Genetics ch.15
What is DNA methylation? When we say that DNA methylation is heritable, what do we mean? How is it passed from a mother to a daughter cell?
DNA methylation is the attachment of a methyl group to a base within the DNA. In many eukaryotic species, the attachment occurs on cytosine at a CG sequence. After de novo methylation has occurred, it is passed from mother to daughter cell. Because DNA replication is semiconservative, the newly made DNA contains one strand that is methylated and one that is not. DNA methyltransferase recognizes this hemimethylated DNA and methylates the cytosine in the unmethylated DNA strand; this event is called maintenance methylation.
Discuss the common points of control in eukaryotic gene regulation.
See Figure 15.1. Transcriptional regulation is the most energy-efficient way, because a cell avoids wasting energy making RNA or proteins.
What is meant by the term histone code? With regard to gene regulation, what is the proposed role of the histone code?
The histone code is the pattern of covalent modifications of histones that acts much like a language in specifying alterations in chromatin structure and function. In this way, the modification of histones plays a role in gene regulation.
The gene that encodes the enzyme called tyrosine hydroxylase is known to be activated by the CREB protein. Tyrosine hydroxylase is expressed in nerve cells and is involved in the synthesis of catecholamine, a neurotransmitter. The exposure of cells to adrenaline normally up-regulates the transcription of the tyrosine hydroxylase gene. A mutant cell was identified in which the tyrosine hydroxylase gene was not up-regulated when exposed to adrenaline. List all the possible mutations that could explain this defect. How would you explain the defect if only the tyrosine hydroxylase gene was not up-regulated by the CREB protein, whereas other genes having CREs were properly up-regulated in response to adrenaline in the mutant cell?
The mutation could cause a defect in the following: 1. Adrenaline receptor 2. G protein 3. Adenylyl cyclase 4. Protein kinase A 5. CREB protein 6. CREs of the tyrosine hydroxylase gene If other genes were properly regulated by the CREB protein, you could conclude that the mutation is probably within the tyrosine hydroxylase gene itself. Perhaps a CRE has been mutated and no longer recognizes the CREB protein.
An enhancer, located upstream from a gene, has the following sequence: 5′-GTAG-3′ 3′-CATC-5′ This enhancer is orientation-independent. Which of the following sequences also works as an enhancer? A. 5′-CTAC-3′ 3′-GATG-5′ B. 5′-GATG-3′ 3′-CTAC-5′ C. 5′-CATC-3′ 3′-GTAG-5′
The sequence found in A would work as an enhancer, but the ones in B and C would not. The sequence that is recognized by the transcriptional activator is 5'-GTAG-3' in one strand and 3'-CATC-5' in the opposite strand. This is the same arrangement found in A. In B and C, however, the arrangement is 5'-GATG-3' and 3'-CATC-5'. In the arrangement found in B and C, the two middle bases (i.e., A and T) are not in the correct order.
What is meant by the term transcription factor modulation? List three general ways this can occur.
Transcription factor modulation refers to different ways that the function of transcription factors can be regulated. The three general ways are the binding of an effector molecule, protein-protein interactions, and covalent modifications.
What is a histone variant?
A histone variant is a histone with an amino acid sequence that is slightly different from the sequence of a core histone. Histone variants play specialized roles with regard to chromatin structure and function.
What is the difference between an miRNA and an siRNA. How do these ncRNAs affect mRNAs?
MicroRNAs (miRNAs) are ncRNAs that are transcribed from endogenous eukaryotic genes—genes that are normally found in the genome. They play key roles in regulating gene expression, particularly during embryonic development in animals and plants. Most commonly, a single type of miRNA inhibits the translation of several different mRNAs. By comparison, small-interfering RNAs (siRNAs) are ncRNAs that usually originate from sources that are exogenous, which means they are not normally made by cells. The sources of siRNAs can be viruses that infect a cell, or researchers can make siRNAs to study gene function experimentally. They usually promote mRNA degradation.
Let's suppose that a vertebrate organism carries a mutation that causes some cells that normally differentiate into nerve cells to differentiate into muscle cells. A molecular analysis reveals that this mutation is in a gene that encodes a DNA methyltransferase. Explain how an alteration in a DNA methyltransferase could produce this phenotype.
Perhaps the methyltransferase is responsible for methylating and inhibiting a gene that causes a cell to become a muscle cell. The methyltransferase is inactivated by the mutation.
Explain how phosphorylation affects the function of the CREB protein.
Phosphorylation of the CREB protein causes it to act as a transcriptional activator. Unphosphorylated CREB protein can still bind to CREs, but it does not stimulate transcription.
Let's suppose a mutation in the glucocorticoid receptor does not prevent the binding of the glucocorticoid hormone to the protein but prevents the ability of the receptor to activate transcription. Make a list of all the possible defects that may explain why transcription cannot be activated.
