contents Principles of Biology 52 Eukaryotic Regulation Gene regulation in eukaryotic cells may occur before or during or or after synthesis.

The . Digital model of a nucleosome, the fundamental structural unit of chromosomes in the eukaryotic cell nucleus, derived from X-ray crystallography data. Each nucleosome consists of a core group of (orange) wrapped in chromosomal DNA (green). The nucleosome is a structure responsible for regulating and condensing DNA strands to fit into the cell's nucleus. Researchers once thought that regulated gene activity through their histone tails, but a 2010 study revealed that the structures' core also plays a role. The finding sheds light on how is regulated and how abnormal gene regulation can lead to cancer. Kenneth Eward/Science Source.

Topics Covered in this Module

Mechanisms of Gene Regulation in Eukaryotic Cells

Major Objectives of this Module

Describe the role of in gene regulation. Explain how transcription offers multiple opportunities for gene regulation. Describe mechanisms of gene regulation that occur during or after translation. Describe the variety of mechanisms for gene regulation in eukaryotic cells.

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contents Principles of Biology

52 Eukaryotic Gene Regulation Mechanisms of Gene Regulation in Eukaryotic Cells Most multicellular organisms develop from a single-celled zygote into a number of different cell types by the process of differentiation, the acquisition of cell-specific differences. An animal nerve cell looks very different from a muscle cell, and a muscle cell has little structurally in common with a lymphocyte in the blood. What do all these cells of a particular organism have in common? They all have a nucleus with identical DNA sequences, , and cytoplasmic organelles like the Golgi apparatus and mitochondria. Thinking back to the zygote, what drives the differentiation process? If all the cells have the same DNA, the same "genetic blueprint," why do they become so different? The answer lies in the process of gene expression. All cellular structures are built from structural proteins or are constructed from raw materials that are synthesized using enzymes. Tracing back to the DNA in the genome, it all starts with the transcription of mRNA specific for the polypeptide chains the cell uses to build its components. Building a nerve cell and want to make some neurotransmitter? Need some actin or myosin to build a muscle cell? Or perhaps a lymphocyte needs to produce a cytokine. In all of these cases, the cell needs to express particular genes and coordinate the expression of multiple genes. How does it go about orchestrating this complex process?

Regulation of gene expression involves many different mechanisms. In , regulatory mechanisms are generally simpler than those found in . Prokaryotic regulation is often dependent on the type and quantity of nutrients that surround the cell as well as a few other environmental factors, such as temperature and pH. A combination of activators, and occasionally enhancers control transcription. Prokaryotic gene expression also happens in the same space as translation, reducing the opportunities for compartmentalization of regulation. Multicellular organisms have more complex genomes and the presence of a nucleus and separate cytoplasm provide a more compartmentalized structure. There are a number of different stages at which gene expression may be regulated in eukaryotes (Figure 1). Figure 1: Regulation of gene expression in eukaryotes may take place at several different stages. In the nucleus, the process of regulates the availability of a gene for transcription. Once transcribed, the of mRNA, or pre-mRNA, undergoes RNA processing, which involves splicing and the addition of a 5′ cap and a 3′ poly(A) tail to produce a mature mRNA in the nucleus. The mature mRNA is then exported from the nucleus to the cytoplasm, where its span varies. Once outside the nucleus, localization factors may target mature mRNAs to specific regions of the cytoplasm where they are translated into polypeptides. The resulting polypeptides can undergo post-translational modifications, which can regulate protein folding, glycosylation, intracellular transport, protein activation, and protein degradation. © 2014 Nature Education All rights reserved. Figure Detail

Several gene expression mechanisms involve chromatin structure. When eukaryotic cells aren't dividing, chromosomes exist in an uncondensed state called chromatin. Chromatin consists of DNA wrapped around a histone protein core. The wrapped DNA isn't as available for transcription as the DNA of prokaryotes, and as we'll discuss, mechanisms exist to relieve this repression. Also in eukaryotes, the RNA polymerase doesn't bind directly to the DNA, but instead binds via a set of proteins: the transcription initiation complex. Two different types of chromatin can be seen during interphase: and . Euchromatin, which is a lightly packed form, contains areas of DNA that are undergoing active gene transcription. Not all of the chromatin is undergoing gene transcription, however. Heterochromatin, in contrast, is mostly inactive DNA that is being actively inhibited or repressed in a region-specific manner. The chromatin state can change in response to cellular signals and gene activity. This is facilitated by enzymes that modify by adding methyl and acetyl groups to their N-terminal tails. Acetylation reduces the net positive charge of the histones, loosening their affinity for DNA, and increasing binding. Methylation, in contrast, leads to increased binding of histones to DNA, and decreases the availability of DNA for transcription. Figure 2 shows an example of how acetylation and methylation of histones may affect transcriptional activity in a normal cell compared to a cancer cell with inappropriate gene expression.

