Medical Physiology, 3rd Edition

Transcription Factors

DNA-binding transcription factors recognize specific DNA sequences

The preceding discussion has emphasized the structure of the gene and the cis-acting elements that regulate gene expression. We now turn to the proteins that interact with these DNA elements and thus regulate gene transcription. Because the basal transcriptional machinery—Pol II and the general transcription factors—is incapable of efficient gene transcription alone, additional proteins are required to stimulate the activity of the enzyme complex. The additional proteins include transcription factors that recognize and bind to specific DNA sequences (enhancers) located near their target genes, as well as others (see pp. 83–84) that do not bind to DNA.

Examples of DNA-binding transcription factors are shown in Table 4-1. The general mechanism of action of a specific transcription factor is depicted in Figure 4-6. After the basal transcriptional machinery assembles on the gene promoter, it can interact with a transcription factor that binds to a specific DNA element, the enhancer (or silencer). Looping out of the intervening DNA permits physical interaction between the activator (or repressor) and the basal transcriptional machinery, which subsequently leads to stimulation (or inhibition) of gene transcription. The specificity with which transcription factors bind to DNA depends on the interactions between the amino-acid side chains of the transcription factor and the purine and pyrimidine bases in DNA. Most of these interactions consist of noncovalent hydrogen bonds between amino acids and DNA bases. A peptide capable of a specific pattern of hydrogen bonding can recognize and bind to the reciprocal pattern in the major (and to a lesser extent the minor) groove of DNA. Interaction with the DNA backbone may also occur and involves electrostatic interactions (salt bridge formation) with anionic phosphate groups. The site that a transcription factor recognizes (see Table 4-1) is generally short, usually less than a dozen or so base pairs.

TABLE 4-1

DNA-Binding Transcription Factors and the DNA Sequences They Recognize

NAME

TYPE

RECOGNITION SITE*

BINDS AS

Sp1

Zinc finger

5′-GGGCGG-3′

Monomer

AP-1

bZIP

5′-TGASTCA-3′

Dimer

C/EBP

bZIP

5′-ATTGCGCAAT-3′

Dimer

Heat shock factor

bZIP

5′-NGAAN-3′

Trimer

ATF/CREB

bZIP

5′-TGACGTCA-3′

Dimer

c-Myc

bHLH

5′-CACGTG-3′

Dimer

Oct-1

HTH

5′-ATGCAAAT-3′

Monomer

NF-1

Novel

5′-TTGGCN5GCCAA-3′

Dimer

*N indicates any nucleotide; S indicates G or C.

ATF, activating transcription factor; NF-1, nuclear factor 1.

DNA-binding transcription factors do not recognize single, unique DNA sequences; rather, they recognize a family of closely related sequences. For example, the transcription factor AP-1 (activator protein 1) recognizes the sequences

5′-imageCimageA-3′

5′-imageGimageA-3′

5′-imageGimageT-3′

and so on, as well as each of the complementary sequences. That is, some redundancy is usually built into the recognition sequence for a transcription factor. An important consequence of these properties is that the recognition site for a transcription factor may occur many times in the genome. For example, if a transcription factor recognizes a 6-bp sequence, the sequence would be expected to occur once every 46(or 4096) base pairs, that is, 7 × 105 times in the human genome. If redundancy is permitted, recognition sites will occur even more frequently. Of course, most of these sites will not be relevant to gene regulation but will instead have occurred simply by chance. This high frequency of recognition sites leads to an important concept: transcription factors act in combination. Thus, high-level expression of a gene requires that a combination of multiple transcription factors bind to multiple regulatory elements. Although it is complicated, this system ensures that transcription activation occurs only at appropriate locations. Moreover, this system permits greater fine-tuning of the system, inasmuch as the activity of individual transcription factors can be altered to modulate the overall level of transcription of a gene.

