Chapter 16: Gene Regulation

List and briefly describe five types of molecular events that may underlie epigenetic gene regulation.

1. DNA methylation— the attachment of methyl groups to cytosines in DNA. This often silences transcription. 2. Covalent histone modification— the covalent attachment of groups to the amino terminal tails of histones. This may silence or activate genes. 3. Chromatin remodeling— changes in the positions of nucleosomes. This may lead to a closed or open conformation for transcription. 4. Histone variants— replacement of standard histones for histone variants. This may silence or activate genes. 5. Feedback loop— the activation of a gene encoding a transcription factor. After the transcription factor is made, it continues to activate its own expression as well as the expression of other genes.

A single RNA molecule can encode several different proteins. A) True B) False

A

Methylation of DNA is a heritable trait. A) True B) False

A

Epigenetic modifications may be programmed during development or they be caused by environmental agents. Of the following examples, which are programmed during development and which are caused by environmental agents? A. Imprinting of the Igf2 gene in mammals B. Development of queen and worker honeybees C. X-chromosome inactivation in mammals D. Development of cancer

A and C are programmed during development B and D are caused by environmental agents

Explain how a non-coding RNA could play a role in establishing an epigenetic modification at a specific site in a chromosome.

A non-coding RNA can bind to a specific site in a chromosome due to complementary base pairing, or it may bind to a protein that is already attached to specific chromosomal site. After binding, the ncRNA can act as a bridge by binding to one or more proteins that cause epigenetic modifications such as DNA methylation and covalent histone modifications.

Are the following events best explained by mutation or epimutation? A. Imprinting of the Igf2 gene B. Variation in coat color in mice carrying the Avy allele C. Formation of cancer cells D. Variation in flower color between different strains of pea plants, such as purple versus white E. X-chromosome inactivation

A. Epimutation: due to differences in DNA methylation B. Epimutation: due to differences in DNA methylation C. Usually both mutation and epimutation D. Mutation: due to variation in DNA seq E. Epimutation: due to changes in chromatin structure of the inactivated X chromosome

Following X-chromosome inactivation, most of the genes on the inactivated X chromosome are silenced. Explain how. Name one gene that is not silenced.

After the Xist RNA coats the X chromosome it recruits proteins that silence genes and proteins that make it more compact and form a barr body. One gene on Xi that is not in activated is the Xist gene

Define alternative splicing. What are advantages and disadvantages of this process?

Alternative splicing is the splicing of a pre-mRNA in two or more different ways. An advantage is that an organism can carry fewer genes in its genome and yet express proteins that are specialized to function in particular cell types. A disadvantage is that this process uses energy to remove the introns. Also, energy is used to synthesize DNA that contains introns. In general, having introns and removing them during splicing uses a fair amount of energy.

Figure 16.8 shows the products of alternative splicing for the α-tropomyosin pre-mRNA. Let's suppose that smooth muscle cells produce splicing factors that are not produced in other cell types. Explain where you think such splicing factors bind and how they influence the splicing of the α-tropomyosin pre-mRNA.

As shown in Figure 16.8, the unique feature of the smooth muscle mRNA for -tropomyosin is that it contains exon 2. Splicing factors that are found only in smooth muscle cells may recognize the splice junction at the 3ʹ end of intron 1 and the 5ʹ end of intron 2 and promote splicing at these sites. This would cause exon 2 to be included in the mRNA. Furthermore, because smooth muscle mRNA does not contain exon 3, a splicing suppressor may bind to the 3ʹ end of intron 2. This would promote exon skipping, so that exon 3 would not be contained in the mRNA.

Methylation of CpG islands: A) Enhances binding of regulatory transcription factors. B) Prevents activation of enhancers. C) Prevents binding of chromatin remodeling proteins. D) Interferes directly with RNA polymerase binding.

B

Why are high copy cloned genes likely to be silenced?

Because the more copies of the gene are inserted, the more likely that one of them will be inserted in reverse orientation next to a promoter, leading to the production of antisense RNA.

The best explanation for interference of DNA methylation with transcription factor binding is: A) Methyl-CpG binding protein competes with the transcription factor for binding to methylated DNA. B) Methylation of the CpG island modifies the shape of the transcription factor. C) Methylation of the CpG island modifies the shape of the response element to which the transcription factor normally binds. D) Methylation prevents dimerization of DNA binding proteins.

C

Two different proteins produced by alternative splicing are most likely to: A) Have completely different functions. B) Have identical functions. C) Have similar functions under different forms of regulation. D) Have different functions under similar forms of regulation.

