance; the two crosses produce offspring with the same genotype (Igf2 Igf2-), yet have different phenotypes.

13.2_Example_of_Genomic_Imprinting_with_Normal_and_Dwarf_Mice.png
Figure 15.2 An example of genomic imprinting.  A normal (right) and a dwarf (left) mouse

Key Questions

  • What is meant by monoallelic expression?
  • In the case of body size in mice, which Igf2 allele is expressed? Which allele is silenced?

Genomic Imprinting Stages

Genomic imprinting has three stages (see figure 15.3):

  1. Establishment of the imprint. Imprinting occurs during egg formation, silencing the maternal allele (Igf2- in figure 15.3) for a gene. The maternal allele remains silent through fertilization. During sperm formation, the paternal allele (Igf2) remains active, so the offspring will be normal in size. The two heterozygote (Igf2 Igf2-) offspring mice in figure 15.3 express the paternal allele.
  2. Maintenance of the imprint. After fertilization and subsequent cell divisions in the offspring mouse (Igf2 Igf2- genotype), the maternal allele is maintained in a silenced form. The organism only expresses the paternally inherited allele.
  3. Erasure and reestablishment. In both the male and female offspring produced by this initial cross, the imprint is erased when these offspring form their own gametes. After erasing the imprint, the imprint can then be reestablished depending on the sex of the animal:
EDI_15.3_Stages_of_genomic_imprinting-01_1.jpg
Figure 15.3 Stages of Genomic Imprinting --- Image created by SL

Key Questions

  • Describe the events that are occurring during the three stages of genomic imprinting.

Genomic Imprinting Mechanism

Genomic imprinting involves DNA methylation patterns established during gametogenesis.  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 is located adjacent to the H19 gene. This enhancer functions to regulate the transcription of the Igf2 gene. DNA methylation occurs at two DNA sequences the flank the Igf2 gene. The first DNA sequence is called 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 oogenesis (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 CTC sequences (cytosine-thymine-cytosine trinucleotide sequences) within both the ICR and the DMR. The CTCF proteins bound to the ICR sequence and the CTCF proteins bound to the DMR sequence also bind to each other, forcing a loop to form in the DNA. This loop contains the Igf2 gene. When the Igf2 gene is found within a DNA loop, an activator protein fails to bind to the enhancer next to H19. As a result, the Igf2 gene is transcriptionally silenced in egg cells.

During spermatogenesis (formation of sperm cells), the ICR and DMR sequences are methylated by de novo methylation. CTCF proteins do not bind to methylated ICR and DMR sequences, preventing the formation of a DNA loop containing the Igf2 gene. In the absence of loop formation, an activator protein binds to the enhancer next to the H19 gene. As a result, the Igf2 gene is transcribed in sperm cells. Note that even though DNA methylation usually silences a gene (see Part 14); in the case of the Igf2 gene, DNA methylation leads to transcriptional activation!

EDI_15.4_Genomic_Mechanism-01_1.jpg
Figure 15.4 Genomic Imprinting Mechanism --- Image created by SL

Key Questions

  • How do the ICR sequence, DMR sequence, CTCF proteins, and a DNA loop contribute to the silencing of the maternal Igf2 allele?
  • How does DNA methylation, an enhancer, and an activator protein activate the paternal Igf2 allele?

Angelman and Prader-Willi Syndromes

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.

Both AS and PWS involve an identical deletion in the long 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 spermatogenesis, 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 UBE3A) from the mother, the offspring has no active UBE3A alleles. The absence of an active 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 oogenesis. If an offspring inherits the silenced alleles from the mother and inherits a deletion copy of chromosome 15 (missing SNRPN and NDN) 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.

13.5_Mechanism_of_AS_and_PWS.jpg
Figure 15.5 Mechanism of AS and PWS --- Image created by SL

Key Questions

  • What defect in chromosome structure contributes to both AS and PWS?
  • How does genomic imprinting of the UBE3A gene contribute to AS?
  • How does genomic imprinting of the SNRPN and NDN alleles contribute to PWS?

X Chromosome Inactivation Mechanism

We learned previously 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 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. Xist is expressed preferentially from the X chromosome that will be inactivated, while Tsix is expressed from the X chromosome that will remain active.