An important general feature of DNA-binding transcription factors is their modular construction (Fig. 4-8A). Transcription factors may be divided into discrete domains that bind DNA (DNA-binding domains) and domains that activate transcription (transactivation domains).imageN4-6 This property was first directly demonstrated for a yeast transcription factor known as GAL4, which activates certain genes when yeast grows in galactose-containing media. GAL4 has two domains. One is a so-called zinc finger (see p. 82) that mediates sequence-specific binding to DNA. The other domain is enriched in acidic amino acids (i.e., glutamate and aspartate) and is necessary for transcriptional activation. This “acidic blob” domain of GAL4 can be removed and replaced with the transactivation domain from a different transcription factor VP16 (see Fig. 4-8B). The resulting GAL4-VP16 chimera binds to the same DNA sequence as normal GAL4 but mediates transcriptional activation via the VP16 transactivation domain. This type of “domain-swapping” experiment indicates that transcription factors have a modular construction in which physically distinct domains mediate binding to DNA and transcriptional activation (or repression).

image

FIGURE 4-8 Modular design of specific transcription factors. A, DNA-binding transcription factors have independent domains for binding to DNA regulatory sequences and for activating transcription. In this example, amino acids 1 through 147 of the GAL4 yeast transcription factor bind to DNA, whereas amino acids 768 through 881 activate transcription. B, Replacement of the transactivation domain of GAL4 with that of VP16 results in a chimera that is a functional transcription factor.

N4-6

Grouping of Transcription Factors According to Transactivation Domain

Contributed by Peter Igarashi

The following table groups some of the transcription factors (described in the text) on the basis of the type of transactivation domain (i.e., the domain that activates transcription).

Type of Transactivation Domain

Transcription Factors with This Domain

Acidic blob (rich in negatively charged amino acids—aspartate and glutamate)

GAL4 (a yeast transcription factor)
VP16 (a herpesvirus transcription factor)

Proline rich

CTF (a family of CCAAT-box–binding transcription factor; also known as NF-1)
NF-1 (nuclear factor 1)

Glutamine rich

Sp1 (stimulating protein 1)

Serine/threonine rich

GHF-1/Pit-1 (growth hormone factor 1, which is the same as pituitary-specific transcription factor 1; an HTH-type transcription factor)

Transcription factors that bind to DNA can be grouped into families based on tertiary structure

On the basis of sequence conservation as well as structural determinations from x-ray crystallography and nuclear magnetic resonance spectroscopy, DNA-binding transcription factors have been grouped into families. Members of the same family use common structural motifs for binding DNA (see Table 4-1). These structures include the zinc finger, basic zipper, basic helix-loop-helix, helix-turn-helix, and β sheet. Each of these motifs consists of a particular tertiary protein structure in which a component, usually an α helix, interacts with DNA, especially the major groove of the DNA.

Zinc Finger

The term zinc finger describes a loop of protein held together at its base by a zinc ion that tetrahedrally coordinates to either two histidine residues and two cysteine residues or four cysteine residues. Sometimes two zinc ions coordinate to six cysteine groups. Figure 4-9A shows a zinc finger in which Zn2+ coordinates to two residues on an α helix and two residues on a β sheet of the protein. The loop (or finger) of protein can protrude into the major groove of DNA, where amino-acid side chains can interact with the base pairs and thereby confer the capacity for sequence-specific DNA binding. Zinc fingers consist of 30 amino acids with the consensus sequences Cys-X2–4-Cys-X12-His-X3–5-His, where X can be any amino acid. Transcription factors of this family contain at least two zinc fingers and may contain dozens. Three amino-acid residues at the tips of each zinc finger contact a DNA subsite that consists of three bases in the major groove of DNA; these residues are responsible for site recognition and binding (see Table 4-1). Zinc fingers are found in many mammalian transcription factors, including several that we discuss in this chapter—Egr-1, Wilms tumor protein (WT-1), and Sp1 (see Table 4-1)—as well as the steroid-hormone receptors (see p. 71).

image

FIGURE 4-9 Structures of transcription factors.