Figure 2: Modifications of methyl and acetyl groups in histones affect transcriptional activity. The grey cylinders represent histone octamers. Acetylation (blue circles) and methylation (green circles) of histone subunits are shown. In normal cells, the promoters of tumor-suppressor genes show acetylation of histone subunits, associated with active transcription. In contrast, in cancer cells, the promoters of tumor-suppressor genes are not acetylated, and the genes are not actively transcribed. In normal cells, the heterochromatic regions at the ends of the chromosomes do not show acetylation, and the genes are not actively transcribed. In cancer cells, the heterochromatic regions at chromosome ends are acetylated and transcriptionally active. © 2007 Nature Publishing Group Esteller, M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nature Reviews Genetics 8, 286-298 (2007) doi:10.1038/nrg2005. Used with permission. Besides the acetylation and methylation of histones, methylation of the DNA itself is also used to transcriptionally inactivate DNA. Some transcriptionally inactive DNA, such as that in inactivated mammalian X chromosomes, shows more methylation than transcriptionally active chromosomal regions. Among individual genes, those that are transcriptionally inactive usually show more methylation than genes that are active, and removal of methyl groups can "turn on" genes. Methylation seems to be important for genes that are to remain inactive for a number of cell divisions. The methylation pattern is preserved during DNA replication, so the daughter cells have the same methylation pattern as the parent cell. Methylation may therefore be an important component in the differentiation process in eukaryotes. If a methylation pattern, and hence the pattern of gene expression, may be inherited in a cell line, is it possible that these kinds of patterns may be inherited through generations? This phenomenon is called "epigenetic inheritance" because it is inheritance "outside of the genes," and it has been observed in a large number of circumstances. The key to the process is the inheritance of epigenetic tags, typically methylation of DNA or methylation or acetylation of histone proteins, instead of the inheritance of mutated or variant sequences of DNA. In eukaryotes, these tags can be passed through the gametes to the next generation.

Transcription is an important stage for gene regulation. Test Yourself

What is necessary before transcription may occur in eukaryotes?

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In eukaryotes, the RNA immediately transcribed from a DNA template, the pre-mRNA, undergoes a number of processing events before it becomes a mature mRNA (Figure 3). The RNA polymerase needs transcription factors to initiate transcription. Some general transcription factors are required for all genes. Some bind to specific sequences of DNA such as the TATA box. Others bind to proteins such as RNA polymerase II. These general transcription factors don't usually produce high rates of transcription, and for that reason, gene-specific transcription factors called activators or repressors are also required. These factors bind to proximal or distal control elements, which are specific DNA sequences that are usually four to eight base pairs long. The rate of gene expression may be greatly affected by binding of specific transcription factors to control elements. Proximal control elements are close to the . Distal control elements may be grouped as enhancers, and may be thousands of nucleotides removed from the gene. Although one gene may have more than one , a given enhancer is usually associated with only one gene.

Figure 3: The structure of a eukaryotic gene and its transcript. Each gene has a promoter, the DNA sequence where RNA polymerase, along with transcription factors, binds and begins transcription. RNA processing removes introns and splices the exons together using structures called spliceosomes, and a 5′ cap and poly(A) 3′ tail are added to the mRNA transcript. The mRNA is then translated into a polypeptide. © 2013 Nature Education All rights reserved.

How does the binding of transcription factors to control elements regulate transcription? There seem to be two structural components in transcription factors: a region that binds to DNA and an activation domain that attaches to other proteins or components of the transcription apparatus itself. There are only a few different kinds of binding regions in control elements: these are called DNA sequence motifs. The binding of transcription factors that function as activators to control elements in an enhancer may cause the DNA to bend. This bending brings the enhancer complex into contact with the at the proximal promoter, creating a large complex that promotes RNA polymerase binding. RNA polymerase II is then recruited and transcription can begin (Figure 4). Some transcription factors function as repressors that bind to control elements, effectively blocking the binding of activators.

