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Cell_Biology_Alberts. Related to the leucine zipper, the helix–loop–helix motif consists of a short ˜ helix connected by a loop (red) to a second, longer ˜ helix. The fexibility of the loop allows one helix to fold back and park against the other thereby forming the dimerization surface. As shown, this two-helix structure binds both to DNA and to the two-helix structure of a second protein to create either a homodimer or a heterodimer. Two ˜helices that extend from the dimerization interface make specifc contacts with the major groove of DNA. CCNNDNAloopHELIX–LOOP–HELIX PROTEINS recognized by the protein has increased from approximately 6 nucleotide pairs to 12 nucleotide pairs, there are many fewer random occurrences of matching sequences.


Cell_Biology_Alberts

Cell_Biology_Alberts. Heterodimers are often formed from two different transcription regulators. Transcription regulators may form heterodimers with more than one partner protein; in this way, the same transcription regulator can be “reused” to create several distinct DNA-binding specificities (see Figure 7–9C). transcription regulators Bind Cooperatively to DnA In the simplest case, the collection of noncovalent bonds that holds the above dimers or heterodimers together is so extensive that these structures form obligatorily, and never fall apart. In this case, the unit of binding is the dimer or heterodimer, and the binding curve for the transcription regulator (the fraction of DNA bound as a function of protein concentration) has a standard exponential shape (Figure 7–10A).


Cell_Biology_Alberts

Cell_Biology_Alberts. In many cases, however, the dimers and heterodimers are held together very weakly; they exist predominantly as monomers in solution, and yet dimers are observed on the appropriate DNA sequence. Here, the proteins are said to bind to DNA cooperatively, and the curve describing their binding is sigmoidal in shape (Figure 7–10B). Cooperative binding means that, over a range of concentrations of the transcription regulator, binding is more of an all-or-none phenomenon than Figure 7–9 Transcription regulators and cis-regulatory sequences. (A) Depiction of the cis-regulatory sequence for nanog, a homeodomain family member that is a key regulator in embryonic stem cells. this “logo” form (see figure 6–12) shows that the protein can recognize a collection of closely related DnA sequences and gives the preferred nucleotide pair at each position. Cis-regulatory sequences are “read” as double-stranded DnA, but only one strand typically is shown in a logo.


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Cell_Biology_Alberts. (B) representation of the cis-regulatory sequence as a colored box. (C) Many transcription regulators form dimers (homodimers) and heterodimers. in the example shown, three different DnAbinding specificities are formed from two transcription regulators. Figure 7–10 Occupancy of a cisregulatory sequence by a transcription regulator. (A) noncooperative binding by a stable heterodimer. (B) Cooperative binding by components of a heterodimer that are predominantly monomers in solution. the shape of the curve differs from that of (A) because the fraction of protein in a form competent to bind DnA (the heterodimer) increases with increasing protein concentration. for noncooperative binding; that is, at most protein concentrations, the cis-regulatory sequence is either nearly empty or nearly fully occupied and rarely is somewhere in between. A discussion of the mathematics behind cooperative binding is given in Chapter 8 (see Figure 8–79A).


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Cell_Biology_Alberts. nucleosome Structure promotes Cooperative Binding of transcription regulators As we have just seen, cooperative binding of transcription regulators to DNA often occurs because the monomers have only a weak affinity for each other. However, there is a second, indirect mechanism for cooperative binding, one that arises from the nucleosome structure of eukaryotic chromosomes.


Cell_Biology_Alberts

Cell_Biology_Alberts. In general, transcription regulators bind to DNA in nucleosomes with lower affinity than they do to naked DNA. There are two reasons for this difference. First, the surface of the cis-regulatory sequence recognized by the transcription regulator may be facing inward on the nucleosome, toward the histone core, and therefore not be readily available to the regulatory protein. Second, even if the face of the cis-regulatory sequence is exposed on the outside of the nucleosome, many transcription regulators subtly alter the conformation of the DNA when they bind, and these changes are generally opposed by the tight wrapping of the DNA around the histone core. For example, many transcription regulators induce a bend or kink in the DNA when they bind.


Cell_Biology_Alberts

Cell_Biology_Alberts. We saw in Chapter 4 that nucleosome remodeling can alter the structure of the nucleosome, allowing transcription regulators access to the DNA. Even without remodeling, however, transcription regulators can still gain limited access to DNA in a nucleosome. The DNA at the end of a nucleosome “breathes,” transiently exposing the DNA and allowing regulators to bind. This breathing happens at a much lower rate in the middle of the nucleosome; therefore, the positions where the DNA exits the nucleosome are much easier to occupy (Figure 7–11).


Cell_Biology_Alberts

Cell_Biology_Alberts. These properties of the nucleosome promote cooperative DNA binding by transcription regulators. If a regulatory protein enters the DNA of a nucleosome and prevents the DNA from tightly rewrapping around the nucleosome core, it will increase the affinity of a second transcription regulator for a nearby cis-regulatory sequence. If the two transcription regulators also interact with each other (as described above), the cooperative effect is even greater. In some cases, the combined action of the regulatory proteins can eventually displace the histone core of the nucleosome altogether. Figure 7–11 How nucleosomes effect the binding of transcription regulators.


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Cell_Biology_Alberts. Figure 7–11 How nucleosomes effect the binding of transcription regulators. “breathing” cis-regulatory sequence this open form occurs about 1/20th of the time (A) (B) compared to its affnity for naked DNA, a typical transcription regulator will bind with 20 times lower affnity if its cis-regulatory sequence is located near the end of a nucleosome a typical transcription regulator will bind with roughly 200-fold less affnity + + transcription regulatorhistone core + + + (C) if its cis-regulatory sequence is located in the middle of a nucleosome (D) one transcription regulator can destabilize the nucleosome, facilitating binding of another


Cell_Biology_Alberts

Cell_Biology_Alberts. The cooperation among transcription regulators can become much greater when nucleosome remodeling complexes are involved. If one transcription regulator binds its cis-regulatory sequence and attracts a chromatin remodeling complex, the localized action of the remodeling complex can allow a second transcription regulator to efficiently bind nearby. Moreover, we have discussed how transcription regulators can work together in pairs; in reality, larger numbers often cooperate by repeated use of the same principles. A highly cooperative binding of transcription regulators to DNA probably explains why many sites in eukaryotic genomes that are bound by transcription regulators are “nucleosome free.”


Cell_Biology_Alberts

Cell_Biology_Alberts. Transcription regulators recognize short stretches of double-helical DNA of defined sequence called cis-regulatory sequences, and thereby determine which of the thousands of genes in a cell will be transcribed. Approximately 10% of the protein-coding genes in most organisms produce transcription regulators, and they control many features of cells. Although each of these transcription regulators has unique features, most bind to DNA as homodimers or heterodimers and recognize DNA through one of a small number of structural motifs. Transcription regulators typically work in groups and bind DNA cooperatively, a feature that has several underlying mechanisms, some of which exploit the packaging of DNA in nucleosomes.


Cell_Biology_Alberts

Cell_Biology_Alberts. Having seen how transcription regulators bind to cis-regulatory sequences embedded in the genome, we can now discuss how, once bound, these proteins influence the transcription of genes. The situation in bacteria is simpler than in eukaryotes (for one thing, chromatin structure is not an issue), and we therefore discuss it first. Following this, we turn to the more complex situation in eukaryotes. the tryptophan repressor Switches Genes Off


Cell_Biology_Alberts

Cell_Biology_Alberts. the tryptophan repressor Switches Genes Off The genome of the bacterium E. coli consists of a single, circular DNA molecule of about 4.6 × 106 nucleotide pairs. This DNA encodes approximately 4300 proteins, although only a fraction of these are made at any one time. Bacteria regulate the expression of many of their genes according to the food sources that are available in the environment. For example, in E. coli, five genes code for enzymes that manufacture the amino acid tryptophan. These genes are arranged in a cluster on the chromosome and are transcribed from a single promoter as one long mRNA molecule; such coordinately transcribed clusters are called operons (Figure 7–12). Although operons are common in bacteria, they are rare in eukaryotes, where genes are typically transcribed and regulated individually (see Figure 7–3).


Cell_Biology_Alberts

Cell_Biology_Alberts. When tryptophan concentrations are low, the operon is transcribed; the resulting mRNA is translated to produce a full set of biosynthetic enzymes, which work in tandem to synthesize tryptophan from much simpler molecules. When tryptophan is abundant, however—for example, when the bacterium is in the gut of a mammal that has just eaten a protein-rich meal—the amino acid is imported into the cell and shuts down production of the enzymes, which are no longer needed. E. coli chromosome series of enzymes required for tryptophan biosynthesis


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Cell_Biology_Alberts. E. coli chromosome series of enzymes required for tryptophan biosynthesis Figure 7–12 A cluster of bacterial genes can be transcribed from a single promoter. each of these five genes encodes a different enzyme, and all of these enzymes are needed to synthesize the amino acid tryptophan from simpler molecules. the genes are transcribed as a single mrnA molecule, a feature that allows their expression to be coordinated. Clusters of genes transcribed as a single mrnA molecule are common in bacteria. each of these clusters is called an operon because its expression is controlled by a cis-regulatory sequence called the operator (green), situated within the promoter. (in this and subsequent figures, the yellow blocks in the promoter represent DnA sequences that bind rnA polymerase; see figure 6–12). start of transcription


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Cell_Biology_Alberts. start of transcription We now understand exactly how this repression of the tryptophan operon comes about. Within the operon’s promoter is a cis-regulatory sequence that is recognized by a transcription regulator. When this regulator binds to this sequence, it blocks access of RNA polymerase to the promoter, thereby preventing transcription of the operon (and thus production of the tryptophan-producing enzymes). The transcription regulator is known as the tryptophan repressor and its cis-regulatory sequence is called the tryptophan operator. These components are controlled in a simple way: the repressor can bind to DNA only if it has also bound several molecules of tryptophan (Figure 7–13).


