genetics: Chapter 21

paramutation

*"this" is defined as an interaction between two alleles that leads to a heritable change in the expression of one of the alleles* - Surprisingly, "this" produces these changes in phenotype without any alteration in the DNA base sequence of the converted allele.

Epigenetics

*changes in gene expression or phenotype that are potentially heritable without alteration of the DNA base sequence* -Many of "these" effects are caused by changes in gene expression that result from alterations in chromatin structure or other aspects of DNA structure, such as DNA methylation

Xist

1 of the 4 major genes involved in X inactivation *encodes:* lncRNA *action of gene:* Coats inactive X chromosome and leads to silencing of transcription of many genes on the inactive X

Tsix

1 of the 4 major genes involved in X inactivation *encodes:* lncRNA *action of gene:* Inhibits transcription of Xist on active X chromosome

Jpx

1 of the 4 major genes involved in X inactivation *encodes:* lncRNA *action of gene:* Stimulates transcription of Xist on inactive X chromosome

Xite

1 of the 4 major genes involved in X inactivation *encodes:* lncRNA *action of gene:* Sustains Tsix expression on active X, which inhibits Xist and maintains transcription of genes on active X chromosome

1) changes in patterns of DNA methylation 2) chemical modifications of histone proteins 3) RNA molecules that affect chromatin structure and gene expression

3 types of molecular mechanisms alter chromatin structure and underlie many epigenetic phenotypes, which are?

inactivation center

A great deal of research has demonstrated that which X chromosome is inactivated within a cell is controlled by a particular segment of the X chromosome called "this" which is 100,000 to 500,000 bp in length

rmr1 gene

Another gene required for paramutation, called "this", *encodes a chromatin-remodeling protein*

Although all details of this process are not yet understood, recent research has demonstrated that there are several additional genes in the X-inactivation center that encode other lncRNAs. These lncRNAs help bring about inactivation of the inactive X while not silencing the active X (see Figure 21.10). -One of these genes is the *Tsix gene, which is transcribed on the active X chromosome. Tsix is antisense to Xist, which means that it overlaps with the Xist gene and is transcribed from the opposite strand (see Figure 21.11), producing a Tsix lncRNA that is complementary to Xist lncRNA. Through several mechanisms, Tsix represses the expression of Xist on the active X chromosome.*

As mentioned, X inactivation is brought about by the transcription of the Xist gene on the inactive X chromosome to produce Xist lncRNA, which coats the inactive X chromosome and leads to changes in chromatin structure that silence transcription. But what happens on the active X chromosome? Why isn't it coated by Xist RNA and silenced?

X inactivation

Early in the development of female mammals, one X chromosome in each cell is randomly inactivated to provide equal expression of X-linked genes in males and females -*Through this process, termed "this", many genes on the inactivated X chromosome are permanently silenced and are not transcribed.* -Once a particular X chromosome is inactivated in a cell, that same X chromosome remains inactivated when the DNA is replicated, and the inactivation mark is passed on to daughter cells through mitosis

-A mother rat licks and grooms her offspring (Figure 21.9), usually while she arches her back and nurses them -The offspring of mothers who display more licking and grooming behavior are less fearful as adults and show reduced hormonal responses to stress compared with the offspring of mothers who lick and groom less -These long-lasting differences in the offspring are not due to genetic differences inherited from their mothers—at least not genetic differences in the base sequences of their DNA. -Offspring exposed to more licking and grooming develop a different pattern of DNA methylation than offspring exposed to less licking and grooming. These differences in DNA methylation affect the acetylation pattern of histone proteins -these differences in DNA methylation affect the acetylation pattern of histone proteins. The altered acetylation pattern persists into adulthood and alters the expression of the glucocorticoid receptor gene, which plays a role in hormonal responses to stress. The expression of other stress-response genes is also affected.

Explain epigenetic changes induced by maternal behavior

-Numerous studies have demonstrated that stress during childhood and adolescence produces a number of adverse effects that persist into adult life -For example, childhood abuse increases the probability that the child will experience depression, anxiety, and suicide as an adult. In one study, researchers examined the brains of 24 people who had committed suicide, half of whom had experienced childhood abuse. They found that those who had experienced childhood abuse had a greater degree of methylation of the glucocorticoid receptor gene, a gene involved in the stress response, than those who had not. Although the number of brains studied was small, the study suggests that early childhood stress can indeed cause epigenetic modifications to chromatin structure in humans.

