Harper's Biochemistry Chapter 38 Regulation of Gene Expression - PDF

C H A P T E R Regulation of Gene Expression P. Anthony Weil, PhD 38 OBJ EC T IVES Explain that the many steps involved in the vectorial processes of gene...

C H A P T E R Regulation of Gene Expression P. Anthony Weil, PhD 38 OBJ EC T IVES Explain that the many steps involved in the vectorial processes of gene expression: targeted modulation of gene copy number, gene rearrangement, After studying this chapter, transcription, mRNA processing and transport from the nucleus, translation, you should be able to: protein subcellular compartmentalization, posttranslational modification, and degradation are all subject to regulatory control, both positive and negative. Changes in any, or multiple of these processes, can increase or decrease the amount and/or activity of the cognate gene product. Appreciate that DNA-binding transcription factors, proteins that bind to specific DNA sequences that are physically linked to their target transcriptional promoter elements, can either activate or repress gene transcription. Recognize that DNA-binding transcription factors are often modular proteins composed of structurally and functionally distinct domains. These transcription factors can directly or indirectly control messenger RNA (mRNA) gene transcription, either through contacts with RNA polymerase and its cofactors, or through interactions with coregulators that modulate nucleosome occupancy, position, structure, composition, and histone covalent modifications. Understand that nucleosome-directed regulatory events typically increase or decrease the accessibility of the underlying DNA such as enhancer or promoter sequences, although some nucleosome modifications can also create new binding sites for other coregulators. Describe how the processes of gene transcription, RNA processing, and nuclear export of RNA are all coupled. Describe the phenomenon of epigenetic gene regulation and how such processes occur at the molecular level. BIOMEDICAL IMPORTANCE and gene regulation can be influenced by a range of physio- logic, biologic, environmental, and pharmacologic agents. Organisms alter expression of genes in response to genetic In addition to transcription level controls, gene expression developmental cues or programs, environmental challenges, can also be modulated by gene amplification, gene rearrange- or disease, by modulating the amount, the spatial, and/or the ment, posttranscriptional modifications, RNA stabilization, temporal patterns of gene expression. The mechanisms con- translational control, protein modification, protein compart- trolling gene expression have been studied in detail and often mentalization, and protein stabilization or degradation. Many involve changes in gene transcription. Control of transcription of the mechanisms that control gene expression are utilized ultimately results from changes in the mode of interaction of to respond to developmental cues, growth factors, hormones, specific regulatory molecules, usually proteins, with various environmental agents, and therapeutic drugs. Dysregulation regions of DNA in the regulated gene. Such interactions can of gene expression can lead to human disease. Thus, a molecu- either have a positive or negative effect on transcription. Tran- lar understanding of these processes will lead to development scription control can result in tissue-specific gene expression, 420  CHAPTER 38 Regulation of Gene Expression 421 of therapeutics that can alter pathophysiologic mechanisms activator. However, a double negative has the effect of acting or inhibit the function or arrest the growth of pathogenic as a positive. Thus, an effector that inhibits the function of a organisms. negative regulator will appear to bring about a positive regula- tion. Many regulated systems that appear to be induced are in fact derepressed at the molecular level. (See Chapter 9 for REGULATED EXPRESSION additional discussion of these terms.) OF GENES IS REQUIRED FOR DEVELOPMENT, BIOLOGIC SYSTEMS EXHIBIT DIFFERENTIATION, & THREE TYPES OF TEMPORAL ADAPTATION RESPONSES TO A REGULATORY The genetic information present in each normal somatic cell SIGNAL of a metazoan organism is practically identical. The geneti- Figure 38–1 depicts the extent or amount of gene expression cally reproducible, hardwired exceptions are found in those in three types of temporal responses to an inducing signal. few cells that have amplified or rearranged genes in order to A type A response is characterized by an increased extent of perform specialized cellular functions. Of course, in various gene expression that is dependent on the continued presence disease states chromosome integrity is altered (ie, cancer; see of the inducing signal. When the inducing signal is removed, Figure 56–11) sometimes even at the whole chromosome level the amount of gene expression diminishes to its basal (eg, trisomy 21, that causes Down syndrome). Expression of the level, but the amount repeatedly increases in response to genetic information must be regulated during ontogeny and differentiation of the organism and its cellular components. Furthermore, in order for the organism to adapt to its envi- Type A ronment and to conserve energy and nutrients, the expression Gene expression of genetic information must be cued to extrinsic signals and respond only when necessary. As organisms have evolved, more sophisticated regulatory mechanisms have appeared which provide the organism and its cells with the responsiveness nec- essary for survival in a complex environment. Mammalian cells possess about 1000 times more genetic information than does the bacterium Escherichia coli. Much of this additional genetic Time Signal information is likely involved in regulation of gene expression during the differentiation of tissues and biologic processes in Type B the multicellular organism and in ensuring that the organism can respond to complex environmental challenges. Gene expression In simple terms, there are only two types of gene regulation: positive regulation and negative regulation (Table 38–1). When the expression of genetic information is quantitatively increased by the presence of a specific regulatory element, reg- ulation is said to be positive; when the expression of genetic Recovery information is diminished by the presence of a specific regula- Time tory element, regulation is said to be negative. The element or Signal molecule mediating negative regulation is said to be a negative Type C regulator, a silencer or repressor; while the element mediat- ing positive regulation is a positive regulator, an enhancer or Gene expression TABLE 38–1 Effects of Positive & Negative Regulation on Gene Expression Rate of Gene Expression Negative Positive Time Regulation Regulation Signal Regulator present Decreased Increased FIGURE 38–1 Diagrammatic representations of the responses Regulator absent Increased Decreased of the extent of expression of a gene to specific regulatory signals as a function of time. 422 SECTION VII Structure, Function, & Replication of Informational Macromolecules the reappearance of the specific signal. This type of response an operon, for example, the lac operon. An operon can be is commonly observed in prokaryotes in response to sudden regulated by a single promoter or regulatory region. The cistron changes of the intracellular concentration of a nutrient. It is also is the smallest unit of genetic expression. A single mRNA observed in many higher organisms after exposure to inducers that encodes more than one separately translated protein is such as hormones, nutrients, or growth factors (see Chapter 42). referred to as a polycistronic mRNA. For example, the poly- A type B response exhibits an increased amount of gene cistronic lac operon mRNA is translated into three separate expression that is transient even in the continued presence of proteins (see following discussion). Operons and polycistronic the regulatory signal. After the regulatory signal has terminated mRNAs are common in bacteria but not in eukaryotes. and the cell has been allowed to recover, a second transient An inducible gene is one whose expression increases in response to a subsequent regulatory signal may be observed. response to an inducer or activator, a specific positive regu- This phenomenon of response-desensitization recovery char- latory signal. In general, inducible genes have relatively low acterizes the action of many pharmacologic agents, but it is also basal rates of transcription. By contrast, genes with high basal a feature of many naturally occurring processes. This type of rates of transcription are often subject to downregulation by response commonly occurs during development of an organ- repressors. ism, when only the transient appearance of a specific gene The expression of some genes is constitutive, meaning that product is required although the signal persists. they are expressed at a reasonably constant rate and not known to The type C response pattern exhibits, in response to the be subject