Cell_Biology_Alberts

Cell_Biology_Alberts. The tryptophan repressor is an allosteric protein, and the binding of tryptophan causes a subtle change in its three-dimensional structure so that the protein can bind to the operator sequence. Whenever the concentration of free tryptophan in the bacterium drops, tryptophan dissociates from the repressor, the repressor no longer binds to DNA, and the tryptophan operon is transcribed. The repressor is thus a simple device that switches production of a set of biosynthetic enzymes on and off according to the availability of the end product of the pathway that the enzymes catalyze. The tryptophan repressor protein itself is always present in the cell. The gene that encodes it is continuously transcribed at a low level, so that a small amount of the repressor protein is always being made. Thus the bacterium can respond very rapidly to a rise or fall in tryptophan concentration.


Cell_Biology_Alberts

Cell_Biology_Alberts. The tryptophan repressor, as its name suggests, is a transcriptional repressor protein: in its active form, it switches genes off, or represses them. Some bacterial transcription regulators do the opposite: they switch genes on, or activate them. These transcriptional activator proteins work on promoters that—in contrast to the promoter for the tryptophan operon—are only marginally able to bind and position RNA polymerase on their own. However, these poorly functioning promoters can be made fully functional by activator proteins that bind to nearby cis-regulatory sequences and contact the RNA polymerase to help it initiate transcription (Figure 7–14).


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Cell_Biology_Alberts. Figure 7–13 Genes can be switched off by repressor proteins. if the concentration of tryptophan inside a bacterium is low (left), rnA polymerase (blue) binds to the promoter and transcribes the five genes of the tryptophan operon. however, if the concentration of tryptophan is high (right), the repressor protein (dark green) becomes active and binds to the operator (light green), where it blocks the binding of rnA polymerase to the promoter. whenever the concentration of intracellular tryptophan drops, the repressor falls off the DnA, allowing the polymerase to again transcribe the operon. Although not shown in the figure, the repressor is a stable dimer.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 7–14 Genes can be switched on by activator proteins. An activator protein binds to its cis-regulatory sequence on the DnA and interacts with the rnA polymerase to help it initiate transcription. without the activator, the promoter fails to initiate transcription efficiently. in bacteria, the binding of the activator to DnA is often controlled by the interaction of a metabolite or other small molecule (red triangle) with the activator protein. the Lac operon works in this manner, as we discuss shortly.


Cell_Biology_Alberts

Cell_Biology_Alberts. DNA-bound activator proteins can increase the rate of transcription initiation as much as 1000-fold, a value consistent with a relatively weak and nonspecific interaction between the transcription regulator and RNA polymerase. For example, a 1000-fold change in the affinity of RNA polymerase for its promoter corresponds to a change in ∆G of ≈18 kJ/mole, which could be accounted for by just a few weak, noncovalent bonds. Thus, many activator proteins work simply by providing a few favorable interactions that help to attract RNA polymerase to the promoter. To provide this assistance, however, the activator protein must be bound to its cis-regulatory sequence, and this sequence must be positioned, with respect to the promoter, so that the favorable interactions can occur.


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Cell_Biology_Alberts. Like the tryptophan repressor, activator proteins often have to interact with a second molecule to be able to bind DNA. For example, the bacterial activator protein CAP has to bind cyclic AMP (cAMP) before it can bind to DNA. Genes activated by CAP are switched on in response to an increase in intracellular cAMP concentration, which rises when glucose, the bacterium’s preferred carbon source, is no longer available; as a result, CAP drives the production of enzymes that allow the bacterium to digest other sugars. An Activator and a repressor Control the Lac Operon


Cell_Biology_Alberts

Cell_Biology_Alberts. In many instances, the activity of a single promoter is controlled by several different transcription regulators. The Lac operon in E. coli, for example, is controlled by both the Lac repressor and the CAP activator that we just discussed. The Lac operon encodes proteins required to import and digest the disaccharide lactose. In the absence of glucose, the bacterium makes cAMP, which activates CAP to switch on genes that allow the cell to utilize alternative sources of carbon— including lactose. It would be wasteful, however, for CAP to induce expression of the Lac operon if lactose itself were not present. Thus the Lac repressor shuts off the operon in the absence of lactose. This arrangement enables the control region of the Lac operon to integrate two different signals, so that the operon is highly expressed only when two conditions are met: glucose must be absent and lactose must be present (Figure 7–15). This genetic circuit thus behaves much like


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA-cis-regulatory polymerase-start of transcription sequence binding site for CAP (promoter) Figure 7–15 The Lac operon is controlled by two transcription regulators, the Lac repressor and CAP. LacZ, the first gene of the operon, encodes the enzyme β-galactosidase, which breaks down lactose to galactose and glucose. when lactose is absent, the lac repressor binds to a cisregulatory sequence, called the Lac operator, and shuts off expression of the operon (Movie 7.4). Addition of lactose increases the intracellular concentration of a related compound, allolactose; allolactose binds to the lac repressor, causing it to undergo a conformational change that releases its grip on the operator DnA (not shown). when glucose is absent, cyclic AMp (red triangle) is produced by the cell, and CAp binds to DnA.


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Cell_Biology_Alberts. a switch that carries out a logic operation in a computer. When lactose is present AND glucose is absent, the cell executes the appropriate program—in this case, transcription of the genes that permit the uptake and utilization of lactose. All transcription regulators, whether they are repressors or activators, must be bound to DNA to exert their effects. In this way, each regulatory protein acts selectively, controlling only those genes that bear a cis-regulatory sequence recognized by it. The logic of the Lac operon first attracted the attention of biologists more than 50 years ago. The way it works was uncovered by a combination of genetics and biochemistry, providing some of the first insights into how transcription is controlled in any organism.


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Cell_Biology_Alberts. We have seen that transcription activators help RNA polymerase initiate transcription and repressors hinder it. However, the two types of proteins are very similar to one another. For example, to occupy their cis-regulatory sequences, both the tryptophan repressor and the CAP activator protein must bind a small molecule; moreover, they both recognize their cis-regulatory sequences using the same structural motif (the helix–turn–helix shown in Panel 7–1). Indeed, some proteins (for example, the CAP protein) can act as both a repressor and an activator, depending on the exact placement of their cis-regulatory sequence relative to the promoter: for some genes, the CAP cis-regulatory sequence overlaps the promoter, and CAP binding thereby prevents the assembly of RNA polymerase at the promoter.


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Cell_Biology_Alberts. Most bacteria have small, compact genomes, and the cis-regulatory sequences that control the transcription of a gene are typically located very near to the start point of transcription. But there are some exceptions to this generalization— cis-regulatory sequences can be located hundreds and even thousands of nucleotide pairs from the bacterial genes they control (Figure 7–16). In these cases, the intervening DNA is looped out, allowing a protein bound at a distant site along the DNA to contact RNA polymerase. Here, the DNA acts as a tether, enormously increasing the probability that the proteins will collide, compared with the situation where one protein is bound to DNA and the other is free in solution. We will see shortly that, although it is the exception in bacteria, DNA looping occurs in the regulation of nearly every eukaryotic gene.


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Cell_Biology_Alberts. A possible explanation for this difference is based on evolutionary considerations. It has been proposed that the compact, simple genetic switches found in bacteria evolved in response to large population sizes where competition for growth put selective pressure on bacteria to maintain small genome sizes. In contrast, there appears to have been little selective pressure to “streamline” the genomes of multicellular organisms. Figure 7–16 Transcriptional activation at a distance. (A) the ntrC protein is a bacterial transcription regulator that activates transcription by directly contacting rnA polymerase. (B) the interaction of ntrC and rnA polymerase, with the intervening DnA looped out, can be seen in the electron microscope. (B, courtesy of harrison echols and Sydney Kustu.)


Cell_Biology_Alberts

Cell_Biology_Alberts. When compared to the situation in bacteria, transcription regulation in eukaryotes involves many more proteins and much longer stretches of DNA. It often seems bewilderingly complex. Yet many of the same principles apply. As in bacteria, the time and place that each gene is to be transcribed is specified by its cis-regulatory sequences, which are “read” by the transcription regulators that bind to them. Once bound to DNA, positive transcription regulators (activators) help RNA polymerase begin transcribing genes, and negative regulators (repressors) block this from happening. In bacteria, as we have seen, most of the interactions between DNA-bound transcription regulators and RNA polymerases (whether they activate or repress transcription) are direct. In contrast, these interactions are almost always indirect in eukaryotes: many intermediate proteins, including the his-tones, act between the DNA-bound transcription regulator and RNA polymerase. Moreover, in multicellular organisms, it


Cell_Biology_Alberts

Cell_Biology_Alberts. always indirect in eukaryotes: many intermediate proteins, including the his-tones, act between the DNA-bound transcription regulator and RNA polymerase. Moreover, in multicellular organisms, it is common for dozens of transcription regulators to control a single gene, with cis-regulatory sequences spread over tens of thousands of nucleotide pairs. DNA looping allows the DNA-bound regulatory proteins to interact with each other and ultimately with RNA polymerase at the promoter. Finally, because nearly all of the DNA in eukaryotic organisms is compacted by nucleosomes and higher-order structures, transcription initiation in eukaryotes must overcome this inherent block.


Cell_Biology_Alberts

Cell_Biology_Alberts. In the next sections, we discuss these features of transcription initiation in eukaryotes, emphasizing how they provide extra levels of control not found in bacteria. A eukaryotic Gene Control region Consists of a promoter plus Many cis-regulatory Sequences In eukaryotes, RNA polymerase II transcribes all the protein-coding genes and many noncoding RNA genes, as we saw in Chapter 6. This polymerase requires five general transcription factors (27 subunits in toto; see Table 6–3, p. 311), in contrast to bacterial RNA polymerase, which needs only a single general transcription factor (the σ subunit). As we have seen, the stepwise assembly of the general transcription factors at a eukaryotic promoter provides, in principle, multiple steps at which the cell can speed up or slow down the rate of transcription initiation in response to transcription regulators.