Figure 4: The eukaryotic transcription initiation complex. When proteins bind to distal control elements called enhancers, the bound activators are brought closer to the promoter by a DNA-bending protein. The activators bind to the Mediator protein complex, which forms an active transcription initiation complex on the promoter together with RNA polymerase and the general transcription factors. © 2014 Nature Education All rights reserved. Figure Detail

The number of different DNA sequence motifs found in control elements is quite small and is thought to be around ten. It is believed that the combination of control elements in an enhancer provides the specificity for gene regulation. The availability of ten different sequences gives a very large set of available combinations — much like the lock on a bicycle, only the correct combination will "unlock" and allow activation of an enhancer.

How do eukaryotic cells coordinate gene expression? Eukaryotic cells frequently need to activate combinations of genes, but unlike prokaryotes, genes involved in the same metabolic pathway or another set of coordinated events may be scattered across different chromosomes. How is their expression coordinated? The answer lies in the combination of control elements for each gene. If all the genes that need to work together have the same set of control elements, the same activators will work on all the control elements, and transcription will be simultaneous for all the genes with the same set of control elements. Similar to the induction of gene activity in a bacterial in response to nutrient levels in the surrounding medium, coordinated gene activity in a tissue is often in response to chemical or mechanical signals from outside the cell.

Future perspectives. By using the FISH (fluorescence in situ hybridization) technique with probes that bind to RNA, we now know that nascent transcription and active RNA polymerase II are often concentrated at foci in the nucleus. These foci have been named transcription factories. The fascinating thing about transcription factories is that it looks as if genes on different chromosomes are brought together into proximity to coordinate transcription. For example, globin genes in developing erythrocytes have been shown to consistently associate with transcription factories that are sites of active transcription of a number of coexpressed genes. Transcription factories can only exist in interphase, as the chromosomes have to condense to undergo mitosis during prophase.

Additional mechanisms of gene expression occur after transcription. So far we have concentrated on gene expression up until the stage of mRNA transcription, but what happens after that? Are there other mechanisms for controlling gene expression? And if so, why do they exist? One post-transcriptional control mechanism is alternative RNA splicing, which produces different mRNA molecules from the same primary transcript. The primary mRNA transcript is composed of both non-coding introns and protein-coding exons. Regulatory proteins remove the introns and control the exon choices by binding to regulatory sequences within the primary transcript. Alternative RNA splicing may be a major reason for the diversity of proteins in higher animals over those found in simpler organisms. For example, the number of genes in a human is remarkably similar to those in a nematode or sea urchin. However, there are many more genes with multiple exons in higher animals, so alternative RNA splicing may provide a way of making more types of proteins from the same amount of genomic DNA (Figure 5).

Figure 5: of RNA. Different splicing patterns result in the production of two different mRNAs and translation of two different proteins from the same gene. In this example, a gene gives rise to a primary transcript, or pre-mRNA, that is spliced differently in two different tissues, A and B. In both tissues, the three blue exons are present in the mature mRNA, but in tissue A (left), the orange exon is spliced out, leaving the purple exon, and in tissue B (right), the purple exon is spliced out, leaving the orange exon. Translation of the respective mature mRNAs results in two structurally and functionally distinct proteins derived from the same gene. © 2014 Nature Education All rights reserved.

Eukaryotic mRNA molecules are longer-lasting than prokaryotic mRNAs, and mRNA life span is a key parameter in protein synthesis in a cell. Rapid mRNA degradation is a useful feature of prokaryotes because they may need to change their protein synthesis rapidly in response to environmental changes. In eukaryotes, some mRNAs may exist for periods ranging from days to weeks, and they may be repeatedly translated, such as the mRNAs that produce hemoglobin molecules in red blood cells. mRNA stability seems to be associated with changes in the poly(A) tail length. If the tail is shortened, enzymes may be triggered that remove the 5′ cap of the RNA. Once the cap is removed, nuclease enzymes degrade the mRNA. Before a polypeptide is produced, translation has to occur. Therefore, another stage where control of gene expression can occur is by blocking the initiation of translation. One place where this is often seen is in unfertilized eggs. Eggs have many mRNA molecules that are not translated until fertilization occurs. These mRNAs will produce many proteins that are important in development, but they are not needed while the egg waits to be fertilized. Translation of the mRNAs may be blocked by the binding of regulatory proteins to sequences in the leader region at the 5′ end of the mRNA, preventing the attachment of . Finally, regulation may occur post-translationally. In eukaryotes, polypeptides must often be processed to create functional proteins. For example, proteins may need to be folded, modified by adding carbohydrate groups, or activated by . Regulation may occur by modifying any of these steps. Protein degradation also occurs in the cell, facilitated by a protein called ubiquitin, so named because it is ubiquitous in cells. Once a protein is "tagged" by adding ubiquitin to it, a protein-degrading enzyme complex called a proteasome cuts the protein up, effectively destroying it and making the amino acids available for synthesis of new polypeptides.