to extensive regulation. Such genes are often referred regulatory signal, an increased extent of gene expression that to as housekeeping genes. As a result of mutation, some induc- persists indefinitely even after termination of the signal. The ible gene products become constitutively expressed. A mutation signal acts as a trigger in this pattern. Once expression of the resulting in constitutive expression of what was formerly a regu- gene is initiated in the cell, it cannot be terminated even in lated gene is called a constitutive mutation. the daughter cells; it is therefore an irreversible and inherited alteration. This type of response typically occurs during the Analysis of Lactose Metabolism in development of differentiated function in a tissue or organ. E. coli Led to the Discovery of the Basic Principles of Gene Transcription Simple Unicellular & Multicellular Activation & Repression Organisms Serve as Valuable Models Jacob and Monod in 1961 described their operon model in for the Study of Gene Expression in a classic paper. Their hypothesis was to a large extent based Human Cells on observations on the regulation of lactose metabolism by Analysis of the regulation of gene expression in prokaryotic the intestinal bacterium E. coli. The molecular mechanisms cells helped establish the principle that information flows responsible for the regulation of the genes involved in the from the gene to a messenger RNA to a specific protein metabolism of lactose are now among the best-understood in molecule. These studies were aided by the advanced genetic any organism. β-Galactosidase hydrolyzes the β-galactoside analyses that could be performed in prokaryotic and lower lactose to galactose and glucose. The gene encoding eukaryotic organisms such as the baker’s yeast, Saccharomyces β-galactosidase (lacZ) is clustered with the genes encoding cerevisiae, and the fruit fly, Drosophila melanogaster, among lactose permease (lacY) and thiogalactoside transacetylase others. In recent years, the principles established in these (lacA). The genes encoding these three enzymes, along with studies, coupled with a variety of physical, optical, biochemi- the lac promoter and lac operator (a regulatory region), and cal, informatic, and molecular biological techniques, have led the lacI gene encoding the LacI repressor are physically linked to remarkable progress in the analysis of gene regulation in and constitute the lac operon as depicted in Figure 38–2. higher eukaryotic organisms, including humans. In this chapter, the initial discussion will center on prokaryotic systems. The Promoter Pro P rro omot mot mo oter er CRE CR C RE R E Operator O Op Ope pe pera ato tto tor or impressive genetic studies will not be described, but the physi- cI lacI lacZ lacY lacA ology of gene expression will be discussed. However, nearly TSS all of the conclusions about this physiology have been derived lac operon from genetic studies and confirmed by molecular biological and biochemical experiments. FIGURE 38–2 The positional relationships of the protein coding and regulatory elements of the ~6kbp lac operon. lacZ Some Features of Prokaryotic Gene encodes β-galactosidase, lacY encodes a permease, and lacA encodes a transacetylase. lacI encodes the lac operon repressor protein. Also Expression Are Unique shown is the transcription start site for lac operon transcription (TSS). Note that the binding site for the LacI protein (ie, lac repressor)—the Before the physiology of gene expression can be explained, lac operator (Operator)—overlaps the lac promoter. Immediately a few specialized genetic and regulatory terms must be defined upstream of the lac operon promoter is the binding site (CRE) for the for prokaryotic systems. In prokaryotes, the genes involved in cAMP-binding protein, CAP, the positive regulator of lac operon tran- a metabolic pathway are often present in a linear array called scription. See Figure 38–3 for more detail.  CHAPTER 38 Regulation of Gene Expression 423 This genetic arrangement of the lac operon allows for coordi- region of double-stranded DNA that exhibits a twofold rota- nate expression of the three enzymes concerned with lactose tional symmetry and an inverted palindrome (indicated by metabolism. Each of the linked operon genes is transcribed arrows about the dotted axis) in a region that is 21-bp long, into one large polycistronic mRNA molecule that contains shown as follows: multiple independent translation start (AUG) and stop (UAA) codons for each of the three cistrons. Thus, each protein is translated separately, and they are not processed from a single large precursor protein. It is now conventional to consider that a gene includes regulatory sequences as well as the region that encodes the At any one time, only two of the four subunits of the repres- primary transcript. Although there are many historical excep- sor appear to bind to the operator; within the 21-base-pair tions, a gene is generally italicized in lower case and the operator region nearly every base of each base pair is involved encoded protein, when abbreviated, is expressed in roman in LacI recognition and binding. Binding occurs mostly in the type with the first letter capitalized. For example, the gene lacI major groove without interrupting the base-paired, double- encodes the repressor protein LacI. When E. coli is presented helical nature of the operator DNA. The operator locus with lactose or some specific lactose analogs under appro- (ie, LacI binding site) is between the promoter, the site where priate nonrepressing conditions (eg, high concentrations of the DNA-dependent RNA polymerase attaches to commence lactose, no or very low glucose in media; see following dis- transcription, and the transcription initiation site of the lacZ cussion), the expression of the activities of β-galactosidase, gene, the structural gene for β-galactosidase (see Figures 38–2 galactoside permease, and thiogalactoside transacetylase is and 36–3). When bound to the operator locus, the LacI repres- increased 100-fold to 1000-fold. This is a type A response, as sor molecule prevents transcription of the distal structural depicted in Figure 38–1. The kinetics of induction can be quite genes, lacZ, lacY, and lacA by interfering with the binding of rapid; lac-specific mRNAs are fully induced within ~5 minutes RNA polymerase to the promoter; RNA polymerase and LacI after addition of lactose to a culture; β-galactosidase protein is repressor cannot be effectively bound to the lac operon at the maximal within 10 minutes. Under fully induced conditions, same time. Thus, the LacI repressor molecule is a negative reg- there can be up to 5000 β-galactosidase molecules per cell, an ulator, and in its presence (and in the absence of inducer; see amount about 1000 times greater than the basal, uninduced following discussion), expression from the lacZ, lacY, and lacA level. Upon removal of the signal, that is, the inducer, the syn- genes is very, very low. There are normally about 30 repressor thesis of these three enzymes declines. tetramer molecules in the cell, a concentration (3 × 10−8 mol/L) When E. coli is exposed to both lactose and glucose as of tetramer sufficient to effect, at any given time, more than 95% sources of carbon, the cells first metabolize the glucose and occupancy of the one lac operator element in a bacterium, thus then temporarily stop growing until the genes of the lac ensuring low (but not zero) basal lac operon gene transcription operon become induced to provide the ability to metabolize in the absence of inducing signals. lactose as a usable energy source. Although lactose is present A lactose analog that is capable of inducing the lac operon from the beginning of the bacterial growth phase, the cell does while not itself serving as a substrate for β-galactosidase is an not induce those enzymes necessary for catabolism of lac- example of a gratuitous inducer. An example is isopropyl- tose until glucose has been exhausted. This phenomenon was thiogalactoside (IPTG). The addition of lactose or of a gra- first thought to be attributable to repression of the lac operon tuitous inducer such as IPTG to bacteria growing on a poorly by some catabolite of glucose; hence, it was termed catabo- utilized carbon source (such as succinate) results in prompt lite repression. It is now known that catabolite repression is induction of the lac operon enzymes. Small amounts of the in fact mediated by a catabolite activator protein (CAP) in gratuitous inducer or of lactose are able to enter the cell even conjunction 3′,5′ cyclic Adenosine monophosphate (cAMP; in the absence of permease. The LacI repressor molecules— see Figure 18–5). This protein is also referred to as the cAMP both those attached to the operator loci and those free in regulatory protein (CRP). The expression of many inducible the cytosol—have a high affinity for the inducer. Binding enzyme systems or operons in E. coli and other prokaryotes of the inducer to repressor molecule induces a conforma- is sensitive to catabolite repression, as