Cell_Biology_Alberts

Cell_Biology_Alberts. Because the many cis-regulatory sequences that control the expression of a typical gene are often spread over long stretches of DNA, we use the term gene control region to describe the whole expanse of DNA involved in regulating and initiating transcription of a eukaryotic gene. This includes the promoter, where the general transcription factors and the polymerase assemble, plus all of the cis-regulatory sequences to which transcription regulators bind to control the rate of the assembly processes at the promoter (Figure 7–17). In animals and plants, it is not unusual to find the regulatory sequences of a gene dotted over stretches of DNA as large as 100,000 nucleotide pairs. Some of this DNA is transcribed (but not translated), and we discuss these long noncoding RNAs (lncRNAs) later in this chapter. For now, we can regard much of this DNA as “spacer” sequences that transcription regulators do not directly recognize. It is important to keep in mind that, like other regions of


Cell_Biology_Alberts

Cell_Biology_Alberts. in this chapter. For now, we can regard much of this DNA as “spacer” sequences that transcription regulators do not directly recognize. It is important to keep in mind that, like other regions of eukaryotic chromosomes, most of the DNA in gene control regions is packaged into nucleosomes and higher-order forms of chromatin, thereby compacting its overall length and altering its properties.


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Cell_Biology_Alberts. In this chapter, we shall loosely use the term gene to refer to a segment of DNA that is transcribed into a functional RNA molecule, one that either codes for a protein or has a different role in the cell (see Table 6–1, p. 305). However, the classical view of a gene includes the gene control region as well, since mutations in it can produce an altered phenotype. Alternative RNA splicing further complicates the definition of a gene—a point we shall return to later.


Cell_Biology_Alberts

Cell_Biology_Alberts. In contrast to the small number of general transcription factors, which are abundant proteins that assemble on the promoters of all genes transcribed by the gene control region for gene X TATA TATA promoter “spacer” DNA coactivatorstranscription regulators general transcription factors RNA polymerase II gene X cis-regulatory sequence gene X Mediator Figure 7–17 The gene control region for a typical eukaryotic gene. the promoter is the DnA sequence where the general transcription factors and the polymerase assemble (see figure 6–15). the cis-regulatory sequences are binding sites for transcription regulators, whose presence on the DnA affects the rate of transcription initiation. these sequences can be located adjacent to the promoter, far upstream of it, or even within introns or entirely downstream of the gene. the broken stretches of DnA signify that the length of DnA between the cis-regulatory sequences and the start of transcription


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA polymerase II, there are thousands of different transcription regulators devoted to turning individual genes on and off. In eukaryotes, operons—sets of genes transcribed as a unit—are rare, and, instead, each gene is regulated individually. Not surprisingly, the regulation of each gene is different in detail from that of every other gene, and it is difficult to formulate simple rules for gene regulation that apply in every case. We can, however, make some generalizations about how transcription regulators, once bound to gene control regions on DNA, set in motion the series of events that lead to gene activation or repression.


Cell_Biology_Alberts

Cell_Biology_Alberts. In bacteria, we saw that proteins such as the tryptophan repressor, the Lac repressor, and the CAP protein bind to DNA on their own and directly affect RNA polymerase at the promoter. Eukaryotic transcription regulators, in contrast, usually assemble in groups at their cis-regulatory sequences. Often two or more regulators bind cooperatively, as discussed earlier in the chapter. In addition, a broad class of multisubunit proteins termed coactivators and co-repressors assemble on DNA with them. Typically, these coactivators and co-repressors do not recognize specific DNA sequences themselves; they are brought to those sequences by the transcription regulators. Often the protein–protein interactions between transcription regulators and between regulators and coactivators are too weak for them to assemble in solution; however, the appropriate combination of cis-regulatory sequences can “crystallize” the assembly of these complexes on DNA (Figure 7–18).


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Cell_Biology_Alberts. As their names imply, coactivators are typically involved in activating transcription and co-repressors in repressing it. In the following sections, we will see that coactivators and co-repressors can act in a variety of different ways to influence transcription after they have been localized on the genome by transcription regulators.


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Cell_Biology_Alberts. As shown in Figure 7–18, an individual transcription regulator can often participate in more than one type of regulatory complex. A protein might function, varies, sometimes reaching tens of thousands of nucleotide pairs in length. the TATA box is a DnA recognition sequence for the general transcription factor tfiiD. As shown in the lower panel, DnA looping allows transcription regulators bound at any of these positions to interact with the proteins that assemble at the promoter. Many transcription regulators act through Mediator (described in Chapter 6), while some interact with the general transcription factors and rnA polymerase directly. transcription regulators also act by recruiting proteins that alter the chromatin structure of the promoter (not shown, but discussed below).


Cell_Biology_Alberts

Cell_Biology_Alberts. whereas Mediator and the general transcription factors are the same for all rnA polymerase ii-transcribed genes, the transcription regulators and the locations of their binding sites relative to the promoter differ for each gene. for example, in one case as part of a complex that activates transcription and in another case as part of a complex that represses transcription. Thus, individual eukaryotic transcription regulators function as regulatory parts that are used to build complexes whose function depends on the final assembly of all of the individual components. Each eukaryotic gene is therefore regulated by a “committee” of proteins, all of which must be present to express the gene at its proper level. Activator proteins promote the Assembly of rnA polymerase at the Start point of transcription


Cell_Biology_Alberts

Cell_Biology_Alberts. Activator proteins promote the Assembly of rnA polymerase at the Start point of transcription The cis-regulatory sequences to which eukaryotic transcription activator proteins bind were originally called enhancers because their presence “enhanced” the rate of transcription initiation. It came as a surprise when it was discovered that these sequences could be found tens of thousands of nucleotide pairs away from the promoter; as we have seen, DNA looping, which was not widely appreciated at the time, can now explain this initially puzzling observation. Once bound to DNA, how do assemblies of activator proteins increase the rate of transcription initiation? At most genes, mechanisms work in concert. Their function is both to attract and position RNA polymerase II at the promoter and to release it so that transcription can begin.


Cell_Biology_Alberts

Cell_Biology_Alberts. Some activator proteins bind directly to one or more of the general transcription factors, accelerating their assembly on a promoter that has been brought in proximity—through DNA looping—to that activator. Most transcription activators, however, attract coactivators that then perform the biochemical tasks needed to initiate transcription. One of the most prevalent coactivators is the large Mediator protein complex, composed of more than 30 subunits. About the same size as RNA polymerase itself, Mediator serves as a bridge between DNA-bound transcription activators, RNA polymerase, and the general transcription factors, facilitating their assembly at the promoter (see Figure 7–17). eukaryotic transcription Activators Direct the Modification of local Chromatin Structure


Cell_Biology_Alberts

Cell_Biology_Alberts. eukaryotic transcription Activators Direct the Modification of local Chromatin Structure The eukaryotic general transcription factors and RNA polymerase are unable, on their own, to assemble on a promoter that is packaged in nucleosomes. Thus, in addition to directing the assembly of the transcription machinery at the promoter, eukaryotic transcription activators promote transcription by triggering changes to the chromatin structure of the promoters, making the underlying DNA more accessible. The most important ways of locally altering chromatin are through covalent histone modifications, nucleosome remodeling, nucleosome removal, and his-tone replacement (discussed in Chapter 4). Eukaryotic transcription activators use all four of these mechanisms: thus they attract coactivators that include his-tone modification enzymes, ATP-dependent chromatin remodeling complexes, and histone chaperones, each of which can alter the chromatin structure of


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Cell_Biology_Alberts. Figure 7–18 Eukaryotic transcription regulators assemble into complexes on DNA. (A) Seven transcription regulators are shown. the nature and function of the complex they form depend on the specific cis-regulatory sequences that seed their assembly. (B) Some assembled complexes activate gene transcription, while another represses transcription. note that the light green and dark green proteins are shared by both activating and repressing complexes. proteins that do not themselves bind DnA but assemble on other DnA-bound transcription regulators are termed coactivators or co-repressors. in some cases (lower right), rnA molecules are found in these assemblies. As described later in this chapter, these rnAs often act as scaffolds to hold a group of proteins together.


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Cell_Biology_Alberts. promoters (Figure 7–19). These local alterations in chromatin structure provide greater access to DNA, thereby facilitating the assembly of the general transcription factors at the promoter. In addition, some histone modifications specifically attract these proteins to the promoter. These mechanisms often work together during transcription initiation (Figure 7–20). Finally, as discussed earlier in this chapter, the local chromatin changes directed by one transcriptional regulator can allow the binding of additional regulators. By repeated use of this principle, large assemblies of proteins can form on control regions of genes to regulate their transcription.


Cell_Biology_Alberts

Cell_Biology_Alberts. The alterations of chromatin structure that occur during transcription initiation can persist for different lengths of time. In some cases, as soon as the transcription regulator dissociates from DNA, the chromatin modifications are rapidly reversed, restoring the gene to its pre-activated state. This rapid reversal is especially important for genes that the cell must quickly switch on and off in response to external signals. In other cases, the altered chromatin structure persists, even after the transcription regulator that directed its establishment has dissociated from DNA. In principle, this memory can extend into the next cell generation because, as discussed in Chapter 4, chromatin structure can be self-renewing (see Figure 4–44). The fact that different histone modifications persist for different times provides the cell with a mechanism that makes possible both longerand shorter-term memory of gene expression patterns.