Future perspectives. Post-translational modifications to proteins may result in specific functions. For example, histone polypeptides, which are intimately associated with DNA in chromatin, are subject to many post-translational modifications. It has been suggested that there may be a "histone code" in which histone tails are "read" by effector molecules and that this coding system may provide a way that epigenetic processes are coded.

BIOSKILL Control Regions of Genes Specify Developmental Patterns In the fruit fly Drosophila melanogaster, a group of mutations was analyzed by Walther Gehring's laboratory in Switzerland in the 1970s and 1980s. Gehring's group, together with a group at Indiana University Bloomington, studied fruit fly mutations like Antennapedia and Ultrabithorax. Antennapedia is a mutation in which legs grow where the antennae should be, and Ultrabithorax flies have a second set of wings on the third thoracic segment. Normal flies have a single set of wings on the second thoracic segment. Test Yourself

What effects are these two genes having on the fly's development? How might the genes be working?

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Antennapedia and Ultrabithorax are actually members of a group of related genes that control anterior-posterior organization in an animal. These are called the Hox genes. Hox genes are found in organisms from insects to humans and are characterized by having a 180-base-pair DNA sequence known as the homeobox that encodes a transcriptional activator. The Hox genes are arranged in a cluster along a chromosome, and they show an expression pattern where the linear order of the genes along the chromosome corresponds to the anterior-posterior region of their expression in the developing embryo (Figure 6).

Figure 6: Drosophila melanogaster embryo that has been colored to indicate the approximate domains of transcriptional expression for Hox genes. Here, the expression domain of Antennapedia is colored light brown and that of Ultrabithorax is colored red. The segments are labeled (Md, mandibular; Mx, maxillary; Lb, labial; T1-T3, thoracic segments 1-3; A1-A9, abdominal segments 1-9). © 2011 Nature Education All rights reserved.

Each of the proteins encoded by the Hox genes binds to specific DNA binding sites that regulate the expression of other transcription factors, turning on the hundreds of genes that determine the characteristics of that region of the embryo. It is thought that the Hox genes regulate whole series of other genes — for example, the gene pathway that produces the proteins needed to make an appendage.

BIOSKILL

IN THIS MODULE

Mechanisms of Gene Regulation in Eukaryotic Cells Summary Test Your Knowledge

WHY DOES THIS TOPIC MATTER?

Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential?

Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.

Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete?

PRIMARY LITERATURE

Using sterile mates and engineered toxins to beat bugs Suppressing resistance to Bt cotton with sterile insect releases. View | Download

Growing new heart cells to treat damaged hearts Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. View | Download Interfering with to control gene expression Silencing of microRNA families by seed-targeting tiny LNAs. View | Download

Classic paper: How scientists cloned the first mammal (1997) Viable offspring derived from fetal and adult mammalian cells. View | Download

Inhibitors may block entry of hepatitis C into cells EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. View | Download

page 265 of 989 2 pages left in this module

contents Principles of Biology

52 Eukaryotic Gene Regulation Summary OBJECTIVE Describe the variety of mechanisms for gene regulation in eukaryotic cells. Eukaryotic cells have several mechanisms for gene regulation, involving regulation of transcription, post-transcriptional modifications, control of translation, control of mRNA degradation and post-translational controls.

OBJECTIVE Describe the role of chromatin in gene regulation. Chromatin plays an important role in gene expression by controlling the availability of genes and their regulatory sequences for transcription.

OBJECTIVE Explain how transcription offers multiple opportunities for gene regulation. Transcription offers multiple opportunities for gene regulation using general and specific transcription factors. Transcription factories may allow simultaneous regulation of genes on different chromosomes.

OBJECTIVE Describe mechanisms of gene regulation that occur during or after translation. Gene regulation that occurs during or after translation includes controlling the initiation of translation and post-translational modifications, which include protein folding, glycosylation, intracellular transport, proteolytic activation, and degradation.