discussed in following tional change in the structure of the repressor that causes a discussion. decrease in operator DNA occupancy because its affinity for The physiology of induction of the lac operon is well the operator is now 104 times lower (Kd about 10−9 mol/L) understood at the molecular level (Figure 38–3). Expression than that of LacI in the absence of IPTG. DNA-dependent of the normal lacI gene of the lac operon is constitutive; it is RNA polymerase can now more efficiently compete with LacI expressed at a constant rate, resulting in formation of the sub- and bind to the promoter (ie, Figures 36–3 and 36–8), and units of the lac repressor. Four identical subunits with molec- transcription will begin, although this process is relatively ular weights of 38,000 assemble into a tetrameric Lac repressor inefficient (see following discussion). In such a manner, an molecule. The LacI repressor protein molecule, the product of inducer derepresses the lac operon and allows transcrip- lacI, has a very high affinity (dissociation constant, Kd about tion of the genes encoding β-galactosidase, galactoside per- 10−13 mol/L) for the operator locus. The operator locus is a mease, and thiogalactoside transacetylase. Translation of the 424 SECTION VII Structure, Function, & Replication of Informational Macromolecules FIGURE 38–3 The mechanism of repression, derepression, and activation of the lac operon. When no inducer is present (A), the con- stitutively synthesized lacI gene products form a repressor tetramer that binds to the operator. Repressor-operator binding prevents the binding of RNA polymerase and consequently prevents transcription of the lacZ, lacY, and lacA genes into a polycistronic mRNA. When inducer is present, but glucose is also present in the culture medium (B), the tetrameric repressor molecules are conformationally altered by inducer, and cannot efficiently bind to the operator locus (affinity of binding reduced >1000-fold). However, RNA polymerase will not efficiently bind the promoter and initiate transcription because positive protein–protein interactions between CRE-bound CAP protein and RNA polymerase fail to occur; thus, the lac operon is not efficiently transcribed. However, when inducer is present, and glucose is depleted from the medium (C), adenylyl cyclase is activated and cAMP is produced. This cAMP binds with high affinity to its binding protein the cyclic AMP activator protein, or CAP. The CAP-cAMP complex binds to its recognition sequence (CRE, the cAMP response element) at lac operon nucleotide coordinate −50. Direct protein–protein contacts between the CRE-bound CAP and the RNA polymerase increases promoter binding more than 20-fold; hence RNAP will efficiently tran- scribe the lac operon and the polycistronic lacZ-lacY-lacA mRNA molecule formed can be translated into the corresponding protein molecules β-galactosidase, permease, and transacetylase as shown. This protein production enables cellular catabolism of lactose as the sole carbon source for growth.  CHAPTER 38 Regulation of Gene Expression 425 polycistronic mRNA can occur even before transcription is The Genetic Switch of Bacteriophage completed. Derepression of the lac operon allows the cell to synthesize the enzymes necessary to catabolize lactose as an Lambda (λ) Provides Another Paradigm energy source. Based on the physiology just described, IPTG- for Understanding the Role of induced expression of transfected plasmids bearing the lac Conditional Regulatory Protein-DNA operator–promoter ligated to appropriate bioengineered con- structs is commonly used to express mammalian recombinant Interactions in Transcriptional Control proteins in E. coli. in Eukaryotic Cells In order for the RNA polymerase to form a PIC at the pro- Like some eukaryotic viruses (eg, herpes simplex virus and HIV), moter site most efficiently, the cAMP-CAP complex must also certain bacterial viruses can either reside in a dormant state be present in the cell. By an independent mechanism, the bac- within the host chromosomes or can replicate within the bac- terium accumulates cAMP only when it is starved for a source terium and eventually lead to lysis and killing of the bacterial of carbon. In the presence of glucose—or of glycerol in con- host. Some E. coli harbor such a “temperate” virus, bacterio- centrations sufficient for growth—the bacteria will lack suf- phage lambda (λ). When lambda infects an organism of that ficient cAMP to bind to CAP because glucose inhibits adenylyl species, it injects its 48,490-bp, double-stranded, linear DNA cyclase, the enzyme that converts ATP to cAMP (see Chap- genome into the cell (Figure 38–4). Depending on the nutri- ter 42). Thus, in the presence of glucose or glycerol, cAMP- tional state of the cell, the lambda DNA will either integrate into saturated CAP is lacking, so that the DNA-dependent RNA the host genome (lysogenic pathway) and remain dormant polymerase cannot initiate transcription of the lac operon at until activated (see following discussion), or it will commence the maximal rate. However, in the presence of the CAP-cAMP replicating until it has made about 100 copies of complete, complex, which binds to CAP Response Element (CRE) protein-packaged virus, at which point it causes lysis of its host DNA just upstream of the promoter site, transcription occurs (lytic pathway). The newly generated virus particles can then at maximal levels (see Figure 38–3). Studies indicate that a infect other susceptible host cells. Poor growth conditions favor region of CAP directly contacts the RNA polymerase (RNAP) lysogeny while good growth conditions promote the lytic path- α subunit, and these protein–protein interactions facilitate way of lambda growth. the binding of RNAP to the promoter. Thus, the CAP-cAMP When integrated into the host genome in its dormant state, regulator is acting as a positive regulator because its pres- lambda will remain in that state until activated by exposure ence is required for optimal gene expression. The lac operon is of its bacterial host to DNA-damaging agents. In response to therefore controlled by two distinct, ligand-modulated DNA- such a noxious stimulus, the dormant bacteriophage becomes binding trans-factors; one that acts positively (cAMP-CRP “induced” and begins to transcribe and subsequently translate complex) to facilitate productive binding of RNA polymerase those genes of its own genome that are necessary for its exci- to the promoter and one that acts negatively (LacI repressor) sion from the host chromosome, its DNA replication, and the that antagonizes RNA polymerase promoter binding. Maximal synthesis of its protein coat and lysis enzymes. This event acts activity of the lac operon occurs when glucose levels are low like a trigger or type C (see Figure 38–1) response; that is, once (high cAMP with CAP activation) and lactose is present dormant lambda has committed itself to induction, there is no (LacI is prevented from binding to the operator) as shown in turning back until the cell is lysed and the replicated bacte- Figure 38–3, panel C. riophage released. This switch from a dormant or prophage With the above information in hand, it becomes relatively state to a lytic infection is well understood at the genetic and to predict the effects of mutations in various components of molecular levels and will be described in detail here; though the lac-system upon lac operon expression. When the lacI less well understood at the molecular level, HIV and herpes gene has been mutated so that its product, LacI, is not capable viruses can behave similarly, transitioning from dormant to of binding to operator DNA, the organism will exhibit con- active states in infected humans. stitutive expression of the lac operon. In a contrary manner, The lytic/lysogenic genetic switching event in lambda is an organism with a lacI gene mutation that produces a LacI centered around an 80-bp region in its double-stranded DNA protein which prevents the binding of lactose or other small genome referred to as the “right operator” (OR) (Figure 38–5A). molecule inducer to the repressor will remain repressed even The right operator is flanked on its left side by the gene for in the presence of the inducer molecule, because such ligands the lambda repressor protein, cI, and on its right side by the cannot bind to the repressor on the operator locus in order to gene encoding another regulatory protein, the cro gene. When derepress the operon. Similarly, bacteria harboring mutations lambda is in its prophage state—that is, integrated into the in their lac operator locus such that the operator sequence host genome—the cI repressor gene is the only lambda gene will not bind a normal repressor molecule will constitutively that is expressed. When the bacteriophage is undergoing express the lac operon genes. Mechanisms of positive and lytic growth, the cI repressor gene is not expressed, but the negative regulation comparable to those described here for cro gene—as well as many other lambda genes—is expressed. the lac system