The XCI process is thought to occur as follows (see figure 15.6, zoom in to view details):

  1. Prior to XCI, a group of activator proteins called pluripotency factors bind to enhancer sequences on both X chromosomes and activate the transcription of both Tsix genes. Tsix expression produces Tsix RNA molecules, which inhibit the expression of the Xist genes on both X chromosomes. The two X chromosomes are active at this point.
  2. The Xic regions on the two X chromosomes interact, causing the X chromosomes to pair. The pairing of the X chromosomes is very brief (for less than an hour) and involves the pluripotency factors and CTCF proteins (see above) binding to both X chromosomes.
  3. The pluripotency factors and CTCF proteins shift from both X chromosomes to just one of the two X chromosomes. The X chromosome that now contains the pluripotency factors and CTCF proteins will continue to express the Tsix RNA and will remain active. The other X chromosome (without the pluripotency factors and CTCF proteins) turns off Tsix expression and begins to express the Xist RNA. As a result, the X chromosome that expresses Xist will be inactivated.
  4. The Xist RNA molecules begin to bind to each other and to the X chromosome destined for inactivation. The Xist RNA binds initially to the Xic but later spreads in both directions along the X chromosome.
  5. The Xist RNA produces the following epigenetic changes to the inactivated X chromosome:

13.6_Mechanism_of_X_Chromosome_Inactivation.jpg

Key Questions

  • How do pluripotency factor proteins and CTCF proteins contribute to XCI?
  • What is the function of the Tsix RNA?
  • How is the Xist gene activated?
  • How does the Xist RNA contribute to the formation of a Barr body that contains silenced genes?

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 does not stain well and looks like a gap (see figure 15.7). This gap region tends to be susceptible to chromosome breakage and is therefore called a fragile site.

A type of mutation called a trinucleotide repeat expansion (TNRE) is thought to be responsible for 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 is also responsible for Huntington’s disease (see Part 7).

In fragile X syndrome, the trinucleotide repeats on the X chromosome are found in the beginning portion 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 that contains many CpG sites. These numerous CpG sites near the beginning of the FMR1 gene can become methylated, 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 lead to disease symptoms.

13.7_Fragile_X_Chromosome_Structure.jpg

13.7_Fragile_X_Syndrome_Patient.jpg
Figure 15.7 Top- Fragile X Chromosome Structure Bottom- Fragile X Syndrome Patient --- top) image created by SL Bottom) Image by Peter Saxon and used under license CC BY-SA 4.0

Key Questions

  • What is a TNRE?
  • How does TNRE and DNA methylation contribute to fragile X syndrome?

Epigenetics in Development

Epigenetic processes are important in the 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 gene 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. In muscle cells, a group of muscle-specific genes is actively transcribed, whereas genes that specify another fate (neuron, epithelial cell) are silenced. Epigenetic factors permanently silence neuron-specific genes or epithelial cell-specific genes in cells destined to become 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 tissue types. The TrxG complex is involved in gene activation processes, while the PcG 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.

We will now consider how a PcG complex silences transcription, by describing how the Hox genes are inactivated after the Hox gene products have been used to help determine the body plan during embryogenesis (see figure 15.8).

  1. A silencer DNA sequence called a polycomb response element (PRE) near the Hox gene is recognized by a repressor protein called PRE-binding protein.
  2. The PRE-binding protein recruits the PcG complex PRC2 to the Hox gene.
  3. PRC2 trimethylates lysine 27 of histone H3 within multiple nucleosomes near the Hox gene.
  4. Trimethylation inhibits transcription of the Hox gene directly by releasing TFIID and RNA polymerase II from the core promoter.
  5. Transcription of the Hox gene is inhibited.

It is important to note that the epigenetic silencing of the Hox gene is maintained during subsequent cell divisions, ensuring that epigenetic changes that occur during embryonic development are transmitted to the cells of the adult organism.

13.8_Silencing_Transcription_of_the_Hox_Genes.jpg
Figure 15.8 - PcG histone methyltransferase silences the Hox gene --- image created by SL

Key Questions

  • What is the function of the Hox genes?
  • How do the TrxG and PcG protein complexes contribute to embryonic development and tissue differentiation?
  • Describe how trimethylation of histone H3 can lead to gene activation in some cases and to gene silencing in other cases.
  • Describe how the PRE sequence, PRE-binding proteins, and the PRC2 protein complex contributes to the silencing of the Hox gene.