Key Terms acetylation The modification of a histone by the addition of an acetyl (–COCH3) functional group, which reduces the net positive charge of the histone, loosens its binding to DNA, and improves access and binding of transcription factors and RNA polymerase to the DNA.

alternative RNA splicing An RNA processing event that produces different mRNA molecules from the same primary mRNA transcript.

control element A DNA sequence, such as an enhancer, , or binding site, that controls the rate of gene expression. differentiation The acquisition of cell-specific differences during a multicellular organism's embryonic development or adult life. enhancer A DNA sequence that is used by a transcriptional activator to increase the expression of a particular gene; this sequence is not necessarily located close to the gene in question and may even be very far from the gene or on a different chromosome altogether.

euchromatin A lightly compacted form of chromatin that is usually under active gene transcription.

heterochromatin A densely compacted form of chromatin that is usually being actively repressed from gene expression. histone protein One of eight proteins that form the histone, a protein structure around which DNA is compacted to form chromatin. methylation The addition of a methyl (–CH3) functional group; methylation of histones increases the compaction between histones and DNA, decreasing the availability of DNA for transcription; methylation of DNA bases can permanently and heritably alter gene expression without changing the DNA sequence. mRNA degradation The destruction of an mRNA molecule; exists as a normal mechanism to regulate gene expression; additionally, in eukaryotes, can be caused by the removal of the 3′ poly(A) tail and/or the 5′ cap of the mRNA. primary transcript The mRNA molecule immediately transcribed from a DNA transcript in eukaryotes; subject to post-transcriptional modification, such as splicing, 5′ capping and 3′ , after its synthesis. protein degradation The decomposition of unneeded polypeptides into their constituent amino acids, freeing the amino acids for use in synthesizing new polypeptides. transcription factor A protein that regulates the transcription of specific genes. transcription factory Sites of active transcription in eukaryotic cells where multiple genes are simultaneously transcribed to maximize efficiency; often involves genes that perform related functions.

IN THIS MODULE

Mechanisms of Gene Regulation in Eukaryotic Cells Summary Test Your Knowledge

WHY DOES THIS TOPIC MATTER?

Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential?

Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.

Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete?

PRIMARY LITERATURE

Using sterile mates and engineered toxins to beat bugs Suppressing resistance to Bt cotton with sterile insect releases. View | Download

Growing new heart cells to treat damaged hearts Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. View | Download

Interfering with microRNAs to control gene expression Silencing of microRNA families by seed-targeting tiny LNAs. View | Download

Classic paper: How scientists cloned the first mammal (1997) Viable offspring derived from fetal and adult mammalian cells. View | Download Inhibitors may block entry of hepatitis C into cells EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. View | Download

page 266 of 989 1 pages left in this module

contents Principles of Biology

52 Eukaryotic Gene Regulation

Test Your Knowledge

1. How do eukaryotes and prokaryotes differ?

Eukaryotes contain RNA, while prokaryotes do not. Prokaryotes contain DNA, while eukaryotes do not. Eukaryotes contain a nucleus, while prokaryotes do not. Prokaryotes contain ribosomes, while eukaryotes do not. Eukaryotes contain proteins, while prokaryotes do not.

2. Which of the following reduces the net positive charge of a histone?

binding to a promoter RNA polymerase lactose binding acetylation methylation

3. Which of the following statements is true of an enhancer?

Enhancers are downstream of the promoter. Enhancers are usually specific for many genes. Enhancers may be thousands of nucleotides from the gene they are associated with. One gene cannot have more than one enhancer. None of the answers are correct.

4. Which of these is NOT a process that may be involved in post-transcriptional regulation?

methylation blocking translation phosphorylation protein degradation protein folding

5. What is a transcription factory?

a supergene that transcribes many different mRNA molecules a physical grouping of genes on different chromosomes that come together to coordinate transcription mitochondrial genes a repeated sequence of genes a group of genes clustered together on the same chromosome

6. What is the function of the proteasome?

mRNA degradation ubiquitin production protein degradation stitching exons together modifying the folding structure of proteins Submit

IN THIS MODULE

Mechanisms of Gene Regulation in Eukaryotic Cells Summary Test Your Knowledge

WHY DOES THIS TOPIC MATTER?

Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential?

Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.

Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete?

PRIMARY LITERATURE

Using sterile mates and engineered toxins to beat bugs Suppressing resistance to Bt cotton with sterile insect releases. View | Download

Growing new heart cells to treat damaged hearts Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. View | Download

Interfering with microRNAs to control gene expression Silencing of microRNA families by seed-targeting tiny LNAs. View | Download

Classic paper: How scientists cloned the first mammal (1997) Viable offspring derived from fetal and adult mammalian cells. View | Download

Inhibitors may block entry of hepatitis C into cells EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. View | Download

page 267 of 989