Explain epigenetic effects of early stress

The b1 locus helps to determine the amount of purple anthocyanin that a corn plant produces. The locus actually encodes a transcription factor that regulates genes involved in anthocyanin production. Plants homozygous for the B-I allele (B-I B-I) show high expression of the b1 locus and are dark purple (Figure 21.6). Plants homozygous for the B′ allele (B′ B′) show a lower expression of the b1 locus and are lightly pigmented. However, the DNA sequences of the B-I and B′ alleles are identical. Genetically identical alleles such as these, which produce heritable differences in phenotypes through epigenetic processes, are referred to as *epialleles*.

Explain how paramutation in corn of the B-I and B' alleles affects the phenotype of the corn

- During the process of DNA replication, nucleosomes are disrupted, and the original histone proteins are distributed randomly between the two new DNA molecules. Newly synthesized histones are then added to complete the formation of new nucleosomes (see Section 12.4). Some models assume that *after replication, the epigenetic marks remain on the original histones, and that these marks recruit enzymes that make similar changes in the new histones.* For example, PRC2, which adds the H3K27me3 epigenetic mark to histones, preferentially targets histones in chromatin that already contains an H3K27me3 mark, ensuring that any new nucleosomes that are added after replication also become methylated. In this way, the histone modifications can be maintained across cell division - An alternative model, supported by experimental evidence in Drosophila embryos, *proposes that the epigenetic marks are lost during replication, but that the enzymes that bring about histone modifications remain attached to the original histones during the replication process and reestablish the marks on the original and new histones after replication is completed.*

Explain how specific epigenetic marks are maintained.

A number of techniques have been developed for examining levels of DNA methylation. Some of these techniques rely on *restriction endonucleases*, enzymes that make double-stranded cuts in the DNA at specific base sequences (see Section 19.2). Some restriction enzymes are sensitive to methylation and will not cut a sequence that contains 5-methylcytosine, whereas other restriction enzymes are insensitive to methylation. By cutting DNA with enzymes that are sensitive to methylation and with enzymes that are not, and then analyzing the resulting fragments, overall patterns of methylation can be determined. -A more precise and widely used technique for analyzing DNA methylation is *bisulfite sequencing* (Figure 21.14). In this technique, genomic DNA is first treated with sodium bisulfite, which chemically converts unmethylated cytosine to uracil. Uracil is then detected as thymine during sequencing. However, 5-methylcytosine is not chemically altered by treatment with bisulfite, and it is detected as cytosine during sequencing (see Section 19.5 for a discussion of DNA sequencing). By sequencing genomic DNA with and without bisulfite treatment, researchers are able to determine the locations of all copies of 5-methylcytosine in the DNA.

Explain the Detecting DNA methylation

epialleles

Genetically identical alleles such as these, which produce heritable differences in phenotypes through epigenetic processes, are referred to as "this"

mop1 gene

Geneticists have isolated several genes in corn that are required for paramutation to take place; inactivating these genes eliminates paramutation. One of these genes is "this" -*which encodes an RNA-directed RNA polymerase (an enzyme that synthesizes RNA from an RNA template). This gene is required to generate the siRNAs encoded by the tandem repeats*, although it does not appear to be the enzyme that actually transcribes the DNA copies of the tandem repeats.

imprinted X-chromosome inactivation

In mice, there are two separate inactivation events. Soon after fertilization, when the embryo reaches the eight-cell stage, the X chromosome from the male parent is inactivated, while the maternal X chromosome remains active. This event is called "this" -In the developing embryo, the paternal X chromosome is then reactivated during blastocyst maturation. Inactivation occurs again in early development, but now which X is inactivated is random: the X from the male parent and the X from the female parent are equally likely to be inactivated. From this point on, whichever X is inactivated remains silenced through subsequent cell divisions. However, some genes on the inactivated X chromosome escape inactivation and continue to be transcribed.

induced pluripotent stem cells (iPSCs)