have been observed in eukaryotic cells (see fol- Thus, when the cI repressor gene is on, the cro gene is off, and lowing discussion). when the cro gene is on, the cI repressor gene is off. As we shall 426 SECTION VII Structure, Function, & Replication of Informational Macromolecules 1 The 80-bp lambda right operator, OR, can be subdivided into three discrete, evenly spaced, 17-bp cis-active DNA ele- ments that represent the binding sites for either of two bac- teriophage lambda regulatory proteins. Importantly, the nucleotide sequences of these three tandemly arranged sites are 2 similar but not identical (Figure 38–5B). The three related cis- elements, termed operators OR1, OR2, and OR3, can be bound by either cI or cro proteins. However, the relative affinities of cI and cro for each of the sites vary, and this differential binding affinity is central to the appropriate operation of the lambda phage lytic or lysogenic “molecular switch.” The DNA region 3 between the cro and repressor genes also contains two pro- moter sequences that direct the binding of RNA polymerase in a specified orientation, where it commences transcribing Lysogenic Lytic pathway pathway adjacent genes. One promoter directs RNA polymerase to transcribe in the rightward direction and, thus, to transcribe 4 6 cro and other distal genes, while the other promoter directs the transcription of the cI repressor gene in the leftward direction (see Figure 38–5B). The product of the cI repressor gene, the 236-amino-acid λ cI repressor protein is a two-domain molecule with amino 5 10 7 terminal DNA-binding domain (DBD) and carboxyl-terminal dimerization domain. Association of one repressor protein with another forms a dimer. cI repressor dimers bind to opera- Ultraviolet tor DNA much more tightly than do monomers (Figure 38–6A radiation Induction 9 8 to 38–6C). The product of the cro gene, the 66-amino-acid, 9-kDa cro protein, has a single domain but also binds the operator DNA more tightly as a dimer (Figure 38–6D). The cro protein’s single domain mediates both operator binding and dimerization. FIGURE 38–4 Alternate lytic and lysogenic lifestyles of In a lysogenic bacterium—that is, a bacterium contain- bacteriophage lambda. Infection of the bacterium E. coli by phage ing an integrated, dormant lambda prophage—the lambda lambda begins when a virus particle attaches itself to specific recep- repressor dimer binds preferentially to OR1 but in so doing, tors on the bacterial cell surface (1) and injects its DNA (dark green line) into the cell (2), where the phage genome then circularizes (3). by a cooperative interaction, enhances the binding (by a factor Infection can take either of two courses depending on which two of 10) of another repressor dimer to OR2 (Figure 38–7). The sets of viral genes is turned on. In the lysogenic pathway, the viral affinity of repressor for OR3 is the least of the three operator DNA becomes integrated into the bacterial chromosome (red) (4, 5), subregions. The binding of repressor to OR1 has two major where it is replicated passively as part of the bacterial DNA during effects. First, occupancy of OR1 by repressor blocks the bind- E. coli cell division. This dormant, bacterial genome-integrated virus is called a prophage, and the cell that harbors is called a lysogen. ing of RNA polymerase to the rightward promoter and in In the alternative, lytic mode of infection, the viral DNA excises from that way prevents expression of cro. Second, as mentioned the E. coli chromosome and replicates itself (6) in order to direct the earlier, repressor dimer bound to OR1 enhances the binding synthesis of viral proteins; black lines (7). About 100 new virus par- of repressor dimer to OR2. The binding of repressor to OR2 ticles (green hexagons) are formed. The proliferating viruses induce has the important added effect of enhancing the binding of lysis of the cell (8). A prophage can be “induced” by a DNA damaging agent such as ultraviolet radiation (9). The inducing agent throws a RNA polymerase to the leftward promoter that overlaps OR3 switch (see text and Figure 38–5; the λ “molecular switch.”), so that and thereby enhances transcription and subsequent expres- a different set of viral genes is turned on. Viral DNA loops out and is sion of the repressor gene. This enhancement of transcrip- excised from the E. coli chromosome (10) and replicates; the virus tion is mediated through direct protein–protein interactions then proceeds along the lytic pathway. between promoter-bound RNA polymerase and OR2-bound repressor, much as described earlier for CAP protein and RNA polymerase on the lac operon. Thus, the λ cI protein is both a negative regulator, by preventing transcription of cro, and a see, these two genes regulate each other’s expression and thus, positive regulator, by enhancing transcription of its own gene, ultimately, the decision between lytic and lysogenic growth cI. This dual effect of repressor is responsible for the stable of lambda. This decision between repressor gene transcrip- state of the dormant lambda bacteriophage; not only does the tion and cro gene transcription is a paradigmatic example of a repressor prevent expression of the genes necessary for lysis, molecular transcriptional switch. but it also promotes expression of itself to stabilize this state  CHAPTER 38 Regulation of Gene Expression 427 Gene encoding cI repressor Gene encoding Cro A OR Repressor mRNA OR3 O R2 O R1 B cI repressor promoter cro promoter cro mRNA T A C C T C T G G C G G T G A T A C A T G G A G A C C G C C A C T A T FIGURE 38–5 Genetic organization of the lambda lifestyle “molecular switch.” Right operator (OR) is shown in increasing detail in this series of drawings. The operator is a region of the viral DNA some 80-bp long (A). To its left lies the gene encoding lambda repressor (cI), to its right the gene (cro) encoding the regulator protein Cro. When the operator region is enlarged (B), it is seen to include three subregions termed operators: OR1, OR2, and OR3, each 17-bp long. These three DNA elements are recognition sites to which both λ cI repressor and Cro proteins can bind. The recognition sites overlap two divergent promoters—sequences of bases to which RNA polymerase binds in order to transcribe these genes into mRNA (wavy lines) that are translated into protein. Site OR1 is enlarged (C) to show its base sequence. (Reproduced with permission from Alan D. Iselin, artist.) of differentiation. In the event that intracellular repressor pro- recA protease hydrolyzes the portion of the repressor pro- tein concentration becomes very high, the excess repressor tein that connects the amino-terminal and carboxyl-terminal will bind to OR3 and by so doing diminish transcription of the domains of that molecule (see Figure 38–6A). Such cleavage repressor gene from the leftward promoter, by blocking RNAP of the repressor domains causes the repressor dimers to dis- binding to the cI promoter, until the repressor concentration sociate, which in turn causes dissociation of the repressor drops and repressor dissociates from OR3. Similar examples of molecules from OR2 and eventually from OR1. The effects of repressor proteins also having the ability to activate transcrip- removal of repressor from OR1 and OR2 are predictable. RNA tion have been observed in eukaryotes. polymerase immediately has access to the rightward promoter With such a stable, repressive, cI-mediated, lysogenic state, and commences transcribing the cro gene, while simultane- one might wonder how the lytic cycle could ever be entered. ously the enhancing effect of the repressor at OR2 on leftward However, this process does occur quite efficiently. When a DNA- transcription is lost as well (see Figure 38–7). damaging signal, such as ultraviolet light, strikes the lysogenic The resulting newly synthesized cro protein also binds to host bacterium, fragments of single-stranded DNA are gener- the operator region as a dimer, but as noted earlier, its order ated that activate a specific co-protease coded by a bacterial of preference is opposite to that of repressor (see Figure 38–7). gene and referred to as recA (see Figure 38–7). The activated That is, cro binds most tightly to OR3, but there is no cooperative A B C D COOH Amino acids COOH COOH COOH COOH 132 – 236 Cro NH2 Amino acids NH2 NH2 NH2 NH2 1 – 92 O R1 O R3 FIGURE 38–6 Schematic molecular structures of lambda regulatory proteins cI and Cro. (A)The lambda repressor protein is a 236-amino-acid polypeptide. The chain folds itself into a dumbbell shape with two substructures: an amino terminal (NH2) domain and a carboxyl-terminal (COOH) domain. The two domains are linked by a region of the chain that is less structured and susceptible to cleavage by proteases (indicated by the two arrows. (B) Single repressor molecules (monomers) tend to reversibly associate to form dimers. A dimer is held together mainly by contact between the carboxyl-terminal domains (green hatching). (C) cI repressor dimers bind to (and can dissociate from) the recognition sites in the operator region; they display differential affinities for the three operator sites, OR1 > OR2 > OR3. The DNA-binding domains (DBD) of the repressor molecule that makes contact with DNA (blue hatching). (D) Cro is a single globular protein that contains both a DNA binding domain (blue hatching) and a cro-cro dimerization domain, which promotes binding of cro-cro dimers to target operator DNA. It is important that cro exhibits the highest affinity for OR3, opposite the sequence binding preference of the cI protein. (Reproduced with permission from Alan D. Iselin, artist.) 