Cell_Biology_Alberts

Cell_Biology_Alberts. A special type of chromatin modification occurs as RNA polymerase II transcribes through a gene. The histones just ahead of the polymerase can be acetylated by enzymes carried by the polymerase, removed by histone chaperones, and deposited behind the moving polymerase. These histones are then rapidly deacetylated and methylated, also by complexes that are carried by the polymerase, leaving behind nucleosomes that are especially resistant to transcription. This remarkable process seems to prevent spurious transcription reinitiation Figure 7–19 Eukaryotic transcription activator proteins direct local alterations in chromatin structure. nucleosome remodeling, nucleosome removal, histone replacement, and certain types of histone modifications favor transcription initiation (see figure 4–39). these alterations increase the accessibility of DnA and facilitate the binding of rnA polymerase and the general transcription factors.


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Cell_Biology_Alberts. Figure 7–20 Successive histone modifications during transcription initiation. in this example, taken from the human interferon gene promoter, a transcription activator binds to DnA packaged into chromatin and attracts a histone acetyl transferase that acetylates lysine 9 of histone h3 and lysine 8 of histone h4. then a histone kinase, also attracted by the transcription activator, phosphorylates serine 10 of histone h3 but it can only do so after lysine 9 has been acetylated. this serine modification signals the histone acetyl transferase to acetylate position K14 of histone h3. next, the general transcription factor tfiiD and a chromatin remodeling complex bind to the chromatin to promote the subsequent steps of transcription initiation. tfiiD and the remodeling complex both recognize acetylated histone tails through a bromodomain, a protein domain specialized to read this particular mark on histones; a bromodomain is carried in a subunit of each protein complex.


Cell_Biology_Alberts

Cell_Biology_Alberts. the histone acetyl transferase, the histone kinase, and the chromatin remodeling complex are all coactivators. the order of events shown applies to a specific promoter; at other genes, the steps may occur in a different order or individual steps may be omitted altogether. (Adapted from t. Agalioti, G. Chen and D. thanos, Cell 111:381–392, 2002. with permission from elsevier.) behind a moving polymerase, which, in essence, must clear a path through chromatin as it transcribes. Later in this chapter, when we discuss RNA interference, the potential dangers to the cell of such inappropriate transcription will become especially obvious. The modification of nucleosomes behind a moving RNA polymerase also plays an important role in RNA splicing (see p. 323).


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Cell_Biology_Alberts. In some cases, transcription initiation requires that a DNA-bound transcription activator releases RNA polymerase from the promoter so as to allow it to begin transcribing the gene. In other cases, the RNA polymerase halts after transcribing about 50 nucleotides of RNA, and further elongation requires a transcription activator bound behind it (Figure 7–21). These paused polymerases are common in humans, where a significant fraction of genes that are not being transcribed have a paused polymerase located just downstream from the promoter.


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Cell_Biology_Alberts. The release of RNA polymerase can occur in several ways. In some cases, the activator brings in a chromatin remodeling complex that removes a nucleosome block to the elongating RNA polymerase. In other cases, the activator communicates with RNA polymerase (typically through a coactivator), signaling it to move ahead. Finally, as we saw in Chapter 6, RNA polymerase requires elongation factors to effectively transcribe through chromatin. In some cases, the key step in gene activation is the loading of these factors onto RNA polymerase, which can be directed by DNA-bound transcription activators. Once loaded, these factors allow the polymerase to move through blocks imposed by chromatin structure and begin transcribing the gene in earnest. Having RNA polymerase already poised on a promoter in the beginning stages of transcription bypasses the step of assembling many components at the promoter, which is often slow. This mechanism can therefore allow cells to begin transcribing a gene as a


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Cell_Biology_Alberts. the beginning stages of transcription bypasses the step of assembling many components at the promoter, which is often slow. This mechanism can therefore allow cells to begin transcribing a gene as a rapid response to an extracellular signal.


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Cell_Biology_Alberts. We have seen that complexes of transcription activators and coactivators assemble cooperatively on DNA. We have also seen that these assemblies can promote different steps in transcription initiation. In general, where several factors work together to enhance a reaction rate, the joint effect is not merely the sum of the enhancements that each factor alone contributes, but the product. If, for example, factor A lowers the free-energy barrier for a reaction by a certain amount and thereby speeds up the reaction 100-fold, and factor B, by acting on another aspect of the reaction, does likewise, then A and B acting in parallel will lower the barrier


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Cell_Biology_Alberts. Figure 7–21 Transcription activators can act at different steps. in addition to (A) promoting binding of additional transcription regulators and (B) assembling rnA polymerase at promoters, transcription activators are often needed (C) to release already assembled rnA polymerases from promoters or (D) to release rnA polymerase molecules that become stalled after transcribing about 50 nucleotides of rnA. the activities shown in figure 7–19 can affect each of these four steps. by a double amount and speed up the reaction 10,000-fold. Even if A and B work simply by attracting the same protein, the affinity of that protein for the reaction site increases multiplicatively. Thus, transcription activators often exhibit transcriptional synergy, where several DNA-bound activator proteins working together produce a transcription rate that is much higher than the sum of their transcription rates working alone (Figure 7–22).


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Cell_Biology_Alberts. An important point is that a transcription activator protein must be bound to DNA to influence transcription of its target gene. And the rate of transcription of a gene ultimately depends upon the spectrum of regulatory proteins bound upstream and downstream of its transcription start site, along with the coactivator proteins they bring to DNA. Although the “default” state of eukaryotic DNA packaged into nucleosomes is resistant to transcription, eukaryotes nonetheless use transcription regulators to Figure 7–22 Transcriptional synergy. this experiment compares the rate of transcription produced by three experimentally constructed regulatory regions in a eukaryotic cell and reveals transcriptional synergy, a greater than additive effect of multiple activators working together. for simplicity, coactivators have been omitted from the diagram.


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Cell_Biology_Alberts. Such transcriptional synergy is not only observed between different transcription activators from the same organism; it is also seen between activator proteins from different eukaryotic species when they are experimentally introduced into the same cell. this last observation reflects the high degree of conservation of the machinery responsible for eukaryotic transcription initiation. recruitment of chromatin masking the activation surface for activator for repressor binding site recruitment of with the general transcription factors recruitment of TATA histone methyl transferase histone methylation proteins that bind to methylated histones


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Cell_Biology_Alberts. Figure 7–23 Six ways in which eukaryotic repressor proteins can operate. (A) Activator proteins and repressor proteins compete for binding to the same regulatory DnA sequence. (B) Both proteins bind DnA, but the repressor prevents the activator from carrying out its functions. (C) the repressor blocks assembly of the general transcription factors. (D) the repressor recruits a chromatin remodeling complex, which returns the nucleosomal state of the promoter region to its pre-transcriptional form. (e) the repressor attracts a histone deacetylase to the promoter. As we have seen, histone acetylation can stimulate transcription initiation (see figure 7–20), and the repressor simply reverses this modification. (f) the repressor attracts a histone methyl transferase, which modifies certain positions on histones by attaching methyl groups; the methylated histones, in turn, are bound by proteins that maintain the chromatin in a transcriptionally silent form.


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Cell_Biology_Alberts. repress the transcription of genes. These transcription repressors can both depress the rate of transcription below the default value and rapidly shut off genes that were previously activated. We saw in Chapter 4 that large regions of the genome can be shut down by the packaging of DNA into especially resistant forms of chromatin. However, eukaryotic genes are rarely organized along the genome according to function, and this strategy is not generally applicable for shutting off a set of genes that work together. Instead, most eukaryotic repressors work on a geneby-gene basis. Unlike bacterial repressors, eukaryotic repressors do not directly compete with the RNA polymerase for access to the DNA; rather, they use a variety of other mechanisms, some of which are illustrated in Figure 7–23. Although all of these mechanisms ultimately block transcription by RNA polymerase, eukaryotic transcription repressors typically act by bringing co-repressors to DNA. Like transcription activation,


Cell_Biology_Alberts

Cell_Biology_Alberts. Although all of these mechanisms ultimately block transcription by RNA polymerase, eukaryotic transcription repressors typically act by bringing co-repressors to DNA. Like transcription activation, transcription repression can act through more than one mechanism at a given target gene, thereby ensuring especially efficient repression.


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Cell_Biology_Alberts. Gene repression is especially important to animals and plants whose growth depends on elaborate and complex developmental programs. Misexpression of a single gene at a critical time can have disastrous consequences for the individual. For this reason, many of the genes encoding the most important developmental regulatory proteins are kept tightly repressed when they are not needed. sequence domain of actively Figure 7–24 Schematic diagram summarizing the properties of insulators and barrier sequences. (A) insulators directionally block the action of cis-regulatory sequences, whereas barrier sequences prevent the spread of heterochromatin. how barrier sequences likely function is depicted in figure 4–41. (B) insulator-binding proteins (purple) hold chromatin in loops, thereby favoring “correct” cis-regulatory sequence–gene associations. thus, gene B is properly regulated, and gene B’s cisregulatory sequences are prevented from influencing the transcription of gene A.


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Cell_Biology_Alberts. We have seen that all genes have control regions, which dictate at which times, under what conditions, and in what tissues the gene will be expressed. We have also seen that eukaryotic transcription regulators can act across very long stretches of DNA, with the intervening DNA looped out. How, then, are control regions of different genes kept from interfering with one another? For example, what keeps a transcription regulator bound on the control region of one gene from looping in the wrong direction and inappropriately influencing the transcription of an adjacent gene?