In the past, embryos were the only source of stem cells with the capacity to differentiate into adult tissues, but because of ethical concerns about creating and using human embryos for harvesting stem cells, researchers have long sought the ability to induce adult somatic cells to dedifferentiate and revert to stem cells. Such cells are called "this" -Researchers have now successfully created iPSCs by treating fibroblasts (fully differentiated human connective-tissue cells) in culture with a cocktail of transcription factors (Figure 21.12), although less than 1% of the cells that are so treated actually revert to iPSCs.

epigenetic marks

Many additional histone modifications, as well as changes to DNA that do not involve the base sequence, have been shown to be associated with the level of transcription. These types of modifications have been called "this" -*chemical modifications to DNA that can turn genes on or off*

Although the mechanism is not completely understood, recent research demonstrates that the communication between B′ and B-I probably occurs through the action of small RNA molecules -The tandem repeats that are required for paramutation encode* 25-nucleotide-long siRNAs*. Some *siRNAs* are known to modify chromatin structure by directing DNA methylation to specific DNA sequences.

The different chromatin states of B-I and B′ may explain their different levels of expression, but how does the B′ allele convert the B-I allele to B′*?

Before replication, cytosine bases on both strands are methylated (Figure 21.3). Immediately after semiconservative replication, the cytosine base on the template strand is methylated, but the cytosine base on the newly replicated strand is unmethylated. Special methyltransferase enzymes recognize the hemimethylated state of CpG dinucleotides and add methyl groups to the unmethylated cytosine bases, resulting in two new DNA molecules that are fully methylated. In this way, the methylation pattern of DNA is maintained across cell division

The fact that epigenetic changes are passed to other cells and sometimes to future generations means that the changes in chromatin structure associated with epigenetic phenotypes must be faithfully maintained when chromosomes replicate. *How are epigenetic changes retained and replicated through the process of cell division?*

Xist gene

The key player in X inactivation is a gene called "this" that is 17,000 bp in length. -As its name implies, this RNA molecule does not encode a protein. Instead, Xist lncRNA coats the X chromosome from which it was transcribed. -Xist lncRNA then attracts polycomb repressive complex 2 (PRC2) and, eventually, polycomb repressive complex 1 (PRC1). These proteins produce epigenetic marks, such as H3K27me3, and other histone modifications that repress transcription. Eventually, many CpG dinucleotides are methylated, leading to permanent silencing of the inactivated X chromosome.

epigenome

The overall pattern of chromatin modifications in a genome has been termed "this"

1) the newly established expression pattern of the converted allele is transmitted to future generations, even when the allele that brought about the alteration is no longer present with it. 2) the altered allele is now able to convert other alleles to the new phenotype. 3) The DNA sequence is not changed in the altered allele

The phenomenon of paramutation has several important features, which are?

cross-talk

There is also considerable ""this"" between epigenetic marks: *one histone mark may affect whether additional marks occur nearby and how they function.* -"This" occurs because histone modifications attract enzymes and proteins that modify other histones.

genomic imprinting

This phenomenon, in which the sex of the parent that transmits the allele determines its expression, is termed "this" -For some imprinted genes, when the allele is inherited from the male parent it is expressed, but when it is inherited from the female it is silent; for other genes, when the allele is inherited from the female parent it is expressed, but when it is inherited from the male it is silent. As discussed in Section 5.3, genomic imprinting is thought to be due to different degrees of methylation of the alleles inherited from the two parents.

stem cells

are undifferentiated cells that are capable of forming every type of cell in an organism, a property referred to as *pluripotency.*

Because some chemicals are capable of modifying chromatin structure, researchers have looked for long-term effects of environmental toxicants on chromatin structure and epigenetic traits. -There has been much recent interest in chemicals called *endocrine disruptors*, which mimic or interfere with natural hormones. Endocrine disruptors are capable of interfering with processes regulated by natural hormones, such as sexual development and reproduction. One of these endocrine disruptors is *vinclozolin*, a common fungicide used to control fungal diseases in vegetables and fruits—particularly wine grapes—and to treat turf on golf courses. *Vinclozolin acts as an antagonist at the androgen receptor; that is, vinclozolin and its metabolites mimic testosterone and bind to the androgen receptor, preventing testosterone from binding.* But *vinclozolin and its metabolites do not properly activate the receptor, and in this way, vinclozolin inhibits the action of androgens and prevents sperm production.* -*In one study, researchers found that exposure of embryonic male rats to vinclozolin led to reduced sperm production not only in the treated animals (when they reached puberty), but also in several subsequent generations. Increased DNA methylation was seen in the sperm of the males that were exposed to vinclozolin, and these patterns of methylation were inherited*