428 SECTION VII Structure, Function, & Replication of Informational Macromolecules Prophage OR3 OR2 OR1 RNA polymerase TSS TSS Repressor promoter Cro promoter OR3 OR2 OR1 Induction (1) recA Repressor promoter cro promoter Ultraviolet radiation OR3 OR2 OR1 Induction (2) Repressor promoter cro promoter Early lytic growth OR3 OR2 OR1 Repressor promoter cro promoter FIGURE 38–7 Configuration of the lytic/lysogenic switch is shown at four stages of the lambda phage “life” cycle. The lysogenic pathway (in which the virus remains dormant as a prophage) is selected when a repressor dimer binds to OR1, thereby making it likely that OR2 will be bound immediately by another dimer due to the cooperative nature of cI-OR DNA binding. In the prophage (top), the repressor dimers bound at OR1 and OR2 prevent RNA polymerase from binding to the rightward cro promoter and so block the synthesis of cro (negative control). Simultaneously these DNA-bound cI proteins enhance the binding of polymerase to the leftward promoter (positive control), with the result that the repressor gene is transcribed into RNA (initiation at cI gene transcription start site; TSS) and more repressor is synthesized, maintaining the lysogenic state. The prophage is induced (middle) when ultraviolet radiation activates the protease recA, which cleaves cI repressor monomers. Induction (1) The equilibrium of free monomers, free cI dimers, and bound dimers is thereby shifted by mass action, and cI dimers thus dissoci- ate from the operator sites. RNA polymerase is no longer stimulated to bind to the leftward promoter, so that repressor is no longer synthesized. As induction proceeds, Induction (2) all the operator sites become vacant, thus polymerase can bind to the rightward promoter and cro is syn- thesized (cro TSS shown). During early lytic growth, a single cro dimer binds to OR3 (light blue shaded circles), the site for which it has the highest affinity thereby occluding the cI promoter. Consequently, RNA polymerase cannot bind to the leftward promoter, but the rightward promoter remains accessible. Polymerase continues to bind there, transcribing cro and other early lytic genes. Lytic growth ensues (bottom). (Reproduced with permission from Alan D. Iselin, artist.) effect of cro at OR3 on the binding of cro to OR2. At increas- way prevents any further expression of the cI repressor gene. ingly higher concentrations of cro, the protein will bind to OR2 The molecular switch is thus completely “thrown” in the lytic and eventually to OR1. direction. The cro gene is now expressed, and the repressor Importantly, occupancy of OR3 by cro immediately turns gene is fully turned off. This event is irreversible, and the off transcription from the leftward cI promoter and in that expression of other lambda genes begins as part of the lytic  CHAPTER 38 Regulation of Gene Expression 429 cycle. When cro repressor concentration becomes quite high, chromatin are transcriptionally inactive while others are either it will eventually occupy OR1 and in so doing reduce the active or potentially active. With few exceptions, each cell con- expression of its own gene, a process that is necessary in order tains the same complement of genes; hence, the development to drive transcription of the genes needed for the final stages of specialized organs, tissues, and cells, and their function in of the lytic cycle. the intact organism depend on the differential expression The three-dimensional structures of cro and of the λ cI of genes. repressor protein have been determined by x-ray crystallography, Some of this differential expression is achieved by having and models for their binding and driving the above-described different regions of chromatin available for transcription in molecular and genetic events have been formulated and tested. cells from various tissues. For example, the DNA containing the Both bind DNA using helix-turn-helix DBD motifs (see follow- β-globin gene cluster is in “active” chromatin in the reticulo- ing discussion). Along with regulation of the expression of the cyte but in “inactive” chromatin in muscle cells. All the fac- lac operon, the λ molecular switch described here provides argu- tors involved in the determination of active chromatin have not ably the best understanding of the molecular events involved in been elucidated. The presence of nucleosomes and of complexes gene transcription activation and repression. of histones and DNA (see Chapter 35) certainly provides a bar- Detailed analysis of the λ repressor led to the important rier against the ready association of most transcription factors concept that transcription regulatory proteins have several with specific DNA regions. The dynamics of the formation and functional domains. For example, lambda repressor binds to disruption of nucleosome structure are therefore an important DNA with high affinity. Repressor monomers form dimers part of eukaryotic gene regulation. that cooperatively interact with each other, these proteins can Histone covalent modification, also dubbed the histone interact with RNA polymerase, to enhance or block promoter code, is an important determinant of gene activity. Histones binding or RNAP open complex formation (see Figure 36–3). are subjected to a wide range of specific posttranslational The protein-DNA interface and the three protein–protein modifications (see Table 35–1). These modifications are interfaces all involve separate and distinct domains of the two dynamic and reversible. Histone acetylation and deacetylation molecules. As will be noted later (see Figure 38–19), this is a are best understood. The surprising discovery that histone characteristic that is typical of most molecules that regulate acetylase and other enzymatic activities are associated with the transcription. coregulators involved in regulation of gene transcription (see Chapter 42 for specific examples) has provided a new concept of gene regulation. Acetylation is known to occur on lysine SPECIAL FEATURES ARE residues in the amino terminal tails of histone molecules, INVOLVED IN REGULATION and has been consistently correlated with either active tran- OF EUKARYOTIC GENE scription, or alternatively, transcriptional potential. Histone acetylation reduces the positive charge of these tails and likely TRANSCRIPTION contributes to a decrease in the binding affinity of histones Most of the DNA in prokaryotic cells is organized into genes, for the negatively charged DNA. Moreover, such covalent and since the DNA is not compacted with nucleosomal his- modification of the histones creates new binding, or docking tones bacterial genomes have the potential to be transcribed if sites for additional proteins such as ATP-dependent chroma- appropriate positive and negative trans-factors are present in tin remodeling complexes that contain subunits that carry a given cell in an active form. A very different situation exists structural domains that specifically bind to histones that have in eukaryotic cells for two major reasons: first, in human cells been subjected to coregulator-deposited PTMs. These com- relatively little of the total DNA is organized into mRNA- plexes can increase accessibility of adjacent DNA sequences encoding genes and their associated regulatory regions. The by removing or otherwise altering nucleosomal histones. function of the extra DNA is being actively investigated (ie, Together then coregulators (chromatin modifiers and chro- Chapter 39; the ENCODE Projects). Secondly, as described in matin remodelers), working in conjunction, can “open up” Chapter 35, the DNA in eukaryotic cells is extensively folded gene promoters and regulatory regions, facilitating binding of and packed into the protein-DNA complex called chromatin. other trans-factors such as transcriptional activator proteins, Histones are an important part of this complex since they both RNA polymerase II and the GTFs (see Figures 36–10 and form the structures known as nucleosomes (see Chapter 35) 36–11). Histone deacetylation catalyzed by transcriptional and also factor significantly into gene regulatory mechanisms corepressors would have the opposite effect. Different proteins as outlined in following discussion. with specific acetylase and deacetylase activities are associated with various components of the transcription apparatus. The The Chromatin Template Contributes proteins that catalyze histone PTMs are sometimes referred to as “code writers” while the proteins that recognize, bind, and