1. It could be in the DNA-binding domain, so that the receptor would not recognize GREs. 2. It could be in the HSP90 domain, so that HSP90 would not be released when the hormone binds. 3. It could be in the dimerization domain, so that the receptor would not dimerize. 4. It could be in the nuclear localization domain, so that the receptor would not travel into the nucleus. 5. It could be in the domain that activates RNA polymerase, so that the receptor would not activate transcription, even though it could bind to GREs.
What is a CpG island? Where would you expect one to be located? How does the methylation of CpG islands affect gene expression?
A CpG island is a stretch of 1,000 to 2,000 base pairs that contains a high number of CpG sites. CpG islands are often located near promoters. When an island is methylated, this inhibits transcription. This inhibition may be the result of the inability of the transcriptional activators to recognize the methylated promoter and the effects of methyl-CpG-binding proteins, which may promote a closed chromatin conformation.
Histones are thought to be displaced as RNA polymerase is transcribing a gene. What would be the potentially harmful consequences if histones were not put back onto a gene after RNA polymerase had passed?
A potentially harmful consequence would be that transcription may be initiated at multiple points within a gene, thereby producing many nonfunctional transcripts. The result would be waste of energy.
Transcription factors usually contain one or more motifs that play key roles in their function. What is the function of the following motifs? A. Helix-turn-helix B. Zinc finger C. Leucine zipper
A. DNA binding. B. DNA binding. C. Protein dimerization.
What is an insulator? Describe two different ways that insulators may exert their effects.
An insulator is a segment of DNA that functions as a boundary between two adjacent genes. An insulator may act as a barrier to changes in chromatin structure or block the effects of a neighboring enhancer.
Describe how the binding of iron regulatory protein to an IRE affects the mRNAs for ferritin and the transferrin receptor. How does iron (Fe3+) influence this process?
The binding of IRP to the IRE inhibits the translation of ferritin mRNA and enhances the stability of the 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 IRP to the IRE, leading to a decrease in ferritin protein and 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, and less transferrin receptor is made.
What are the functions of transcriptional activator proteins and repressor proteins? Explain how they work at the molecular level.
Transcriptional activation occurs when a regulatory transcription factor binds to a response element and activates transcription. Such proteins, called activators, may interact with TFIID and/or mediator to promote the assembly of RNA polymerase and general transcription factors at the promoter region. They can also alter the structure of chromatin so that RNA polymerase and transcription factors are able to gain access to the promoter. Transcriptional inhibition occurs when a regulatory transcription factor inhibits transcription. Such repressors also may interact with TFIID and/or mediator to inhibit RNA polymerase.
What is a nucleosome-free region? Where are such regions typically found in a genome? How are nucleosome-free regions thought to be functionally important?
A nucleosome-free region (NFR) is a location in the genome where nucleosomes are missing. They are typically found at the beginning and ends of genes. An NFR at the beginning of a gene is thought to be important so that genes can be activated. The NFR at the end of a gene may be important for the proper termination of transcription.
The glucocorticoid receptor and the CREB protein are two examples of transcriptional activators. These proteins bind to response elements and activate transcription. (Note: The answers to this Page 385 question are not directly described in this chapter. You have to rely on your understanding of the functioning of other proteins that are modulated by the binding of effector molecules, such as lac repressor.) A. How could the function of the glucocorticoid receptor be shut off? B. What type of enzyme would be needed to shut off the activation of transcription by the CREB protein?
A. Eventually, the glucocorticoid hormone will be degraded by the cell. The glucocorticoid receptor binds the hormone with a certain affinity. The binding is a reversible process. Once the concentration of the hormone falls below the affinity of the hormone for the receptor, the receptor will no longer have the glucocorticoid hormone bound to it. When the hormone is released, the glucocorticoid receptor will change its conformation, and it will no longer bind to the DNA. B. An enzyme known as a phosphatase will eventually cleave the phosphate groups from the CREB protein. When the phosphates are removed, the CREB protein will stop activating transcription.
A particular drug inhibits the protein kinase that is responsible for phosphorylating the CREB protein. How would this drug affect the following events? A. The ability of the CREB protein to bind to CREs B. The ability of extracellular hormones to enhance cAMP levels C. The ability of the CREB protein to stimulate transcription D. The ability of the CREB protein to dimerize
A. No effect. B. No effect. C. It would be inhibited. D. No effect.
Briefly describe three ways that ATP-dependent chromatin-remodeling complexes may change chromatin structure.
ATP-dependent chromatin remodeling complexes may change the positions of nucleosomes, evict histones, and/or replace histones with histone variants.
Describe the steps that need to occur for the glucocorticoid receptor to bind to a GRE.
For the glucocorticoid receptor to bind to a GRE, a steroid hormone must first enter the cell. The hormone then binds to the glucocorticoid receptor, which releases HSP90. The release of HSP90 exposes a nuclear localization signal (NLS) within the receptor, which enables it to dimerize and then enter the nucleus. Once inside the nucleus, the dimer binds to a GRE, which activates transcription of the adjacent gene.
Discuss the structure and function of regulatory elements. Where are they located relative to the core promoter?