Basic Zipper

Also known as the leucine zipper family, the basic zipper (bZIP) family consists of transcription factors that bind to DNA as dimers (see Fig. 4-9B). Members include C/EBPβ (CCAAT/enhancer-binding protein-β), c-Fos, c-Jun, and CREB. Each monomer consist of two domains, a basic region that contacts DNA and a leucine zipper region that mediates dimerization. The basic region contains about 30 amino acids and is enriched in arginine and lysine residues. This region is responsible for sequence-specific binding to DNA via an α helix that inserts into the major groove of DNA. The leucine zipper consists of a region of about 30 amino acids in which every seventh residue is a leucine. Because of this spacing, the leucine residues align on a common face every second turn of an α helix. Two protein subunits that both contain leucine zippers can associate because of hydrophobic interactions between the leucine side chains; they form a tertiary structure called a coiled coil. Proteins of this family interact with DNA as homodimers or as structurally related heterodimers. Dimerization is essential for transcriptional activity because mutations of the leucine residues abolish both dimer formation and the ability to bind DNA and activate transcription. The crystal structure reveals that these transcription factors resemble scissors in which the blades represent the leucine zipper domains and the handles represent the DNA-binding domains (see Fig. 4-9B).

Basic Helix-Loop-Helix

Similar to the bZIP family, members of the basic helix-loop-helix (bHLH) family of transcription factors also bind to DNA as dimers. Each monomer has an extended α-helical segment containing the basic region that contacts DNA, linked by a loop to a second α helix that mediates dimer formation (see Fig. 4-9C). Thus, the bHLH transcription factor forms by association of four amphipathic α helices (two from each monomer) into a bundle. The basic domains of each monomer protrude into the major grooves on opposite sides of the DNA. bHLH proteins include the MyoD family, which is involved in muscle differentiation, and E proteins (E12 and E47). MyoD and an E protein generally bind to DNA as heterodimers. imageN4-7

N4-7

Dimerization of bHLH Transcription Factors

Contributed by Peter Igarashi

The MyoD family of transcription factors includes MyoD itself as well as myogenin, myf5, and MRF4. All are involved in controlling the differentiation of muscle. MyoD and an E protein generally bind to DNA as heterodimers.

Some bHLH transcription factors contain additional domains—located immediately adjacent to the HLH domain—that mediate protein dimerization. A leucine zipper motif is contained in bHLH-Zip proteins such as c-Myc and SREBP, and a PAS domain is contained in bHLH-PAS proteins such as HIF-1α.

Helix-Turn-Helix

Homeodomain proteins that regulate embryonic development are members of the helix-turn-helix (HTH) family (see Fig. 4-9D). The homeodomain consists of a 60–amino-acid sequence that forms three α helices. Helices 1 and 2 lie adjacent to one another, and helix 3 is perpendicular and forms the DNA-recognition helix. Particular amino acids protrude from the recognition helix and contact bases in the major groove of the DNA. Examples of homeodomain proteins include the Hox proteins, which are involved in mammalian pattern formation; engrailed homologs, which are important in nervous system development; and the POU family members Pit-1, Oct-1, and unc-86. imageN4-8

N4-8

Novel Families of Transcription Factors

Contributed by Peter Igarashi

On pages 82–83 we describe four families of transcription factors: zinc finger, bZIP, bHLH, and HTH. In each case, an α helix in the transcription factor binds in the major groove of the DNA. Some transcription factors use an antiparallel β-pleated sheet for DNA binding. The β sheet fills the major groove of DNA, and amino-acid side chains that are exposed on the face of the β sheet contact the DNA bases.

In addition to transcription factors that bind to DNA via β-pleated sheets, there are several other transcription factors that do not appear to fall into one of the four structural families listed on pages 82–83. Thus, it seems likely that other structural motifs can also mediate DNA binding. One example is the forkhead domain. It is also important to note that some transcription factors bind to DNA through more than one domain. Examples include the POU family, in which the POU-specific domain is required in addition to the POU homeodomain for DNA binding.