explain epigenetic effects on environmental chemicals

In the introduction to this chapter, we saw that nutrition during prenatal development can have effects on health in later life. These types of epidemiological studies on humans are supported by laboratory studies of mice and rats - *In one study, researchers fed inbred male mice either a normal (control) diet or a diet low in protein. They then bred mice in both groups to control females fed a normal diet.* The males were then separated from the females and never had any contact with their offspring; their only contribution to the offspring was a set of paternal genes transferred through the sperm. -*The offspring were raised and their lipid and cholesterol levels examined. The offspring of males fed a low-protein diet exhibited increased expression of genes involved in lipid and cholesterol metabolism, and a corresponding decrease in levels of cholesterol, compared with the offspring of males fed a normal diet. The researchers also observed numerous differences in DNA methylation in the offspring of the two groups of fathers*, although no differences could be found in the methylation patterns of the sperm of the two groups of fathers. These results suggest that epigenetic changes altered the cholesterol metabolism of the offspring -In another study, researchers fed male rats a high-fat diet and, not surprisingly, the rats gained weight. The researchers then bred these males to females that had been fed a normal diet. The offspring were also fed a normal diet. The daughters of the male rats on the high-fat diet had normal weight, but as adults they developed a diabetes-like condition of impaired glucose tolerance and insulin secretion

explain epigenetic effects on metabolism

A number of research studies have shown that abnormalities in DNA methylation are associated with disorders of development and intellectual ability in humans. -These findings prompted researchers to look for effects of chromatin structure on learning, memory, and cognitive ability in mice and rats. -*One study found that training mice to avoid an aversive stimulus at a specific location reduced DNA methylation of the Bdnf gene, which encodes a growth factor that stimulates the growth of connections between neurons. When demethylated, the Bdnf gene was more active. When researchers injected a drug that inhibits demethylation into the brains of the trained mice, activity of the Bdnf gene was decreased, and the mice's memory of where the adverse stimulus occurred also decreased.* -*another study found that a drug that promotes the acetylation of histone proteins improved learning and memory in mice that have a disorder similar to Alzheimer disease.* Recall that acetylation of histones alters chromatin structure by loosening the association of DNA with histone proteins and stimulates transcription of many genes.

explain epigenetics in cognition

Monozygotic (identical) twins develop from a single egg fertilized by a single sperm that divides and gives rise to two zygotes (see Section 6.3). Thus, monozygotic twins are genetically identical, in the sense that they possess identical DNA sequences, but they often differ somewhat in appearance, health, and behavior -The nature of these differences in the phenotypes of identical twins is not well understood, but recent evidence suggests that at least some of these differences may be *due to epigenetic changes.* - In one study, Mario Fraga, at the Spanish National Cancer Center, and his colleagues examined 80 pairs of identical twins and compared the degree and location of their DNA methylation and histone acetylation. They found that DNA methylation and histone acetylation in identical twin pairs were similar early in life, *but that older twin pairs had remarkable differences in their overall content and distribution of DNA methylation and histone acetylation*. Furthermore, these differences affected gene expression in the twins. This research suggests that identical twins do differ epigenetically and that phenotypic differences between them may be caused by differential gene expression.

explain the epigenetic effects in monozygotic twins

Histone modifications can be detected by breaking the chromatin into fragments and applying an antibody specific to a particular histone modification, a process called *chromatin immunoprecipitation* -The antibody mediates the precipitation of chromatin fragments with the histone modification, causing them to separate from chromatin fragments without the modification. The histones are then removed by digestion with an enzyme that degrades protein, but not DNA. The genomic location of the precipitated DNA fragment with which the histone was associated is then determined.

how do researchers detect histone modifications?

pluripotency

stem cells are undifferentiated cells that are capable of forming every type of cell in an organism, a property referred to as "this"

DNA methylation

the 1st mechanism that alters chromatin structure and underlie many epigenetic phenotypes -*The best-understood mechanism of epigenetic change is "this". "This" refers to the addition of methyl groups to the nucleotide bases. In eukaryotes, the predominant type of DNA methylation is the methylation of cytosine to produce 5-methylcytosine (Figure 21.2a). DNA methylation is often associated with repression of transcription.* -Cells repress and activate genes by methylating and demethylating cytosine bases.