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Cell_Biology_Alberts. To avoid such cross-talk, several types of DNA elements compartmentalize the genome into discrete regulatory domains. In Chapter 4, we discussed barrier sequences that prevent the spread of heterochromatin into genes that need to be expressed. A second type of DNA element, called an insulator, prevents cis-regulatory sequences from running amok and activating inappropriate genes (Figure 7–24). Insulators function by forming loops of chromatin, an effect mediated by specialized proteins that bind them (see Figures 4–48 and 7–24B). The loops hold a gene and its control region in rough proximity and help to prevent the control region from “spilling over” to adjacent genes. Importantly, these loops can be in different in different cell types, depending on the particular proteins and chromatin structures that are present.


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Cell_Biology_Alberts. The distribution of insulators and barrier sequences in a genome is thought to divide it into independent domains of gene regulation and chromatin structure (see pp. 207–208). Aspects of this organization can be visualized by staining whole chromosomes for the specialized proteins that bind these DNA elements (Figure 7–25). Figure 7–25 Localization of a Drosophila insulator-binding protein on polytene chromosomes.


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Cell_Biology_Alberts. Figure 7–25 Localization of a Drosophila insulator-binding protein on polytene chromosomes. A polytene chromosome (discussed in Chapter 4) was stained with propidium iodide (red) to show its banding patterns, with bands appearing bright red and interbands as dark gaps in the pattern (top). the positions on this polytene chromosome that are bound by a particular insulator protein are stained bright green using antibodies directed against the protein (bottom). this protein is preferentially localized to interband regions, reflecting its role in organizing chromosomes into structural, as well as functional, domains. for convenience, these two micrographs of the same polytene chromosome are arranged as mirror images. (Courtesy of Uli laemmli, from K. Zhao et al., Cell 81:879–889, 1995. with permission from elsevier.)


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Cell_Biology_Alberts. Although chromosomes are organized into orderly domains that discourage control regions from acting indiscriminately, there are special circumstances where a control region located on one chromosome has been found to activate a gene located on a different chromosome. Although there is much we do not understand about this mechanism, it indicates the extreme versatility of transcriptional regulation strategies.


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Cell_Biology_Alberts. Transcription regulators switch the transcription of individual genes on and off in cells. In prokaryotes, these proteins typically bind to specific DNA sequences close to the RNA polymerase start site and, depending on the nature of the regulatory protein and the precise location of its binding site relative to the start site, either activate or repress transcription of the gene. The flexibility of the DNA helix, however, also allows proteins bound at distant sites to affect the RNA polymerase at the promoter by the looping out of the intervening DNA. The regulation of higher eukaryotic genes is much more complex, commensurate with a larger genome size and the large variety of cell types that are formed. A single eukaryotic gene is typically controlled by many transcription regulators bound to sequences that can be tens or even hundreds of thousands of nucleotide pairs from the promoter that directs transcription of the gene. Eukaryotic activators and repressors act by a wide variety


Cell_Biology_Alberts

Cell_Biology_Alberts. to sequences that can be tens or even hundreds of thousands of nucleotide pairs from the promoter that directs transcription of the gene. Eukaryotic activators and repressors act by a wide variety of mechanisms—generally altering chromatin structure and controlling the assembly of the general transcription factors and RNA polymerase at the promoter. They do this by attracting coactivators and co-repressors, protein complexes that perform the necessary biochemical reactions. The time and place that each gene is transcribed, as well as its rates of transcription under different conditions, are determined by the particular spectrum of transcription regulators that bind to the regulatory region of the gene.


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Cell_Biology_Alberts. Although all cells must be able to switch genes on and off in response to changes in their environments, the cells of multicellular organisms have evolved this capacity to an extreme degree. In particular, once a cell in a multicellular organism becomes committed to differentiate into a specific cell type, the cell maintains this choice through many subsequent cell generations, which means that it remembers the changes in gene expression involved in the choice. This phenomenon of cell memory is a prerequisite for the creation of organized tissues and for the maintenance of stably differentiated cell types. In contrast, other changes in gene expression in eukaryotes, as well as most such changes in bacteria, are only transient. The tryptophan repressor, for example, switches off the tryptophan genes in bacteria only in the presence of tryptophan; as soon as tryptophan is removed from the medium, the genes are switched back on, and the descendants of the cell will have no memory that


Cell_Biology_Alberts

Cell_Biology_Alberts. genes in bacteria only in the presence of tryptophan; as soon as tryptophan is removed from the medium, the genes are switched back on, and the descendants of the cell will have no memory that their ancestors had been exposed to tryptophan.


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Cell_Biology_Alberts. In this section, we shall examine not only cell memory mechanisms, but also how gene regulatory devices can be combined to create the “logic circuits” through which cells integrate signals and remember events in their past. We begin by considering one such complex gene control region in detail. We have seen that transcription regulators can be positioned at multiple sites along long stretches of DNA and that these proteins can bring into play coactivators and co-repressors. Here, we discuss how the numerous transcription regulators that are bound to the control region of a gene can cause the gene to be transcribed at the proper place and time.


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Cell_Biology_Alberts. Consider the Drosophila Even-skipped (Eve) gene, whose expression plays an important part in the development of the Drosophila embryo. If this gene is inactivated by mutation, many parts of the embryo fail to form, and the embryo dies early in development. As discussed in Chapter 21, at the stage of development when Eve begins to be expressed, the embryo is a single giant cell containing multiple nuclei in a common cytoplasm. This cytoplasm contains a mixture of transcription regulators that are distributed unevenly along the length of the embryo, thus providing positional information that distinguishes one part of the embryo from another (Figure 7–26). Although the nuclei are initially identical, they rapidly begin to express different genes because they are exposed to different transcription regulators. For example, the nuclei near the anterior end of the developing embryo are exposed to a set of transcription regulators that is distinct from the set that influences nuclei at the


Cell_Biology_Alberts

Cell_Biology_Alberts. regulators. For example, the nuclei near the anterior end of the developing embryo are exposed to a set of transcription regulators that is distinct from the set that influences nuclei at the middle or at the posterior end of the embryo.


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Cell_Biology_Alberts. The regulatory DNA sequences that control the Eve gene have evolved to “read” the concentrations of transcription regulators at each position along the length of the embryo, and they cause the Eve gene to be expressed in seven precisely positioned stripes, each initially five to six nuclei wide (Figure 7–27). How is this remarkable feat of information processing carried out? Although there is still much to learn, several general principles have emerged from studies of Eve and other genes that are similarly regulated.


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Cell_Biology_Alberts. The regulatory region of the Eve gene is very large (approximately 20,000 nucleotide pairs). It is formed from a series of relatively simple regulatory modules, each of which contains multiple cis-regulatory sequences and is responsible for specifying a particular stripe of Eve expression along the embryo. This modular organization of the Eve gene control region was revealed by experiments in which a particular regulatory module (say, that specifying stripe 2) is removed from its normal setting upstream of the Eve gene, placed in front of a reporter gene, and reintroduced into the Drosophila genome. When developing embryos derived from flies carrying this genetic construct are examined, the reporter gene is found to be expressed in precisely the position of stripe 2 (Figure 7–28). Similar experiments reveal the existence of other regulatory modules, each of which specifies other stripes.


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Cell_Biology_Alberts. Figure 7–27 The seven stripes of the protein encoded by the Even-skipped (Eve) gene in a developing Drosophila embryo. two and one-half hours after fertilization, the egg was fixed and stained with antibodies that recognize the eve protein (green) and antibodies that recognize the Giant protein (red). where eve and Giant proteins are both present, the staining appears yellow. At this stage in development, the egg contains approximately 4000 nuclei. the eve and Giant proteins are both located in the nuclei, and the eve stripes are about four nuclei wide. the pattern for the Giant protein is also shown in figure 7–26. (Courtesy of Michael levine.)


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Cell_Biology_Alberts. Figure 7–26 The nonuniform distribution of transcription regulators in an early Drosophila embryo. At this stage, the embryo is a syncytium; that is, multiple nuclei are contained in a common cytoplasm. Although not shown in these drawings, all of these proteins are concentrated in the nuclei. how such differences are established is discussed in Chapter 21. start of segment INSERT start of transcription


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Cell_Biology_Alberts. start of segment INSERT start of transcription Figure 7–28 Experiment demonstrating the modular construction of the Eve gene regulatory region. (A) A 480-nucleotidepair section of the Eve regulatory region was removed and (B) inserted upstream of a test promoter that directs the synthesis of the enzyme β-galactosidase (the product of the E. coli LacZ gene—see figure 7–15). (C, D) when this artificial construct was reintroduced into the genome of Drosophila embryos, the embryos (D) expressed β-galactosidase (detectable by histochemical staining) precisely in the position of the second of the seven Eve stripes (C). β-Galactosidase is simple to detect and thus provides a convenient way to monitor the expression specified by a gene control region. As used here, β-galactosidase is said to serve as a reporter, since it “reports” the activity of a gene control region. (C and D, courtesy of Stephen Small and Michael levine.) the Drosophila Eve Gene is regulated by Combinatorial Controls


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Cell_Biology_Alberts. A detailed study of the stripe 2 regulatory module has provided insights into how it reads and interprets positional information. The module contains recognition sequences for two transcription regulators (Bicoid and Hunchback) that activate Eve transcription and for two transcription regulators (Krüppel and Giant) that repress it (Figure 7–29). The relative concentrations of these four proteins determine whether the protein complexes that form at the stripe 2 module activate transcription of the Eve gene. Figure 7–30 shows the distributions of the four transcription regulators across the region of a Drosophila embryo where stripe 2 forms. It is thought that either of the two repressor proteins, when bound to the DNA, will turn off the stripe 2 module, whereas both Bicoid and Hunchback must bind for this module’s maximal activation. This simple regulatory scheme suffices to turn on the stripe 2 module (and therefore the expression of the Eve gene) only in those nuclei located where


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Cell_Biology_Alberts. must bind for this module’s maximal activation. This simple regulatory scheme suffices to turn on the stripe 2 module (and therefore the expression of the Eve gene) only in those nuclei located where the levels of both Bicoid and Hunchback are high and both Krüppel and Giant are absent—a combination that occurs in only one region of the early embryo. It is not known exactly how these four transcription regulators interact with coactivators and co-repressors to specify the final level of transcription across the stripe, but the outcome very likely relies on competition between activators and repressors that act by the mechanisms outlined in Figures 7–17, 7–19, and 7–23.