Coactivators and corepressors are transcription factors that do not bind to DNA

Some transcription factors that are required for the activation of gene transcription do not directly bind to DNA. These proteins are called coactivators. Coactivators work in concert with DNA-binding transcriptional activators to stimulate gene transcription. They function as adapters or protein intermediaries that form protein-protein interactions between activators bound to enhancers and the basal transcriptional machinery assembled on the gene promoter (see Fig. 4-6). Coactivators often contain distinct domains, one that interacts with the transactivation domain of an activator and a second that interacts with components of the basal transcriptional machinery. Transcription factors that interact with repressors and play an analogous role in transcriptional repression are called corepressors.

One of the first coactivators found in eukaryotes was the VP16 herpesvirus protein discussed above (see Fig. 4-8B). VP16 has two domains. The first is a transactivation domain that contains a region of acidic amino acids that in turn interacts with two components of the basal transcriptional machinery, general transcription factors TFIIB and TFIID. The other domain of VP16 interacts with the ubiquitous activator Oct-1, which recognizes a DNA sequence called the octamer motif (see Table 4-1). Thus, VP16 activates transcription by bridging an activator and the basal transcriptional machinery.

Some coactivators play a general role in the activation of transcription. One example is Mediator, a multiprotein complex consisting of 28 to 30 subunits, which is not required for basal transcription but is essential for transcriptional activation by most activator proteins. Consistent with its essential role, Mediator is present in the basal transcriptional machinery or preinitiation complex.

Another type of coactivator is involved in transcriptional activation by specific transcription factors. This type of coactivator is not a component of the basal transcriptional machinery. Rather, these coactivators are recruited by a DNA-binding transcriptional activator through protein-protein interactions. An example is the coactivator CBP (CREB-binding protein), which interacts with a DNA-binding transcription factor called CREB (see Table 4-1).

Transcriptional activators stimulate transcription by three mechanisms

Once transcriptional activators bind to enhancers (i.e., positive regulatory elements on the DNA) and recruit coactivators, how do they stimulate gene transcription? We discuss three mechanisms by which transactivation might be achieved. These mechanisms are not mutually exclusive, and more than one mechanism may be involved in the transcription of a particular gene.

Recruitment of the Basal Transcriptional Machinery

We have already introduced the concept by which looping out of DNA (see pp. 79–80) permits proteins that are bound to distant DNA enhancer elements to become physically juxtaposed to proteins that are bound to the gene promoter (Fig. 4-10, pathway 1). The interaction between the DNA-binding transcription factor and general transcription factors, perhaps with coactivators as protein intermediaries, enhances the recruitment of the basal transcriptional machinery to the promoter. Two general transcription factors, TFIID and TFIIB, are targets for recruitment by transcriptional activators. For example, the acidic transactivation domain of VP16 binds to TFIIB, and mutations that prevent the interaction between VP16 and TFIIB also abolish transcriptional activation. Conversely, mutations of TFIIB that eliminate the interaction with an acidic activator also abolish transactivation but have little effect on basal transcription.

image

FIGURE 4-10 Mechanisms of transcriptional activation. The transcriptional activator binds to the enhancer and directly or indirectly (via coactivators) activates transcription by recruiting RNA polymerase to the promoter (1), recruiting HATs that remodel chromatin (2), or stimulating the phosphorylation of the CTD of RNA polymerase (3).

Chromatin Remodeling

A second mechanism by which transcriptional activators may function is alteration of chromatin structure. Gene transcription first requires local disruption of the regular nucleosome structure of chromatin (see p. 76) so that the basal transcriptional machinery can access the promoter and initiate RNA synthesis. One mechanism of chromatin remodeling involves histone acetylation. Acetylation of lysine residues in histones (see p. 88) weakens the electrostatic interaction between histones and the negatively charged DNA. Histone acetyltransferases (HATs) are enzymes that acetylate histones and thus produce local alterations in chromatin structure that are more favorable for transcription. Transcriptional activators may recruit HATs either directly or indirectly through coactivators (see Fig. 4-10, pathway 2). Alternatively, some coactivator proteins that mediate transcriptional activation possess intrinsic histone acetylase activity.