C

What is the key difference between a cis- and trans-epigenetic mechanism that maintains an epigenetic modification? In Chapter 5, we considered genomic imprinting of the Igf2 gene in which offspring express the copy of the gene they inherit from their father, but not the copy they inherit from their mother. Is this a cis- or trans-epigenetic mechanism?

Cis-epigenetic mechanism- the pattern of gene modification is maintained when two or more copies of a gene are found in the same cell. One copy may be silenced and other is active. Trans-epigenetic mechanism- soluble proteins, like transcription factors, are responsible for maintaining gene activation. In this case, all copies of the gene will be active. The imprinting of Igf2 is a cis-epigenetic mechanism.

List at least three ways that concentrations of a particular functional mRNA can be regulated.

Control of rate of transcription; regulation of pre-mRNA processing; increased stability of the RNA molecule; increased degradation of the RNA molecule; control of translational rate.

Control of alternative splicing involves: A) Repressors, which cause exon skipping. B) Enhancers, which increase recognition of weak splice sites. C) Splicing factors, which modulate spliceosome function. D) All of these. E) None of these.

D

Using coat color in mice and the development of female honeybees as examples, how can dietary factors cause epigenetic modifications, leading to phenotypic effects?

Dietary factors can affect the occurrence of epigenetic changes, particularly those that happen during early stages of development. - Ex. Dietary factors that affect the level of DNA methylation appear to be responsible or partly responsible for altering coat color in mice and female bee development. -These dietary factors may affect the function of DNA methyltransferase and thereby influence if particular genes are methylated or not. If they are methylated, this typically inactivates genes.

Explain how DNA methylation and the formation of a DNA loop control the expression of the Igf2 gene in mammals. How is this gene imprinted so that only the paternal copy is expressed in offspring?

During oogenesis, the ICR and DMR are not methylated. This allows CTCF to bind to these sites and to each other to form a loop. This loop prevents a nearby enhancer from activating the Igf2 gene. During sperm formation, methylation of these sites prevents CTCF binding, thereby preventing loop formation. The nearby enhancer activates the Igf2 gene. Therefore, the copy of the Igf2 gene inherited from the father via active sperm

In general, explain how epigenetic modifications are an important mechanism for developmental changes that lead to specialized body parts and cell types. How do the trithorax and polycomb group complexes participate in this process?

Epigenetic modifications lead to changes in gene expression that persist from early development to adulthood. This allows some genes to be expressed in certain cell types but not in others. - The trithorax and polycomb group complexes are involved w/ regulating gene expression.

Define epigenetics. Are all epigenetic changes passed from parent to offspring? Explain

Epigenetics- study of mechanisms that lead to changes in gene expression that can be passed from cell to cell and are reversible but don't involve a change in the seq of DNA. Not all epigenetic changes are passed from parent to offspring. - Example: those that occur in somatic cells would not be passed to offspring

With regard to RNAi, what are three possible sources for double stranded RNA?

First the double-stranded miRNA could come from the transcription of a gene as in pri-miRNA. Second it could come from a virus. Third double-stranded RNA could be made experimentally

What is the relationship between mRNA stability and mRNA concentration? What factors affect mRNA stability?

If mRNA stability is low, this means that it is degraded more rapidly. Therefore, low stability results in a low mRNA concentration. The length of the polyA tail is one factor that affects stability. A longer tail makes mRNA more stable. Certain mRNAs have sequences that affect their half-lives. For example, AU-rich elements (AREs) are found in many short-lived mRNAs. The AREs are recognized by cellular proteins that cause the mRNAs to be rapidly degraded.

With regard to development, what would be the dire consequences if polycomb group complexes did not function properly?

Many genes would be expressed in cell types where they should not be expressed. This would cause abnormalities in development and that would likely be lethal.

Outline the molecular steps in the process of X-chromosome inactivation. Which step plays a key role in choosing the X chromosome that will remain active versus the one that will become inactivated?

Pluripotent bind and stimulate transcription from Tsix and inhibit transcription from Xist - Both chromosomes are active CTCF binds to Xic and the X chromosomes pair w/ each other at Six gene Pluripotency factors and CTCF shift to one of the x chromosomes. This chromosome expresses Tsix and remains active. Other chromosome expresses Xist which makes it inactive. The symmetry break is the step that chooses which X chromosome will be active and which one will be inactivated.

What is the phenomenon of RNA interference (RNAi)? During RNAi, explain how the double-stranded RNA is processed and how it leads to the silencing of a complementary mRNA.