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Cell_Biology_Alberts. The stripe 2 element is autonomous, inasmuch as it specifies stripe 2 when isolated from its normal context (see Figure 7–28). The other stripe regulatory modules are thought to be constructed similarly, reading positional information provided by other combinations of transcription regulators. The entire Eve gene control region binds more than 20 different transcription regulators. Seven combinations of regulators—one combination for each stripe—specify Eve expression, while many other combinations (all those found in the interstripe regions of


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Cell_Biology_Alberts. Figure 7–29 The Eve stripe 2 unit. the segment of the Eve gene control region identified in figure 7–28 contains cisregulatory sequences for four transcription regulators. it is known from genetic experiments that these four regulatory proteins are responsible for the proper expression of Eve in stripe 2. flies that are deficient in the two gene activators Bicoid and hunchback, for example, fail to efficiently express Eve in stripe 2. in flies deficient in either of the two gene repressors, Giant and Krüppel, stripe 2 expands and covers an abnormally broad region of the embryo. As indicated, in some cases the binding sites for the transcription regulators overlap, and the proteins can compete for binding to the DnA. for example, binding of Krüppel and binding of Bicoid to the site at the far right is mutually exclusive.


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Cell_Biology_Alberts. the embryo) keep the stripe elements silent. A large and complex control region is thereby built from a series of smaller modules, each of which consists of a unique arrangement of short cis-regulatory sequences recognized by specific transcription regulators. The Eve gene itself encodes a transcription regulator, which, after its pattern of expression is set up in seven stripes, controls the expression of other Drosophila genes. As development proceeds, the embryo is thus subdivided into finer and finer regions that eventually give rise to the different body parts of the adult fly, as discussed in Chapter 21.


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Cell_Biology_Alberts. Eve exemplifies the complex control regions found in plants and animals. As this example shows, control regions can respond to many different inputs, integrate this information, and produce a complex spatial and temporal output as development proceeds. However, exactly how all these mechanisms work together to produce the final output is understood only in broad outline (Figure 7–31).


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Cell_Biology_Alberts. The above example from Drosophila clearly illustrates the power of combinatorial control, but this case is unusual in that the nuclei are exposed directly to positional cues in the form of concentrations of transcription regulators. In embryos of most other organisms and in all adults, individual nuclei are in separate cells, and extracellular information (including positional cues) must be passed across the plasma membrane so as to generate signals in the cytosol that cause different transcription regulators to become active in different cell types. Some of the different mechanisms that are known to be used to activate transcription regulators are diagrammed in Figure 7–32, and in Chapter 15, we discuss how extracellular signals trigger these changes. strongly activating assembly neutral assembly of


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Cell_Biology_Alberts. strongly activating assembly neutral assembly of Figure 7–30 Distribution of the transcription regulators responsible for ensuring that Eve is expressed in stripe 2. the distributions of these proteins were visualized by staining a developing Drosophila embryo with antibodies directed against each of the four proteins. the expression of Eve in stripe 2 occurs only at the position where the two activators (Bicoid and hunchback) are present and the two repressors (Giant and Krüppel) are absent. in fly embryos that lack Krüppel, for example, stripe 2 expands posteriorly. likewise, stripe 2 expands posteriorly if the DnA-binding sites for Krüppel in the stripe 2 module are inactivated by mutation (see also figures 7–26 and 7–27).


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Cell_Biology_Alberts. Figure 7–31 The integration of multiple inputs at a promoter. Multiple sets of transcription regulators, coactivators, and co-repressors can work together to influence transcription initiation at a promoter, as they do in the Eve stripe 2 module illustrated in figure 7–29. it is not yet understood in detail how the cell achieves integration of multiple inputs, but it is likely that the final transcriptional activity of the gene results from a competition between activators and repressors that act by the mechanisms summarized in figures 7–17, 7–19, and 7–23.


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Cell_Biology_Alberts. We have seen that transcription regulators can act in combination to control the expression of an individual gene. It is also generally true that each transcription regulator in an organism contributes to the control of many genes. This point is illustrated schematically in Figure 7–33, which shows how combinatorial gene control makes it possible to generate a great deal of biological complexity even with relatively few transcription regulators. Due to combinatorial control, a given transcription regulator does not necessarily have a single, simply definable function as commander of a particular battery of genes or specifier of a particular cell type. Rather, transcription regulators can be likened to the words of a language: they are used with different meanings in a variety of contexts and rarely alone; it is the well-chosen combination that conveys the information that specifies a gene regulatory event.


Cell_Biology_Alberts

Cell_Biology_Alberts. Combinatorial gene control causes the effect of adding a new transcription regulator to a cell to depend on that cell’s past history, since it is this history that determines which transcription regulators are already present. Thus, during development, a cell can accumulate a series of transcription regulators that need not initially alter gene expression. The addition of the final members of the requisite combination of transcription regulators will complete the regulatory message, and can lead to large changes in gene expression.


Cell_Biology_Alberts

Cell_Biology_Alberts. The importance of combinations of transcription regulators for the specification of cell types is most easily demonstrated by their ability—when expressed artificially—to convert one type of cell to another. Thus, the artificial expression of three neuron-specific transcription regulators in liver cells can convert the liver cells into functional nerve cells (Figure 7–34). In some cases, expression of even a single transcription regulator is sufficient to convert one cell type to another. For example, when the gene encoding the transcription regulator MyoD is artificially introduced into fibroblasts cultured from skin connective tissue, the fibroblasts form muscle-like cells. As discussed in Chapter 22, fibroblasts, which are derived from the same broad class of embryonic cells as muscle cells, have already accumulated many of the other necessary transcription regulators required for the


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Cell_Biology_Alberts. Figure 7–32 Some ways in which the activity of transcription regulators is controlled inside eukaryotic cells. (A) the protein is synthesized only when needed and is rapidly degraded by proteolysis so that it does not accumulate. (B) Activation by ligand binding. (C) Activation by covalent modification. phosphorylation is shown here, but many other modifications are possible (see table 3–3, p. 165). formation of a complex between a DnA-binding protein and a separate protein with a transcription-activating domain. Unmasking of an activation domain by the phosphorylation of an inhibitor protein. Stimulation of nuclear entry by removal of an inhibitory protein that otherwise keeps the regulatory protein from entering the nucleus. (G) release of a transcription regulator from a membrane bilayer by regulated proteolysis.


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Cell_Biology_Alberts. combinatorial control of the muscle-specific genes, and the addition of MyoD completes the unique combination required to direct the cells to become muscle. An even more striking example is seen by artificially expressing, early in development, a single Drosophila transcription regulator (Eyeless) in groups of cells Figure 7–34 A small set of transcription regulators can convert one differentiated cell type into another. in this experiment, (A) liver cells grown in culture were converted into (B) neuronal cells via the artificial expression of three nerve-specific transcription regulators. Both types of cells express an artificial red fluorescent protein, which is used to visualize them. this conversion involves the activation of many nerve-specific genes as well as the repression of many liver-specific genes. (from S. Marro et al., Cell Stem Cell 9:374–382, 2011. with permission from elsevier.)


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Cell_Biology_Alberts. Figure 7–33 The importance of combinatorial gene control for development. Combinations of a few transcription regulators can generate many cell types during development. in this simple, idealized scheme, a “decision” to make one of a pair of different transcription regulators (shown as numbered circles) is made after each cell division. Sensing its relative position in the embryo, the daughter cell toward the left side of the embryo is always induced to synthesize the even-numbered protein of each pair, while the daughter cell toward the right side of the embryo is induced to synthesize the odd-numbered protein. the production of each transcription regulator is assumed to be self-perpetuating once it has become initiated (see figure 7–39). in this way, through cell memory, the final combinatorial specification is built up step by step. in this purely hypothetical example, five different transcription regulators have created eight final cell types (G–n).


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Cell_Biology_Alberts. fy with Eyeless gene artifcially that would normally go on to form leg parts. Here, this abnormal gene expression change causes eye-like structures to develop in the legs (Figure 7–35). Specialized Cell types Can Be experimentally reprogrammed to Become pluripotent Stem Cells


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Cell_Biology_Alberts. Specialized Cell types Can Be experimentally reprogrammed to Become pluripotent Stem Cells Manipulation of transcription regulators can also coax various differentiated cells to de-differentiate into pluripotent stem cells that are capable of giving rise to the different cell types in the body, much like the embryonic stem (ES) cells discussed in Chapter 22. When three specific transcription regulators are artificially expressed in cultured mouse fibroblasts, a number of cells become induced pluripotent stem cells (iPS cells)—cells that look and behave like the pluripotent ES cells that are derived from embryos (Figure 7–36). This approach has been adapted to produce iPS cells from a variety of specialized cell types, including cells taken from humans. Such human iPS cells can then be directed to generate a population of differentiated cells for use in the study or treatment of disease, as we discuss in Chapter 22.


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Cell_Biology_Alberts. Although it was once thought that cell differentiation was irreversible, it is now clear that by manipulating combinations of master transcription regulators, cell types and differentiation pathways can be readily altered. Combinations of Master transcription regulators Specify Cell types by Controlling the expression of Many Genes As we saw in the introduction of this chapter, different cell types of multicellular organisms differ enormously in the proteins and RNAs they express. For example, only muscle cells express special types of actin and myosin that form the contractile Figure 7–35 Expression of the Drosophila Eyeless gene in precursor cells of the leg triggers the development of an eye on the leg. (A) Simplified diagrams showing the result when a fruit fly larva contains either the normally expressed Eyeless gene (left) or an Eyeless gene that is additionally expressed artificially in cells that normally give rise to leg tissue (right).