Another mechanism of chromatin remodeling involves the SWI/SNF family of proteins. SWI/SNF (switching mating type/sucrose nonfermenting) are large multiprotein complexes, initially identified in yeast but evolutionarily conserved in all animals. SWI/SNF chromatin-remodeling complexes can inhibit the association between DNA and histones by using the energy of ATP to peel the DNA away from the histones and thereby make this DNA more accessible to transcription factors. Some transcriptional activators promote chromatin remodeling by binding and recruiting subunits of the SWI/SNF complex.

Stimulation of Pol II

A third mechanism by which transcriptional activators function is by stimulating RNA polymerase II (see Fig. 4-10, pathway 3). The C-terminal domain (CTD) of the largest subunit of Pol II contains 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, which can be phosphorylated at multiple serine and threonine residues. A cyclin-dependent kinase called positive transcription elongation factor b (P-TEFb) phosphorylates the CTD. imageN4-9 Phosphorylation of the CTD occurs coincident with initiation of transcription and is required for chain elongation. Thus, transcriptional activators that interact with P-TEFb may stimulate the conversion of the Pol II holoenzyme from an initiation complex into an elongation complex.

N4-9

Phosphorylation of the CTD

Contributed by Peter Igarashi

The CTD of the largest subunit of Pol II can be phosphorylated in vitro by TFIIH and by cdc2 kinase (a regulatory component of the cell cycle). However, the identity of the kinase that is responsible for phosphorylation of the CTD in vivo remains uncertain. There is some evidence that phosphorylation of the CTD may mediate responses to viral transcriptional activators, such as VP16.

Taken together, these three mechanisms of interaction lead to the following model for activation of transcription. A transcription factor that is bound to an enhancer presents a functional domain (e.g., an acidic domain) that either directly or via coactivators interacts with chromatin-remodeling enzymes and components of the basal transcriptional machinery. These interactions facilitate the assembly of the basal transcriptional machinery on the gene promoter and thereby stimulate the initial transcription of RNA. Subsequent interactions—with the CTD of Pol II, for example—may promote elongation of the nascent RNA transcript.

Transcriptional activators act in combination

Two or more activators may increase the rate of transcription by an amount that is greater than the sum of the amounts for each of the activators alone. Almost all naturally occurring promoters contain more than one site for binding of transcriptional activators. A promoter region that contains only a single copy of an enhancer element shows only weak stimulation, whereas a promoter containing multiple copies of an enhancer exhibits substantial activation (Fig. 4-11). Two mechanisms for synergy have been proposed.

image

FIGURE 4-11 Synergism of transcriptional activators. The promoter contains three DNA enhancer elements A, B, and C. Binding of a transcription factor to only one of the enhancer elements (A, B, or C) causes a modest activation of transcription. Simultaneous binding of different transcription factors to each of the three enhancer elements can produce a supra-additive increase in transcription (i.e., synergy).

In the first, synergy may reflect cooperative binding to DNA; that is, binding of one transcription factor to its recognition site enhances binding of a second transcription factor to a different site. This phenomenon occurs with the glucocorticoid receptor (GR), which binds to a site on DNA known as the glucocorticoid response element (GRE). Binding of GRs to the multiple GREs is cooperative in that binding of the first receptor promotes binding of additional receptors. Thus, the presence of multiple copies of the GRE greatly stimulates gene expression in comparison to a single copy of the GRE.

In the second case, synergy reflects cooperative protein-protein interactions between transcription factors and multiple sites on the basal transcriptional machinery. For example, a transcriptional activator that recruits TFIID could synergize with another activator that recruits TFIIB. Similarly, a transcriptional activator that interacts with a HAT could synergize with another activator that interacts with components of the basal transcriptional machinery. Here, the effect on transcription depends on the cumulative effects of multiple transcription factors, each bound to its cognate recognition site and interacting with chromatin-remodeling proteins and the basal transcriptional machinery.