Regulatory elements are relatively short genetic sequences that are recognized by regulatory transcription factors. After a regulatory transcription factor binds to the regulatory element, it will affect the rate of transcription, either activating it or repressing it, depending on the action of the regulatory protein. Regulatory elements are typically located in the upstream region near the promoter, but they can be located almost anywhere (i.e., upstream and downstream) and even quite far from the promoter.
The binding of a small effector molecule, protein-protein interactions, and covalent modifications are three common ways to modulate the activities of transcription factors. Which of these three mechanisms are used by steroid receptors and by the CREB protein?
Steroid receptors: binding of an effector molecule and protein-protein interactions; CREB protein: covalent modification and protein-protein interactions.
Explain how the acetylation of core histones may loosen chromatin packing.
The attraction between DNA and histones occurs because the histones are positively charged and the DNA is negatively charged. The covalent attachment of acetyl groups decreases the amount of positive charge on the histone proteins and thereby may decrease the binding of the DNA. In addition, histone acetylation may attract proteins to the region that loosen chromatin compaction.
Is each of the following statements true or false? A. An enhancer is a type of regulatory element. B. A core promoter is a type of regulatory element. C. Regulatory transcription factors bind to regulatory elements. D. An enhancer may cause the down regulation of transcription.
A. True. B. False. C. True. D. False, it causes up regulation.
Transcription factors such as the glucocorticoid receptor and the CREB protein form homodimers and activate transcription. Other transcription factors form heterodimers. For example, a transcription factor known as myogenic bHLH forms a heterodimer with a protein called the E protein. This heterodimer activates the transcription of genes that promote muscle cell differentiation. However, when myogenic bHLH forms a heterodimer with a protein called the Id protein, transcriptional activation does not occur. (Note: Id stands for "Inhibitor of differentiation.") Which of the following possibilities best explains this observation? Only one possibility is correct.
Possibility 2 is the correct one. Because we already know that the E protein and the Id protein form heterodimers with myogenic bHLH, we expect all three proteins to have a leucine zipper. Leucine zippers promote dimer formation. We also need to explain why the Id protein inhibits transcription, while the E protein enhances transcription. As seen in possibility 2, the Id protein does not have a DNA-binding domain. Therefore, if it forms a heterodimer with myogenic bHLH, the heterodimer probably will not bind to the DNA very well. In contrast, when the E protein forms a heterodimer with myogenic bHLH, there will be two DNA-binding domains, which would promote good binding to the DNA. (Note: A description of these proteins is found in Chapter 26.)
Briefly describe the method of chromatin immunoprecipitation sequencing (ChIP-Seq). How is it used to determine nucleosome positions within a genome?
The method of ChIP-Seq is described in Figure 15.11. In the method described in this figure, only DNA fragments that are bound to nucleosomes are precipitated and sequenced. By comparing these sequences with the entire genome sequence, this information tells you which DNA sequences within a genome have nucleosomes bound to them.
The DNA-binding domain of each CREB protein subunit recognizes the sequence 5′-TGACGTCA-3′. Due to random chance, how often would you expect this sequence to occur in the human genome, which contains approximately 3 billion base pairs? Actually, only a few dozen genes are activated by the CREB protein. Does the value of a few dozen agree with the number of random occurrences expected in the human genome? If the number of random occurrences of the sequence in the human genome is much higher than a few dozen, provide at least one explanation why the CREB protein is not activating more than a few dozen genes.
There are four types of bases (A, T, G, and C), and this CRE sequence contains 8 bp, so according to random chance, it should occur every 4^8 bp, which equals every 65,536 bp. If we divide 3 billion by 65,536, this sequence is expected to occur approximately 45,776 times. This is much greater than a few dozen. There are several reasons why the CREB protein does not activate over 45,000 genes. 1. To create a functional CRE, there needs to be two of these sequences close together, because the CREB protein functions as a homodimer. 2. CREs might not be near a gene. 3. The conformation of chromatin containing a CRE might not be accessible to binding by the CREB protein.
Researchers can isolate a sample of cells, such as skin fibroblasts, and grow them in the laboratory. This procedure is called a cell culture. A cell culture can be exposed to a sample of DNA. If the Page 386 cells are treated with agents that make their membranes permeable to DNA, the cells may take up the DNA and incorporate it into their chromosomes. This process is called transformation or transfection. Scientists have transformed human skin fibroblasts with methylated DNA and then allowed the fibroblasts to divide for several cellular generations. The DNA in the daughter cells was then isolated, and the segment that corresponded to the transformed DNA was examined. This DNA segment in the daughter cells was also found to be methylated. However, if the original skin fibroblasts were transformed with unmethylated DNA, the DNA found in the daughter cells was also unmethylated. With regard to the transformed DNA, do fibroblasts perform de novo methylation, maintenance methylation, or both? Explain your answer.
These results indicate that the fibroblasts perform maintenance methylation because they can replicate and methylate DNA if it has already been methylated. However, the fibroblasts do not perform de novo methylation, because if the donor DNA was unmethylated, the DNA in the daughter cells remains unmethylated.