Histone modification

the 2nd mechanism that alters chromatin structure and underlie many epigenetic phenotypes -*epigenetic changes can also occur through "this", which includes the addition of phosphates, methyl groups, acetyl groups, and ubiquitin. Many of these modifications take place in the positively charged tails of the histone proteins, which interact with the DNA and affect chromatin structure* -These modifications can occur at different amino acids on different histones and create more than 100 unique potential changes in the histones. Many of these modifications alter chromatin structure and affect transcription of genes

RNA molecules that affect chromatin structure and gene expression

the 3rd mechanism that alters chromatin structure and underlie many epigenetic phenotypes -*Evidence increasingly demonstrates that RNA molecules play an important role in bringing about epigenetic effects*

true

true or false: *However, epigenetic alterations can also be influenced by environmental factors. For example, environmental stress has been shown to alter methylation of the rat Bdnf gene, which encodes a growth factor that plays an important role in brain development.* DNA methylation has been tied to the expression of genes and the phenotypes they produce. As we will see, altered DNA methylation is capable of being replicated across cell division, resulting in progeny with the new phenotype, although there is no corresponding difference in the DNA base sequence of individuals that "inherit" the new phenotype.

true

true or false: *The addition of acetyl groups* to amino acids in the histone tails (histone acetylation) generally destabilizes chromatin structure, causing it to assume a more open configuration, and is associated with increased transcription

true

true or false: *The addition of methyl groups to histones* (histone methylation) also alters chromatin structure, but the effect varies depending on the specific amino acid that is methylated; some types of histone methylation are associated with increased transcription and other types are associated with decreased transcription

true

true or false: Another study compared histone modifications of fibroblasts, iPSCs, and embryonic stem cells. The iPSCs and embryonic stem cells had many fewer H3K27me3 and H3K9me3 marks than did the fibroblasts, but researchers also found significantly more of these marks on the iPSCs than on the embryonic stem cells.

true

true or false: As a stem cell divides and gives rise to a more specialized type of cell, the gene-expression program of the cell becomes progressively fixed, so that each particular cell type expresses only those genes necessary to carry out the functions of that cell type. *Though the control of these cell-specific expression programs is not well understood, changes in DNA methylation and chromatin structure clearly play important roles in silencing some genes and activating others.*

true

true or false: Genomic imprinting has a number of interesting parallels to X inactivation. Most imprinted genes are located in clusters of 3-12 genes that occur in a discrete region of a particular chromosome. Each cluster contains genes that encode proteins as well as genes that produce noncoding RNA.

true

true or false: Histone modifications are added and removed by special proteins. The *polycomb group (PcG)* is a large group of proteins that repress transcription by modifying histones -For example, polycomb repressive complex 2 (PRC2) adds two or three methyl groups to lysine 27 of histone H3, creating the H3K27me3 epigenetic mark, which represses transcription.

true

true or false: In plants that are heterozygous B-I B′, the B-I allele is converted to B′(star), with the result that the heterozygous plants are lightly pigmented (see Figure 21.6), just like the B′ B′ homozygotes. The newly converted allele is usually designated B′(star). Importantly, there is no functional difference between B′ and B′(star) ; the B′(star) allele is now fully capable of converting other B-I alleles into B′(star) alleles in subsequent generations.

true

true or false: In the case of X inactivation, the *Xist RNA* coats one X chromosome and then attracts PRC2, which deposits methyl groups on lysine 27 of histone H3, creating the H3K27me3 epigenetic mark, which alters chromatin structure and represses transcription.