The Agouti Phenotype in Mice

One of the best examples of how environmental changes can influence epigenetics involves the transcription of the Agouti gene in mice. The protein product of the Agouti gene is involved in the formation of yellow pigment in the hairs of developing mice pups.

The Agouti gene has three alleles: A, a, and Avy. If a mouse is homozygous for the A allele (AA genotype), the mouse coat color is agouti (brown). However, if the hairs from this agouti mouse are examined closely, each hair contains a stripe of yellow pigment sandwiched between layers of black pigment. In mice that are homozygous for the a allele (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. If mice are heterozygous for the Avy allele (AAvy or Avya), a variety of phenotypes are possible; some mice are yellow, some are mottled (black and yellow fur patches), and some are pseudo-agouti (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 transcribed, then a yellow coat is produced. Intermediate levels of Avy allele expression produces the mottled phenotype. If the Avy allele displays low levels of transcription, then the pseudo-agouti coat is produced.

Interestingly, mice that have high levels of Avy allele expression also are prone to develop adult-onset obesity, diabetes, and cancer (see figure 15.9). The AA homozygotes, aa homozygotes, and heterozygotes with lower Avy allele expression are lean in appearance and are less susceptible to diabetes and cancer.

13.9_Agouti_and_Wild_Type_Mouse.jpg
Figure 15.9 Yellow and Wild Type Mice.  The yellow phenotype is the result of high Avy allele expression.  The wild-type mouse is homozygous for the A allele.  Yellow mice have a higher incidence of obesity, diabetes, and cancer than wild-type mice. --- image provided by R. Jirtle and D. Dolinoy and used under license CC BY 3.0

Key Questions

  • What are the phenotypes of mice with high levels of Avy allele expression?
  • What are the phenotypes of mice with low levels of Avy allele expression?

The Agouti Phenotype is Influenced by Diet and Bisphenol A

The variation in coat color phenotypes among Avy heterozygotes can be partially explained by their mother’s diet. 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 an non-supplemented (normal) diet (see figure 15.10). 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 the normal 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 expression.

Another environmental agent that affects the Agouti phenotype is the chemical bisphenol A (BPA), a chemical found in some plastics. The exposure of pregnant female mice to BPA produces more heterozygous offspring that have yellow coat color 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 the 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.

13.10_Diet_Can_Affect_the_Agouti_Phenotype.jpg
Figure 15.10 Diet Can Affect the Agouti Phenotype --- Image by SL

Key Questions

  • How does the consumption of folic acid and vitamin B12 by a pregnant mouse influence the expression of the Avy allele and the phenotype of her offspring?
  • How does the exposure of a pregnant mouse to BPA influence the expression of the Avy allele and the phenotype of her offspring?

Epigenetics and Cancer

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 make DNA methyltransferase, histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase, and histone demethylase, 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 certain genes called oncogenes can result in higher rates of cell division, promoting cancer. Alternatively, lower than normal expression of cancer-preventing genes called 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 could 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 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 inhibit cancer genes, the overall effect is a higher rate of cancer formation.

Additionally, 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.

Key Questions

  • What is an oncogene and a tumor-suppressor gene?
  • Explain how mutations in the DNA can have epigenetic consequences, potentially leading to cancer.
  • How do the PAHs in cigarette smoke have epigenetic consequences, potentially leading to cancer?

Epigenetic Therapy

As we have seen, epigenetic processes are associated with several human diseases (AS, PWS, fragile X syndrome, certain cancers). 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 of the cancers mentioned above. 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.

Key Questions

  • What is meant by epigenetic therapy?
  • How do 5-azacytidine and phenylbutyric acid contribute to epigenetic therapy?

The Epigenomes of Identical Twins are Not Identical

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 sequence 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 DNA sequences. 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.

13.11_Astronauts_Scott_and_Mark_Kelly_1.jpg
Figure 15.11 Astronauts Scott and Mark Kelly --- photo taken by NASA

Key Questions

  • What is an epigenome?
  • Are the epigenomes of identical twins the same? Why or why not?

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