Transcription factors may act in combination by binding to DNA as homodimers or heterodimers. This synergism is particularly true for members of the bZIP and bHLH families but also for steroid and thyroid hormone receptors. Often, different combinations of monomers have different DNA-binding affinities. For example, the thyroid hormone receptor (TR) can bind to DNA as a homodimer, but the heterodimer formed from the TR and the retinoid X receptor (TR/RXR) has much higher binding affinity. The transcription factor MyoD (see p. 83), which is involved in muscle differentiation, requires heterodimerization with a ubiquitous E protein (e.g., E12 or E47) for maximal DNA binding. Different combinations of monomers may also have different DNA-binding specificities and thus be targeted to different sites on the DNA. Finally, different combinations of monomers may have different transactivational properties. For example, the c-Myc protein can bind to DNA as a homodimer or as a heterodimer with Max, but c-Myc/Max heterodimers have greater transcriptional activity.

Transcriptional repressors act by competition, quenching, or active repression

Cells can regulate transcription not only positively via transcriptional activators but also negatively via transcriptional repressors. imageN4-10 Repression of transcription is important for tissue specificity in that it allows cells to silence certain genes where they should not be expressed. Repression is also important for regulating inducible gene expression by rapidly turning off transcription after removal of the inducing stimulus.

N4-10

Modes of Action of Transcriptional Repression

Contributed by Peter Igarashi

image

EFIGURE 4-6 Transcriptional repressor modes of action.

Transcriptional repressors may act by three mechanisms. First, some repressors inhibit the binding of transcriptional activators because they compete for DNA-binding sites that are identical to, or overlap with, those for activators. An example is the CCAAT displacement protein (CDP), which binds to the CAAT box in the promoter of the γ-globin gene and thereby prevents binding of the transcriptional activator CP1. This action helps prevent inappropriate expression of the fetal globin gene in adults.

Second, some repressors inhibit the activity of transcriptional activators not by interference with DNA binding but by a direct protein-protein interaction with activators. This form of repression is termed quenching. A classic example in yeast is the GAL80 repressor, which inhibits transcriptional activation by GAL4. By binding to the transactivation domain of GAL4, GAL80 blocks transcriptional activation. Dissociation of GAL80 (which occurs in the presence of galactose) relieves the inhibition of GAL4, which can then induce expression of galactose-metabolizing genes. Transcriptional repression can also be mediated by proteins that prevent transcriptional activators from entering the nucleus. For example, the heat shock protein hsp90 binds to GR and prevents this transcriptional activator from entering the nucleus.

A third class of repressors binds to a silencer (or NRE) and then directly inhibits transcription. This mechanism is referred to as active repression. The opposite of transcriptional activators, these proteins contain domains that mediate repression. Repression domains may directly interact with and inhibit the assembly or activity of the basal transcriptional machinery. Alternatively, transcriptional repressors may inhibit transcription through protein-protein interactions with corepressors. We have previously seen that transcriptional activators may function by recruiting histone acetylases (HATs) that remodel chromatin and facilitate the initiation of RNA synthesis. Conversely, histone deacetylases (HDACs) are enzymes that remove the acetyl groups from histones, which leads to tighter binding to DNA and inhibition of transcription. Some transcriptional corepressors, such as the N-CoR adapter protein that mediates repression by steroid hormone receptors, can interact with HDAC.

The activity of transcription factors may be regulated by post-translational modifications

Cells can regulate the activity of transcription factors by controlling the amount of transcription factor they synthesize. In addition, cells can modulate the activity of preformed transcription factors by three general mechanisms of post-translational modification (Table 4-2).

TABLE 4-2

Post-Translational Modifications of Transcription Factors

MODIFICATION

MODIFYING GROUP

MODIFIED AMINO ACID

TRANSCRIPTION FACTOR (EXAMPLEs)

EFFECTS (EXAMPLE)

Phosphorylation

–PO4

Ser/Thr, Tyr

p53, HIF-1α, GR, Sp1, PPARα, β-catenin, STAT1, CREB, NFAT

Altered affinity for coactivators, promotion of ubiquitination, altered protein-protein or protein-DNA interactions