Importantly to Eukaryotic Gene thus interpret these histone PTMs are termed “code readers” Transcription Control while the enzymes that remove histone PTMs are called “code Chromatin structure provides an additional level of control of erasers.” (The analogy to signal transduction, with its kinases, gene transcription. As discussed in Chapter 35, large regions of phosphatases, and phospho-amino acid binding proteins should 430 SECTION VII Structure, Function, & Replication of Informational Macromolecules be apparent—see Chapter 42.) Collectively then, histone is transcriptionally inactive, that all inactive chromatin is PTMs represent a very dynamic, potentially information-rich methylated, or that active DNA is not methylated. source of regulatory information. The exact rules and mecha- Finally, the binding of specific transcription factors to nisms defining the specificity of these various processes are cognate DNA elements may result in disruption of nucleoso- under investigation. Some specific examples are illustrated in mal structure. Most eukaryotic genes have multiple protein- Chapter 42. A variety of commercial enterprises are working binding DNA elements. The serial binding of transcription to develop drugs that specifically alter the activity of the pro- factors to these elements—in a combinatorial fashion—may teins that orchestrate the presence and composition of the his- either directly disrupt the structure of the nucleosome, pre- tone code, whose relevant PTMs continue to grow at a rapid vent its reformation, or recruit, via protein–protein interac- pace (compare Table 38–2 with Table 35–1). tions, multiprotein coregulator complexes that have the ability In addition to the histone code and its effects on all to covalently modify and/or remodel nucleosomes. These DNA-mediated reactions, the methylation of deoxycyti- reactions result in chromatin-level structural changes that in dine residues, 5meC, (in the sequence 5′-meCpG-3′) in DNA the end increase or decrease DNA accessibility to other factors has important effects on chromatin, some of which lead to a and the transcription machinery. decrease in gene transcription. For example, in mouse liver, Eukaryotic DNA that is in an “active” region of chroma- only the unmethylated ribosomal RNA encoding genes can be tin can be transcribed. As in prokaryotic cells, a promoter expressed, and there is evidence that many animal viruses are dictates where the RNA polymerase will initiate transcrip- not transcribed when their DNA is methylated. Acute demeth- tion, but the promoter in mammalian cells (see Chapter 36) ylation of 5meC residues in specific regions of steroid hormone is more complex. Additional complexity is added by elements inducible genes has been associated with an increased rate or factors that enhance or repress transcription, define tissue- of transcription of the gene. However, as with many histone specific expression, and modulate the actions of many effector PTMs, it is not yet possible to generalize that methylated DNA molecules. Finally, recent results suggest that gene activation TABLE 38–2 Summary of Novel Histone PTMs (2011 to 2020) Physiological Histone PTM Reaction Donor Precursor Writer Eraser Function Relevance Glutarylation Acylation Glutarate Kat2a, Sirt7 Nucleosome Glutaric acidemia intramolecular destabilization, catalysis permissive transcription Lactylation Acylation Lactate p300 Permissive Macrophage response, transcription hypoxia Benzoylation Acylation Benzoate Sirt2 Permissive Sodium benzoate transcription treatment S-palmitoylation S-acylation Palmitic acid Cell signaling O-palmitoylation O-acylation Palmitic acid Lpcat1 Reduced Cell signaling transcription Serotonylation Transamidation Serotonin Tgm2 Permissive Neuronal differentiation transcription Dopaminylation Transmidation Dopamine Tgm2 Altered Drug-seeking behaviors transcription 5-Hydroxylysine Hydroxylation 2-Oxoglutarate Jmjd6 Testes, development Glycation Maillard Methylglyoxal, Nonenzymatic DJ-1 Altered nucleosome Breast cancer, monosaccharides stability hyperglycemia 4-Oxononanoylation Ketoamide 4-Oxo-2-nonenal Nonenzymatic Sirt2 Nucleosome Lipid peroxidation adduction destabilization Acrolein adduct Michael Acrolein Nonenzymatic Nucleosome Cigarette smoke, lipid addition destabilization peroxidation S-glutathionylation Disulfide Glutathione Nonenzymatic Nucleosome Aging formation destabilization Homocysteinylation Thiolation Homocysteine Nonenzymatic Reduced Hyperhomocysteinemia thyolactone transcription Modified with permission from Chan JC, Maze I. Nothing Is Yet Set in (Hi)stone: Novel Post-Translational Modifications Regulating Chromatin Function, Trends Biochem Sci. 2020;45(10):829-844.  CHAPTER 38 Regulation of Gene Expression 431 and repression might occur when particular genes move into gene sequence. This field of study has been termed epigenetics. or out of different subnuclear compartments or locations As mentioned in Chapter 35, one aspect of these mechanisms, wherein variable amounts of transcription proteins and RNA PTMs of histones has been dubbed the histone code or histone either promote or disrupt biomolecular condensate formation epigenetic code. The term “epigenetics” means “above genetics” that stimulate or inhibit transcription. and refers to the fact that these regulatory mechanisms do not change the underlying regulated DNA sequence, but rather Epigenetic Mechanisms Contribute simply the expression patterns, or function, of this DNA. Epigenetic mechanisms play key roles in the establishment, Importantly to the Control of Gene maintenance, and reversibility of transcriptional states. A key Transcription feature of epigenetic mechanisms is that the controlled tran- The molecules and regulatory biology described earlier con- scriptional on/off states can be maintained through multiple tributes importantly to transcriptional regulation. Indeed, in rounds of cell division. This observation indicates that there recent years the role of covalent modification of DNA and must be robust, biochemically based mechanisms to maintain histone (and nonhistone) proteins and the newly discovered and stably propagate these epigenetic states. ncRNAs has received tremendous attention in the field of gene Two forms of epigenetic signals, cis- and trans-epigenetic regulation research, particularly through investigation into signals, can be described; these are schematically illustrated how such chemical modifications and/or molecules stably alter in Figure 38–8. A simple trans-signaling event composed of gene expression patterns without altering the underlying DNA positive transcriptional feedback mediated by an abundant, A Trans epigenetic signal B Cis epigenetic signal FIGURE 38–8 cis- and trans-epigenetic signals. (A) An example of an epigenetic signal that acts in trans. A DNA-binding transactivator protein (yellow circle) is transcribed from its cognate gene (yellow bar) located on a particular chromosome (blue). The expressed protein is freely diffusible between nuclear and cytoplasmic compartments. Note that excess transactivator reenters the nucleus following cell division, binds to its own gene, and activates transcription in both daughter cells. This cycle re-establishes the positive feedback loop that was in effect prior to cell division, and thereby enforces stable expression of this transcriptional activator protein in both cells. (B) A cis-epigenetic signal; a gene (pink) located on a particular chromosome (blue) carries a cis-epigenetic signal (small yellow flag) within the regulatory region upstream of the pink gene transcription unit. In this case, the epigenetic signal is associated with active gene transcription and subsequent gene product production (pink circles). During DNA replication, the newly replicated chromatid serves as a template that both elicits, and templates, the introduction of the same epigenetic signal, or mark, on the newly synthesized, unmarked chromatid. Consequently, both daughter cells contain the pink gene in a similarly cis-epigenetically marked state, which ensures expression in an identical fashion in both cells. See text for more detail. 