RNA interference refers to the phenomenon in which the presence of a double-stranded RNA molecule leads to the silencing of a complementary mRNA. For this to occur, the double-stranded RNA is processed by dicer into small RNA fragments. The miRNA or siRNA associates with a complex called the RNA induced silencing complex (RISC). The RISC complex then binds to a complementary mRNA. This either leads to mRNA degradation or inhibits translation.

Describe the molecular steps by which polycomb group complexes cause epigenetic gene silencing

See Figure 16.6.

Let's suppose a mutation removes the ICR next to the Igf2 gene. If this mutation was inherited from the mother, would the Igf2 gene (from the mother) be silenced or expressed? Explain.

The Igf2 gene inherited from the mother would be active. For silencing to occur a loop must form. The loop cannot form if ICR is missing.

Describe how the binding of the iron regulatory protein affects the mRNAs for ferritin and the transferrin receptor. How does iron influence this process?

The binding of IRP two IRE inhibits the translation of ferritin mRNA and enhances the stability of transferrin receptor mRNA. The Increase in the stability of transferrin receptor mRNA increases the concentration of this mRNA and ultimately leads to more transferrin receptor protein. Conditions of low iron promote the binding of Iribu to IRE leading to a decrease in ferritin protein in an increase in transferrin receptor protein. When the iron concentration is high iron binds to IRP causing it to be released from the IRE. This allows the ferritin mRNA to be translated and also causes a decrease in the stability of transferrin receptor mRNA. Under these conditions more ferritin protein is translated in less transferrin receptor is made.

During embryonic development, what event causes one of the two X chromosomes to be remain active, whereas the other one becomes an inactive X chromosome?

The choosing of the X chromosome that is inactivated occurs just after X-chromosome pairing. The pluripotency factors and CTCFs shift entirely to one of the X chromosomes. Because the pluripotency factors stimulate the expression of the Tsix gene, the X chromosome to which they shift remains active. The other X chromosome is able to express the Xist gene and becomes the inactive X chromosome.

What is the function of a splicing factor? Explain how splicing factors can regulate the cell-specific splicing of mRNAs.

The function of splicing factors is to influence the selection of splice sites in RNA. In certain cell types the concentration of particular splicing factors is higher than others. The high concentration of particular splicing factors in the regulation of their activities may promote the selection of particular splice sites in there by lead to tissue specific splicing

Let's suppose a person is homozygous for a mutation in the IRP gene that changed the structure of the iron regulatory protein in such a way that it could not bind iron, but it could still bind to IREs. How wud this mutation affect the regulation of ferritin and transferrin receptor mRNAs? Do you think such a person would need more iron in his or her diet than normal individuals? Do you think that excess iron in the diet would be more toxic than it would be for normal individuals? Explain.

The person would be unable to make ferratin because the IRP would always be bound to the IRE. The amount of transferrin receptor mRNA would be high even in the presence of high amounts of iron because the IRP would always remain bound to the IRE and stabilize the transferrin receptor mRNA.such a person would not have any problem taking up iron into his her cells. in fact this person would take up a lot of iron via the transferrin receptor even when the iron concentrations were high. Therefore this person would not need more iron in their diet. However excess iron in the diet would be very toxic for two reasons. First the person cannot make ferritin which prevents the toxicbuild up of iron in the cytosol. Second when Iron levels are high the person would continue to synthesize the transferrin receptor which functions in the uptake of iron.

How can environmental agents that do not cause gene mutations contribute to cancer? Would these epigenetic changes be passed to offspring?

Though they don't change the DNA seq, epigenetic modifications can increase gene expression. Resulting in oncogenes or they could inhibit the expression of tumor suppressor genes. Either type of change could contribute to cancer. - Ex. DNA methylation of the tumor suppressor gene could promote cancer.

Explain how epigenetic changes may be targeted to specific genes.

Transcription factors can find to a particular gene and then recruit other proteins that change DNA or chromatin structure. Similarly non-coding RNAs can bind to specific gene and do the same thing

In response to potentially toxic substances, eukaryotic cells often use translational regulatory mechanisms to prevent cell death, rather than using transcriptional regulatory mechanisms. Explain why.

Translational control is faster because it acts closer to the step of protein synthesis. If a cell quickly needs ferritin, translational control will allow its synthesis to occur more quickly. By comparison, transcriptional control would require the synthesis and processing of ferritin mRNA before more ferritin protein could be made.

What are the contrasting roles of trithorax and polycomb group complexes during development in animals and plants?

Tritthorax involved w/ gene activation complexes Polycomb group involved w/ gene repression during development. They cause epigenetic changes that allow genes to remain active or permanently repressed. Such changes occurred during every ionic development and are maintained during following stages.