true

true or false: Inactivation is initiated at the X-inactivation center and then spreads to the remainder of the inactivated X chromosome. Examination of the X-inactivation center led to the discovery of several genes that play a role in inactivating all but one X chromosome in each female cell and keeping the other X chromosome active

true

true or false: Many epigenetic changes are stable, persisting across cell divisions or even generations. However, epigenetic alterations can also be influenced by environmental factors.

true

true or false: Many epigenetic effects are caused by changes in gene expression that result from alterations in chromatin structure or other aspects of DNA structure, such as DNA methylation

true

true or false: Modifications of histone proteins, including the addition of methyl groups, acetyl groups, phosphates, and ubiquitin, alter chromatin structure. Some of these modifications are passed to daughter cells during cell division and to future generations.

true

true or false: Recent research has shown, however, that iPSCs retain a memory of their past and are not completely equivalent to embryonic stem cells (those derived from embryos). One study found that although the DNA methylation patterns of iPSCs differ greatly from those of differentiated somatic cells, the iPSCs retained some methylation marks of the somatic cells, and the methylation of iPSCs was not identical with that of embryonic stem cells

true

true or false: Research indicates that single histone modifications, such as those mentioned here, do not individually determine the transcriptional activity of a gene. Rather, it is the combined presence of multiple epigenetic marks that determines the activity level.

true

true or false: Researchers have now successfully created iPSCs by treating fibroblasts (fully differentiated human connective-tissue cells) in culture with a cocktail of transcription factors (Figure 21.12), although less than 1% of the cells that are so treated actually revert to iPSCs.

true

true or false: Some DNA regions have many CpG dinucleotides and are referred to as CpG islands. In mammalian cells, CpG islands are often located in or near the promoters of genes.

true

true or false: Stem cells provide a potential source of cells for regeneration of tissues, medical treatment, and research

true

true or false: Thus, the current evidence suggests that siRNA molecules convert B-I to B′* and that this conversion involves a change in the chromatin states of the alleles

true

true or false: Transcription factors that induce pluripotency cause extensive epigenetic reprogramming, altering the patterns of DNA methylation and histone modifications that accumulate with cell differentiation

true

true or false: X inactivation is a type of epigenetic effect because it results in a stable change in gene expression that is passed on to other cells.

true

true or flase: Xist lncRNA then attracts polycomb repressive complex 2 (PRC2) and, eventually, polycomb repressive complex 1 (PRC1). These proteins produce epigenetic marks, such as H3K27me3, and other histone modifications that repress transcription. Eventually, many CpG dinucleotides are methylated, leading to permanent silencing of the inactivated X chromosome.

- The first discovered and still best understood example of RNA mediation of epigenetic change is X inactivation (mentioned in Section 4.3), in which a long noncoding RNA called *Xist* suppresses transcription on one of the X chromosomes in female mammals.

what are some examples of how RNA molecules that affect chromatin structure and gene expression?

Xist, Tsix, Jpx, and Xite

what are the 4 major genes involved in X inactivation?

*is the presence of a series of seven tandem repeats of an 853-bp sequence located approximately 100,000 base pairs upstream of the coding sequence for the b1 locus* -The repeats do not encode any protein. Both the B-I and B′ alleles have these tandem repeats, but the chromatin structure of the two alleles differs: the B-I allele has more open chromatin. *The tandem repeats are required for high expression of the B-I allele and high pigment production. It has been suggested that the repeats act like an enhancer (see Chapter 17), stimulating transcription at the b1 locus, but only when the chromatin surrounding the repeats is in an open configuration, as it is in the B-I allele.* The more closed configuration in the B′ allele may prevent the repeats from interacting with the promoter of b1 and stimulating transcription. How the repeats might interact with the B′ allele is not known.

what is a feature required for paramutation at the b1 locus?

-A remarkable example of epigenetics is seen in honeybees. Queen bees and worker bees are both female, but the resemblance ends there How royal jelly affects gene expression has long been a mystery, but research now suggests that it changes an epigenetic mark. In 2008, Ryszard Kucharski and his colleagues demonstrated that royal jelly silences the expression of a key gene called Dnmt3, whose product normally adds methyl groups to DNA (Figure 21.5). With Dnmt3 shut down, bee DNA is less methylated, and many genes that are normally silenced in workers are expressed, leading to the development of queen characteristics.

what is an example of DNA methylation involving epigenetics?