432 SECTION VII Structure, Function, & Replication of Informational Macromolecules diffusible transactivator that efficiently partitions roughly DNA methylases. Thus, the original 5meC methylation mark equally between mother and daughter cell at each division is ultimately results in both DNA daughter strands having the depicted in Figure 38–8A. So long as the indicated, transcription complete cis-epigenetic mark. factor is expressed at a sufficient level to allow all subsequent Both cis- and trans-epigenetic signals can result in stable and daughter cells to inherit the trans-epigenetic signal (transcrip- hereditable expression states, and therefore generally represent tion factor), such cells will have the cellular or molecular phe- type C gene expression responses (ie, Figure 38–1). However, notype dictated by the other target genes of this transcriptional it is important to note that both states can be reversed if either activator. Shown in Figure 38–8 panel B is an example of how the trans- or cis-epigenetic signals are removed by, for example, a cis-epigenetic signal (here as a specific meCpG methylation extinguishing the expression of the enforcing transcription factor mark) can be stably propagated to the two daughter cells fol- (trans-signal) or by completely removing a DNA cis-epigenetic lowing cell division. The hemi-methylated (ie, only one of the signal (via DNA demethylation). Enzymes have been described two DNA strands is 5meC-modified) DNA mark generated dur- that can remove both protein PTMs and 5meC modifications. ing DNA replication directs the methylation of the newly rep- Stable transmission of epigenetic on/off states can be affected licated strand through the action of ubiquitous maintenance by multiple molecular mechanisms. Shown in Figure 38–9 are me A me me me me me me me B Replication machinery RbAP EED EZH2 SUZ12 A RBP CMC C Replication A machinery RBP CMC FIGURE 38–9 Mechanisms for the transmission and propagation of epigenetic signals following a round of DNA replication. (A) Propagation of a 5meC signal (yellow flag; see Figure 38–8B). (B) Propagation of a histone PTM epigenetic signal (H3K27me) that is mediated through the action of the PRC2, a chromatin modifying complex, or CMC. PRC2 is composed of EED, EZH2 histone methylase, RbAP, and SUZ12 subunits. Note that in this context, PRC2 is both a histone code reader (via the methylated histone–binding domain in EED) and histone code writer (via the SET domain histone methylase within EZH2). Location-specific deposition of the histone PTM cis-epigenetic signal is targeted by the recognition of the H3K27me marks in preexisting nucleosomal histones (yellow flag). (C) Another example of the transmission of a histone epigenetic signal (yellow flag) except here signal-targeting is mediated through the action of small ncRNAs that work in concert with an RNA- binding protein (RBP), an adaptor (A) protein, and a CMC. See text for more detail. (Reproduced with permission from Bonasio R, Tu S, Reinberg D. Molecular signals of epigenetic states. Science. 2010;330(6004):612-616.)  CHAPTER 38 Regulation of Gene Expression 433 three ways by which cis-epigenetic marks can be propagated the SV40 enhancer and the β-globin gene on the same plasmid through a round of DNA replication. The first example of epi- (see following discussion and Figure 38–10); in this case the genetic mark transmission involves the propagation of DNA SV40 enhancer-β-globin reporter gene was constructed using 5meC marks, and occurs as described in Figure 38–8. The recombinant DNA technology—see Chapter 39. The enhancer second example of epigenetic state transmission illustrates element does not produce a product that in turn acts on the how a nucleosomal histone PTM (in this example, Lysine K-27 promoter, since it is active only when it exists within the same trimethylated histone H3; H3K27me3) can be propagated. In DNA molecule as the promoter (ie, in cis, or physically linked to). this example immediately following DNA replication, both Enhancer-binding proteins are responsible for this effect. The H3K27me3-marked and H3-unmarked nucleosomes ran- exact mechanism(s) by which these transcription activators domly reform on both daughter DNA strands. The polycomb work is subject to intensive investigation. Enhancer-binding repressive complex 2 (PRC2), composed of EED-SUZ12- trans-factors, some of which are cell-type specific, while oth- EZH2 and RbAP subunits, binds to the nucleosome contain- ers are ubiquitously expressed, have been shown to interact ing the preexisting H3K27me3 mark via the EED subunit. Binding of PRC2 to this histone mark stimulates the methyl- ase activity of the EZH2 subunit of PRC2, which results in the local methylation of nucleosomal H3. Histone H3 methyla- tion thus causes the full, stable transmission of the H3K27me3 epigenetic mark to both chromatids. Finally, locus/sequence- specific targeting of nucleosomal histone epigenetic cis-signals can be attained through the action of lncRNAs as depicted in Figure 38–9, panel C. Here a specific ncRNA interacts with target DNA sequences and the resulting RNA–DNA complex is recognized by RBP, an RNA-binding protein. Then, likely through a specific adaptor protein (A), the RNA-DNA-RBP complex recruits a chromatin modifying complex (CMC) that locally modifies nucleosomal histones. Again, this mecha- nism leads to the transmission of a stable epigenetic mark. Additional work will be required to establish the com- plete molecular details of epigenetic processes, determine FIGURE 38–10 A schematic illustrating the methods used how ubiquitously these mechanisms operate, identify the full to study the organization and action of enhancers and other cis- complement of molecules involved, and genes controlled. acting regulatory elements. These model chimeric genes, all con- Epigenetic signals are critically important to gene regulation structed by recombinant DNA techniques in vitro (see Chapter 39), as evidenced by the fact that mutations and/or overexpression consist of a reporter gene that encodes a protein that can be readily assayed, and that is not normally produced in the cells to be studied, of many of the molecules that contribute to epigenetic control a promoter that ensures accurate initiation of transcription, and the lead to human disease. indicated enhancer (regulatory response) elements. In all cases, high- level transcription from the indicated chimeras depends on the pres- ence of enhancers, which stimulate transcription ≥100-fold over basal Certain DNA Elements Enhance or transcriptional levels (ie, transcription of the same chimeric genes Repress Transcription of Eukaryotic containing just promoters fused to the indicated reporter genes). Examples (A) and (B) illustrate the fact that enhancers (eg, here SV40) Genes work in either orientation and upon a heterologous promoter. Exam- In addition to gross changes in chromatin affecting transcrip- ple (C) illustrates that the metallothionein (mt) regulatory element tional activity, certain DNA elements facilitate or enhance (which under the influence of cadmium or zinc induces transcription of the endogenous mt gene and hence the metal-binding mt protein) initiation at the promoter and hence are termed enhancers. will work through the herpes simplex virus (HSV) thymidine kinase Enhancer elements, which typically contain multiple bind- (tk) gene promoter to enhance transcription of the human growth ing sites for transactivator proteins, differ from the promoter hormone (hGH) reporter gene. In a separate experiment, this engi- in notable ways. Enhancers can exert their positive influence neered genetic construct was introduced into the male pronuclei of on transcription even when separated by tens of thousands of single-cell mouse embryos and the embryos placed into the uterus of a surrogate mother to develop as transgenic animals. Offspring base pairs from a promoter; enhancers work when oriented have been generated under these conditions, and in some the addi- in either direction; and enhancers can work upstream (5′) or tion of zinc ions to their drinking water effects an increase in growth downstream (3′) from the promoter, or even when embedded hormone expression in liver. In this case, these transgenic animals within the transcription unit of a gene. Experimentally, enhanc- have responded to the high levels of growth hormone by becoming ers can be shown to be promiscuous, in that they can stimulate twice as large as their normal litter mates. Example (D) illustrates that a glucocorticoid response element (GRE) enhancer will work through transcription of any promoter in their vicinity, and may act on homologous (PEPCK gene) or heterologous gene promoters (not more than one promoter. The viral SV40 enhancer can exert shown; ie, HSV tk promoter, SV40 promoter, β-globin promoter, etc) an influence on, for example, the transcription of β-globin by to drive expression of the chloramphenicol acetyltransferase (CAT) increasing its transcription 200-fold in cells containing both reporter gene. 434 SECTION VII Structure, Function, & Replication of Informational Macromolecules with a plethora of other transcription proteins. These interactions formation of a unique 3D structure in concert with the afore- include chromatin-modifying coactivators, mediator, as well as mentioned three trans-factors, by inducing a series of criti- the individual components of the basal RNA polymerase II tran- cally spaced DNA bends. Consequently, HMG I(Y) likely scription machinery. Ultimately, transfactor-enhancer DNA- induces the cooperative formation of a unique, stereospecific binding events result in an increase in the binding and/or activity structure within which all four factors are active when viral of the basal transcription machinery on the linked promoter. infection signals are sensed by the cell. The putative structure Enhancer elements and associated binding proteins often con- formed by the