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Cell_Biology_Alberts. (B) photograph of an abnormal leg that contains a misplaced eye (see also figure 21–2). the transcription regulator was named eyeless because its inactivation in otherwise normal flies causes the loss of eyes. (B, courtesy of walter Gehring.) Figure 7–36 A combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell. the artificial expression of a set of three genes, each of which encodes a transcription regulator, can reprogram a fibroblast into a pluripotent cell with embryonic stem (eS)-cell-like properties. like eS cells, such induced pluripotent stem (ipS) cells can proliferate indefinitely in culture and can be stimulated by appropriate extracellular signal molecules to differentiate into almost any cell type found in the body. transcription regulators such as Oct4, Sox2, and Klf4 are often called master transcription regulators because their expression is sufficient to trigger a change in cell identity.


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Cell_Biology_Alberts. apparatus, while nerve cells must make and assemble all the proteins needed to form dendrites and synapses. We have seen that these patterns of cell-type-specific expression are orchestrated by a combination of master transcription regulators. In many cases, these proteins bind directly to cis-regulatory sequences of the genes particular to that cell type. Thus, MyoD binds directly to cis-regulatory sequences located in the control regions of the muscle-specific genes. In other cases, the master regulators control the expression of “downstream” transcription regulators which, in turn, bind to the control regions of other cell-type-specific genes and control their synthesis.


Cell_Biology_Alberts

Cell_Biology_Alberts. The specification of a particular cell type typically involves changes in the expression of several thousand genes. Genes whose protein products are required in the cell type are expressed at high levels, while those not needed are typically down-regulated. As might be imagined, the pattern of binding between the master regulators and all of the regulated genes can be extremely elaborate (Figure 7–37). When we consider that many of these regulated genes have control regions that span tens of thousands of nucleotide pairs, commensurate with the Eve example discussed above, we can begin to appreciate the enormous complexity of cell-type specification. An outstanding question in biology is how the information in a genome is used to specify a multicellular organism. Although we have the general outline of the answer, we are far from understanding how a single cell type is completely specified, let alone a whole organism. Specialized Cells Must rapidly turn Sets of Genes On and Off


Cell_Biology_Alberts

Cell_Biology_Alberts. Although they generally maintain their identities, specialized cells must constantly respond to changes in their environment. Among the most important changes are signals from other cells that coordinate the behavior of the whole organism. Many of these signals induce transient changes in gene transcription, and we discuss the nature of these signals in detail in Chapter 15. Here, we consider how specialized cell types rapidly and decisively switch groups of genes on and off in response to their environment. Even though control of gene expression is combinatorial, the effect of a single transcription regulator can still be decisive in switching any particular gene on or off, simply by completing the combination needed to maximally activate or repress that gene. This situation is analogous to dialing in the final number of a combination lock: the lock will spring open with only this simple addition if all of the other numbers have been previously entered.


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Cell_Biology_Alberts. Figure 7–37 A portion of the transcription network specifying embryonic stem cells. (A) the three master transcription regulators in figure 7–36 are shown as large circles. Genes whose cis-regulatory sequences are bound by each regulator in embryonic stem cells are indicated by a small dot (representing the gene) connected by a thin line (representing the binding reaction). note that many of the target genes are bound by more than one of the regulators. (B) the master regulators control their own expression. As shown here, the three transcriptional regulators bind to their own control regions (indicated by feedback loops), as well as those of the other master regulators (indicated by straight arrows). (Courtesy of trevor Sorrells, based on data from J. Kim et al., Cell 132:1049– 1061, 2008.) Moreover, the same number can complete the combination for many different locks. Likewise, the addition of a particular protein can turn on many different genes.


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Cell_Biology_Alberts. An example is the rapid control of gene expression by the human glucocorticoid receptor protein. To bind to its cis-regulatory sequences in the genome, this transcription regulator must first form a complex with a molecule of a glucocorticoid steroid hormone, such as cortisol (see Figure 15–64). The body releases this hormone during times of starvation and intense physical activity, and among its other activities, it stimulates liver cells to increase the production of glucose from amino acids and other small molecules. To respond in this way, liver cells increase the expression of many different genes that code for metabolic enzymes, such as tyrosine aminotransferase, as we discussed earlier in this chapter (see Figure 7–3). Although these genes all have different and complex control regions, their maximal expression depends on the binding of the hormone–glucocorticoid receptor complex to its cis-regulatory sequence, which is present in the control region of each gene. When the body


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Cell_Biology_Alberts. their maximal expression depends on the binding of the hormone–glucocorticoid receptor complex to its cis-regulatory sequence, which is present in the control region of each gene. When the body has recovered and the hormone is no longer present, the expression of each of these genes drops to its normal level in the liver. In this way, a single transcription regulator can rapidly control the expression of many different genes (Figure 7–38).


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Cell_Biology_Alberts. The effects of the glucocorticoid receptor are not confined to cells of the liver. In other cell types, activation of this transcription regulator by hormone also causes changes in the expression levels of many genes; the genes affected, however, are usually different from those affected in liver cells. As we have seen, each cell type has an individualized set of transcription regulators, and because of combinatorial control, these critically influence the action of the glucocorticoid receptor. Because the receptor is able to assemble with many different sets of celltype-specific transcription regulators, switching it on with hormone produces a different spectrum of effects in each cell type.


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Cell_Biology_Alberts. Once a cell has become differentiated into a particular cell type, it will generally remain differentiated, and all its progeny cells will remain that same cell type. Some highly specialized cells, including skeletal muscle cells and neurons, never divide again once they have differentiated—that is, they are terminally differentiated (as discussed in Chapter 17). But many other differentiated cells—such as receptor in absence of glucocorticoid hormone


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Cell_Biology_Alberts. Figure 7–38 A single transcription regulator can coordinate the expression of many different genes. the action of the glucocorticoid receptor is illustrated schematically. On the left is a series of genes, each of which has various transcription regulators bound to its regulatory region. however, these bound proteins are not sufficient on their own to fully activate transcription. On the right is shown the effect of adding an additional transcription regulator—the glucocorticoid receptor in a complex with glucocorticoid hormone—that has a cisregulatory sequence in the control region of each gene. the glucocorticoid receptor completes the combination of transcription regulators required for maximal initiation of transcription, and the genes are now switched on as a set. when the hormone is no longer present, the glucocorticoid receptor dissociates from DnA and the genes return to their pre-stimulated levels.


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Cell_Biology_Alberts. fibroblasts, smooth muscle cells, and liver cells—will divide many times in the life of an individual. When they do, these specialized cell types give rise only to cells like themselves: smooth muscle cells do not give rise to liver cells, nor liver cells to fibroblasts. For a proliferating cell to maintain its identity—a property called cell memory—the patterns of gene expression responsible for that identity must be remembered and passed on to its daughter cells through subsequent cell divisions. Thus, in the model we discussed in Figure 7–33, the production of each transcription regulator, once begun, has to be continued in the daughter cells of each cell division. How is such perpetuation accomplished?


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Cell_Biology_Alberts. Cells have several ways of ensuring that their daughters “remember” what kind of cells they are. One of the simplest and most important is through a positive feedback loop, where a master cell-type transcription regulator activates transcription of its own gene, in addition to that of other cell-type-specific genes. Each time a cell divides, the regulator is distributed to both daughter cells, where it continues to stimulate the positive feedback loop, making more of itself each division. Positive feedback is crucial for establishing “self-sustaining” circuits of gene expression that allow a cell to commit to a particular fate—and then to transmit that information to its progeny (Figure 7–39).


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Cell_Biology_Alberts. As was previously shown in Figure 7–37B, the master regulators needed to maintain the pluripotency of iPS cells bind to cis-regulatory sequences in their own control regions, providing examples of the type of positive feedback loop. In addition, most of these pluripotent cell regulators also activate transcription of other master regulators, resulting in a complex series of indirect feedback loops. For example, if A activates B, and B activates A, this forms a positive feedback loop where A activates its own expression, albeit indirectly. The series of direct and indirect feedback loops observed in the iPS circuit is typical of other specialized cell circuits. Such a network structure strengthens cell memory, increasing the probability that a particular pattern of gene expression is transmitted through successive generations. For example, if the level of A drops below the critical threshold to stimulate its own synthesis, regulator B can rescue it. By successive application of this


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Cell_Biology_Alberts. through successive generations. For example, if the level of A drops below the critical threshold to stimulate its own synthesis, regulator B can rescue it. By successive application of this mechanism, a complex series of positive feedback loops among multiple transcription regulators can stably maintain a differentiated state through many cell divisions.


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Cell_Biology_Alberts. Figure 7–39 A positive feedback loop can create cell memory. protein A is a master transcription regulator that activates the transcription of its own gene—as well as other cell-type-specific genes (not shown). All of the descendants of the original cell will therefore “remember” that the progenitor cell had experienced a transient signal that initiated the production of protein A.