cooperative assembly of these four factors has vey nuclease hypersensitivity to those regions where they reside been termed the β-interferon enhanceosome (Figure 38–11), (see Chapter 35). Recently, while analyzing regulatory sequences that control cellular identity (and other genes essential for cell function) in mammalian genomes investigators have identified large tandem clusters of various enhancer elements in tandem arrays. These sequence elements have been termed super- enhancers. Not surprisingly, the cis-linked genes modulated by super-enhancers are highly expressed. It is highly likely that such super-enhancers contribute importantly to the formation of the biomolecular condensates described earlier. A summary of the properties of enhancers is presented in Table 38–2. One of the best-understood mammalian enhancer systems is that of the β-interferon gene. This gene is induced upon viral infection of mammalian cells. One goal of the cell, once virally infected, is to attempt to mount an antiviral response—if not to save the infected cell, then to help to save the entire organ- ism from viral infection. Interferon production is one mecha- nism by which this is accomplished. This family of proteins is secreted by virally infected cells. Secreted interferon interacts with neighboring cells to cause an inhibition of viral replica- tion by a variety of mechanisms, thereby limiting the extent of viral infection. The enhancer element controlling induction of the β-interferon gene, which is located between nucleotides −110 and −45 relative to the transcription start site (+1), is well characterized. This enhancer consists of four distinct clustered FIGURE 38–11 Formation and putative structure of the cis-elements, each of which is bound by unique trans-factors. enhanceosome formed on the human β-interferon gene enhancer. Diagrammatically represented at the top is the distribution of the One cis-element is bound by the transacting factor NF-κB (see multiple cis-elements (HMG, PRDIV, PRDI-III, PRDII, NRDI) composing Figures 42–10 and 42–13), one by a member of the interferon the β-interferon gene enhancer. The intact enhancer mediates tran- regulatory factor (IRF) family of transactivator factors, and a scriptional induction of the β-interferon gene (IFNB1) over 100-fold third by the heterodimeric leucine zipper factor ATF-2/c-Jun upon virus infection of human cells. The cis-elements of this modular (see following discussion). The fourth factor is the ubiquitous, enhancer represent the binding sites for the trans-factors HMG I(Y), cJun-ATF-2, IRF3-IRF7, and NF-κB, respectively. The factors interact with abundant architectural transcription factor known as HMG these DNA elements in an obligatory, ordered, and highly coopera- I(Y). Upon binding to its A + T-rich binding sites, HMG I(Y) tive fashion as indicated by the arrow. Initial binding of four HMG I(Y) induces a significant bend in the DNA. There are four such proteins induces sharp DNA bends in the enhancer, causing the entire HMG I(Y) binding sites interspersed throughout the enhancer. 70- to 80-bp region to assume a high level of curvature. This curvature It is believed that these sites play a key role in facilitating the is integral to the subsequent highly cooperative binding of the other trans-factors since bending enables the DNA-bound factors to make critical direct protein–protein interactions that both contribute to the formation and stability of the enhanceosome and generate a unique TABLE 38–3 Summary of the Properties of Enhancers 3D surface that serves to recruit chromatin-modifying coregulators that Work when located both short and long distances from target carry enzymatic activities (eg, Swi/Snf: ATPase, chromatin remodeler promoter and P/CAF: histone acetyltransferase) as well as the general transcrip- Work when upstream or downstream from the promoter tion machinery (RNA polymerase II and GTFs). Although four of the five Work when oriented in either direction cis-elements (PRDIV, PRDI-III, PRDII, NRDI) independently can modestly Work when embedded within target promoter stimulate (~10-fold) transcription of a reporter gene in transfected Can work with homologous or heterologous promoters cells (see Figures 38–10 and 38–12), all five cis-elements, in appropriate Work by binding one or more proteins order, are required to form an enhancer that can appropriately stimu- Can be composed of one to a few binding elements or many mul- late transcription of IFNB1 (ie, ≥100-fold) in response to viral infection of tiples of activation elements (super enhancers) a human cell. This distinction indicates a strict requirement for appro- Work by recruiting chromatin-modifying coregulatory complexes priate enhanceosome architecture for efficient trans-activation. Similar Work by facilitating binding and/or function of the basal transcrip- enhanceosomes, involving distinct cis- and trans-factors and coregula- tion complex at the cis-linked promoter tors, are proposed to form on many other mammalian genes.  CHAPTER 38 Regulation of Gene Expression 435 so named because of its proposed structural similarity to the nucleosome, which is also a unique three-dimensional pro- tein-DNA structure that wraps DNA about a core assembly of proteins (see Figures 35–1 and 35–2). The enhanceosome, once formed, induces a large increase in β-interferon gene transcription upon virus infection. Thus, it is thought that it is not simply the protein occupancy of the linearly apposed cis- element sites that induces β-interferon gene transcription— rather, it is the formation of the enhanceosome proper that provides appropriate surfaces and 3-dimensional organization for the efficient recruitment of coactivators that results in the enhanced formation of the PIC on the cis-linked promoter and thus transcription activation. cis-Acting DNA elements that decrease the expression of specific genes are termed silencers. Silencers have also been identified in a number of eukaryotic genes. However, because fewer of these elements have been intensively studied, it is not possible to formulate accurate generalizations about their mechanism of action. That said, it is clear that as for gene acti- vation, chromatin level covalent modifications of histones, and other proteins, by silencer-recruited repressors and co- recruited multisubunit corepressors likely play central roles in these regulatory events. Tissue-Specific Expression May Result From Either the Action of Enhancers or FIGURE 38–12 The use of reporter genes to define DNA reg- ulatory elements. A DNA fragment bearing regulatory cis-elements Repressors, or a Combination of Both (triangles, square, circles in diagram) from the gene in question—in this example, approximately 2 kb of 5′-flanking DNA and cognate cis-Acting Regulatory Elements promoter—is ligated into a plasmid vector that contains a suitable Most genes are now recognized to harbor enhancer elements reporter gene—in this case, the enzyme firefly luciferase, abbreviated in various locations relative to their coding regions. In addi- LUC. As noted in Figure 38–10 in such experiments, the reporter cannot be present endogenously in the cells transfected. Consequently, any tion to being able to enhance gene transcription, some of these detection of these activities in a cell extract means that the cell was enhancer elements clearly possess the ability to do so in a successfully transfected by the plasmid. Not shown here, but typically tissue-specific manner. By fusing known or suspected tissue- one cotransfects an additional reporter such as Renilla luciferase to specific enhancers or silencers to reporter genes (see following serve as a transfection efficiency control. Assay conditions for the firefly discussion) and introducing these chimeric enhancer-reporter and Renilla luciferases are different, hence the two activities can be independently sequentially assayed using the same cell extract. An constructs via microinjection into single-cell embryos, one increase of firefly luciferase activity over the basal level, for example, can create a transgenic animal (see Chapter 39), and rigorously after addition of one or more hormones, means that the region of test whether a given test enhancer or silencer truly modulates DNA inserted into the reporter gene plasmid contains functional expression in a cell- or tissue-specific fashion. This transgenic hormone response elements (HRE). Progressively shorter pieces of animal approach has proved useful in studying tissue-specific DNA, regions with internal deletions, or regions with point mutations can be constructed and inserted upstream of the reporter gene to gene expression. pinpoint the response element (Figure 38–13). One caveat of this approach is that the transfected plasmid DNAs likely do not form Reporter Genes Are Used to Define “classical” chromatin structures. Enhancers & Other Regulatory Elements That Modulate Gene gene and introduced into a host cell (see Figure 38–12). Expression Expression of the reporter gene will be increased if the DNA By ligating re