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Cell_Biology_Alberts. Positive feedback loops formed by transcription regulators are probably the most prevalent way of ensuring that daughter cells remember what kind of cells they are meant to be, and they are found in all species on Earth. For example, many bacteria and single-cell eukaryotes form different types of cells, and positive feedback loops lie at the heart of mechanisms that maintain their cell types through many rounds of cell division. Plants and animals also make extensive use of transcription feedback loops; as we shall discuss later in the chapter, they have additional, more specialized mechanisms for making cell memory even stronger. But first, we briefly consider how combinations of transcription regulators and cis-regulatory sequences can be combined to create useful logic devices for the cell. transcription Circuits Allow the Cell to Carry Out logic Operations


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Cell_Biology_Alberts. Simple gene regulatory switches can be combined to create all sorts of control devices, just as simple electronic switching elements in a computer can be linked to perform different types of operations. An analysis of gene regulatory circuits reveals that certain simple types of arrangements (called network motifs) are found over and over again in cells from widely different species. For example, positive and negative feedback loops are common in all cells (Figure 7–40). Whereas the former provides a simple memory device, the latter is often used to keep the expression of a gene close to a standard level despite the variations in biochemical conditions inside a cell. Suppose, for example, that a transcription repressor protein binds to the regulatory region of its own gene and exerts a strong negative feedback, such that transcription falls to a very low rate when the concentration of the repressor protein is above some critical value (determined by its affinity for its DNA binding


Cell_Biology_Alberts

Cell_Biology_Alberts. strong negative feedback, such that transcription falls to a very low rate when the concentration of the repressor protein is above some critical value (determined by its affinity for its DNA binding site). The concentration of the protein can then be held close to the critical value, since any circumstance that causes a fall below that value can lead to a steep increase in synthesis, and any that causes a rise above that value will lead to synthesis being switched off. Such adjustments will, however, take time, so that an abrupt change of conditions will cause a disturbance of gene expression that is strong but transient. If there is a delay in the feedback loop, the result may be spontaneous oscillations in the expression of the gene (see Figure 15–18). The different types of behavior produced by a feedback loop will depend on the details of the system; for example, how tightly the transcription regulator binds to its cis-regulatory sequence, its rate of synthesis, and its rate of


Cell_Biology_Alberts

Cell_Biology_Alberts. produced by a feedback loop will depend on the details of the system; for example, how tightly the transcription regulator binds to its cis-regulatory sequence, its rate of synthesis, and its rate of decay. We discuss these issues in quantitative terms and in more detail in Chapter 8.


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Cell_Biology_Alberts. With two or more transcription regulators, the possible range of circuit behaviors becomes more complex. Some bacterial viruses contain a common type of two-gene circuit that can flip-flop between expression of one gene and expression of the other. Another common circuit arrangement is called a feed-forward loop; such a loop can serve as a filter, responding to input signals that are prolonged but disregarding those that are brief (Figure 7–41). These various network motifs resemble miniature logic devices, and they can process information in surprisingly sophisticated ways. The simple types of devices just illustrated are found to be interwoven in eukaryotic cells, creating exceedingly complex circuits (Figure 7–42). Each cell in a developing multicellular organism is equipped with similarly complex control


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Cell_Biology_Alberts. Figure 7–40 Common types of network motifs in transcription circuits. A and B represent transcription regulators, arrows indicate positive transcription control, while lines with bars depict negative transcription control. in the feed-forward loop, A and B represent transcription regulators that both activate the transcription of target gene Z (see also figure 8–86). machinery, and it must, in effect, use its intricate system of interlocking transcription switches to compute how it should behave at each time point in response to the many different past and present inputs received. We are only beginning to understand how to study such complex intracellular control networks. Indeed, without new approaches, coupled with quantitative information that is far more precise and complete than we now possess, it will be impossible to predict the behavior of a system such as that shown in Figure 7–42. As explained in Chapter 8, a circuit diagram by itself is not enough.


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Cell_Biology_Alberts. Figure 7–41 How a feed-forward loop can measure the duration of a signal. in this theoretical example, transcription regulators A and B are both required for transcription of Z, and A becomes active only when an input signal is present. if the input signal to A is brief, A does not stay active long enough for B to accumulate, and the Z gene is not transcribed. (C) if the signal to A persists, B accumulates, A remains active, and Z is transcribed. this arrangement allows the cell to ignore rapid fluctuations of the input signal and respond only to persistent levels. this strategy could be used, for example, to distinguish between random noise and a true signal.


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Cell_Biology_Alberts. the behavior shown here was computed for one particular set of parameter values describing the quantitative properties of A, B, and the product of Z, along with their syntheses. with different values of these parameters, feed-forward loops can in principle perform other types of “calculations.” Many feed-forward loops have been discovered in cells, and theoretical analysis helps researchers to discern—and subsequently test—the different ways in which they may function (see figure 8–86). (Adapted from S.S. Shen-Orr et al., Nat. Genet. 31:64–68, 2002. with permission from Macmillan publishers ltd.)


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Cell_Biology_Alberts. S.S. Shen-Orr et al., Nat. Genet. 31:64–68, 2002. with permission from Macmillan publishers ltd.) Figure 7–42 The exceedingly complex gene circuit that specifies a portion of the developing sea urchin embryo. each colored small box represents a different gene. those in yellow code for transcription regulators and those in green and blue code for proteins that give cells of the mesoderm and endoderm, respectively, their specialized characteristics. Genes depicted in gray are largely active in the mother and provide the egg with cues needed for proper development. As in figure 7–40, arrows depict instances in which a transcription regulator activates the transcription of another gene. lines ending in bars indicate examples of gene repression. (from i.S. peter and e.h. Davidson, Nature 474:635–639, 2011. with permission from Macmillan publishers ltd.)


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Cell_Biology_Alberts. The many types of cells in animals and plants are created largely through mechanisms that cause different sets of genes to be transcribed in different cells. The transcription of any particular gene is generally controlled by a combination of transcription regulators. Each type of cell in a higher eukaryotic organism contains a specific set of transcription regulators that ensures the expression of only those genes appropriate to that type of cell. A given transcription regulator may be active in a variety of circumstances and is typically involved in the regulation of many different genes.


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Cell_Biology_Alberts. Since specialized animal cells can maintain their unique character through many cell-division cycles, and even when grown in culture, the gene regulatory mechanisms involved in creating them must be stable once established and heritable when the cell divides. These features reflect the cell’s memory of its developmental history. Direct or indirect positive feedback loops, which enable transcription regulators to perpetuate their own synthesis, provide the simplest mechanism for cell memory. Transcription circuits also provide the cell with the means to carry out other types of logic operations. Simple transcription circuits combined into large regulatory networks drive highly sophisticated programs of embryonic development that will require new approaches to decipher.


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Cell_Biology_Alberts. Thus far in this chapter, we have emphasized the regulation of gene transcription by proteins that associate either directly or indirectly with DNA. However, DNA itself can be covalently modified, and certain types of chromatin states appear to be inherited. In this section, we shall see how these phenomena also provide opportunities for the regulation of gene expression. At the end of this section, we discuss how, in mice and humans, an entire chromosome can be transcription-ally inactivated using such mechanisms, and how this state can be maintained through many cell divisions. patterns of DnA Methylation Can Be inherited when vertebrate Cells Divide


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Cell_Biology_Alberts. In vertebrate cells, the methylation of cytosine provides a mechanism through which gene expression patterns can be passed on to progeny cells. The methylated form of cytosine, 5-methyl cytosine (5-methyl C), has the same relation to cytosine that thymine has to uracil, and the modification likewise has no effect on base-pairing (Figure 7–43). DNA methylation in vertebrate DNA occurs on cytosine (C) nucleotides largely in the sequence CG, which is base-paired to exactly the same sequence (in opposite orientation) on the other strand of the DNA helix. Consequently, a simple mechanism permits the existing pattern of DNA methylation to be inherited directly by the daughter DNA strands. An enzyme called maintenance methyl transferase acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated. As a result, the pattern of DNA methylation on the parental DNA strand serves as a template for the methylation of the daughter DNA strand, causing


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Cell_Biology_Alberts. with a CG sequence that is already methylated. As a result, the pattern of DNA methylation on the parental DNA strand serves as a template for the methylation of the daughter DNA strand, causing this pattern to be inherited directly following DNA replication (Figure 7–44).


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Cell_Biology_Alberts. Although DNA methylation patterns can be maintained in differentiated cells by the mechanism shown in Figure 7–44, methylation patterns are dynamic during mammalian development. Shortly after fertilization, there is a genome-wide wave of demethylation, when the vast majority of methyl groups are lost from the DNA. This demethylation may occur either by suppression of maintenance DNA methyl transferase activity, resulting in the passive loss of methyl groups during each round of DNA replication, or by demethylating enzymes (discussed below). Later in development, new methylation patterns are established by several de novo DNA methyl transferases that are directed to DNA by sequence Figure 7–43 Formation of 5-methyl cytosine occurs by methylation of a cytosine base in the DNA double helix.


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Cell_Biology_Alberts. Figure 7–43 Formation of 5-methyl cytosine occurs by methylation of a cytosine base in the DNA double helix. in vertebrates, this event is largely confined to selected cytosine (C) nucleotides located in the sequence CG. CG sequences are sometimes denoted as CpG sequences, where the p indicates a phosphate linkage to distinguish it from a CG base pair. in this chapter, we will continue to use the simpler nomenclature CG to indicate this dinucleotide. Figure 7–44 How DNA methylation patterns are faithfully inherited. in vertebrate DnA, a large fraction of the cytosine nucleotides in the sequence CG is methylated (see figure 7–43). Because of the existence of a methyl-directed methylating enzyme (the maintenance methyl transferase), once a pattern of DnA methylation is established, that pattern of methylation is inherited in the progeny DnA, as shown.


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Cell_Biology_Alberts. specific DNA-binding proteins. Once the new patterns of methylation are established, they can be propagated through rounds of DNA replication by the maintenance methyl transferases. DNA methylation has several uses in the vertebrate cell. A very important role is to work in conjunction with other gene expression control mechanisms to establish a particularly efficient form of gene repression. This combination of mechanisms ensures that unneeded eukaryotic genes can be repressed to very high degrees. For example, the rate at which a vertebrate gene is transcribed can vary 106-fold between one tissue and another. The unexpressed vertebrate genes are much less “leaky” in terms of transcription than bacterial genes, in which the largest known differences in transcription rates between expressed and unexpressed gene states are about 1000-fold.