Site-specific proteolysis

None

None

SREBP-1, Notch, NF-κB, ATF-6

Generation of an active transcription factor from an inactive precursor

Acetylation

–COCH3

Lys

Sp3, p53, MEF2, STAT3

Regulation of protein stability, DNA binding, protein-protein interactions

Methylation

–CH3

Arg, Lys

PGC-1α, STAT1, CBP

Inhibition of protein-protein interactions

Glycosylation

GlcNAc

Ser/Thr

Elf-1, c-Myc, Sp1, ER

Stimulation of transcriptional activity, nuclear transport, protein stability

Ubiquitination

Ubiquitin

Lys

SREBP-1, c-Myc, VP16, β-catenin, p53, SMAD2

Targeting for proteasomal degradation (polyubiquitination). Transcriptional activation (monoubiquitination)

Sumoylation

SUMO

Lys

ER, SF-1, AR

Inhibition of transcriptional activity

Hydroxylation

–OH

Pro, Asn

HIF-1α

Altered affinity for coactivators, altered protein-protein interactions

Nitrosylation

–NO

Cys

NF-κB, Sp1, HIF-1α

Inhibition of DNA binding or protein degradation

ATF-6, activating transcription factor 6; GlcNAc, N-acetylglucosamine; MEF2, myocyte enhancer factor; NFAT, nuclear factor of activated T cell; PGC-1α, peroxisome proliferator–activated receptor-γ cofactor 1α; SF-1, steroidogenic factor 1.

Phosphorylation

The best-studied post-translational modification affecting transcription factor activity is phosphorylation, which increases or decreases (1) transport of the transcription factor from the cytoplasm into the nucleus, (2) the affinity with which the transcription factor binds to DNA, and (3) transcriptional activation.

For transcription factors that reside in the cytoplasm under basal conditions, migration from the cytoplasm into the nucleus is a necessary step. Many proteins that are transported into the nucleus contain a sequence that is relatively enriched in basic amino-acid residues (i.e., arginine and lysine). This sequence, the nuclear localization signal, is required for transport of the protein into the nucleus. Phosphorylation at sites within or near the nuclear localization signal can dramatically change the rate of nuclear translocation. Phosphorylation can also modulate import into the nucleus by regulating the binding of transcription factors to cytoplasmic anchors. In the case of the transcription factor nuclear factor κB (NF-κBFig. 4-12A), binding to a cytoplasmic anchor called inhibitor of κB (IκB ) conceals the nuclear localization signal on the p50 and p65 subunits of NF-κB from the nuclear translocation machinery. Only after these two other subunits dissociate from the phosphorylated IκB is the transcription factor dimer free to enter the nucleus.

image

FIGURE 4-12 Regulation of transcription factors by post-translational modification. A, The phosphorylation of the cytoplasmic anchor IκB releases the p50 and p65 subunits of NF-κB, which allows them to translocate into the nucleus. B, Proteolytic cleavage of a 105-kDa precursor releases the p50 subunit of NF-κB. Together with the p65 subunit, the p50 subunit can now translocate to the nucleus.

Phosphorylation can also regulate transcription factor activity by altering the affinity of the transcription factor for its target recognition sequences on DNA. As a result, phosphorylation increases or decreases DNA-binding activity. For example, phosphorylation of SRF (serum response factor), a transcription factor that activates the c-fos gene in response to growth factors, enhances DNA binding. In contrast, phosphorylation of the transcription factor c-Jun by casein kinase II inhibits binding to DNA.

Phosphorylation can greatly influence the transactivation properties of transcription factors. c-Jun is an example of a transcription factor whose transcriptional activity is increased by the phosphorylation of serine residues located within the transactivation domain near the N terminus of the protein. Phosphorylation of a transcriptional activator may stimulate its activity by increasing its binding affinity for a coactivator. Phosphorylation can also inhibit transcriptional activation by reducing transcriptional activation or stimulating active transcriptional repression.

Effects of phosphorylation on nuclear translocation, DNA binding, and transactivation are not mutually exclusive. Moreover, not only phosphorylation by protein kinases but also dephosphorylation by protein phosphatases may regulate transcriptional activity.

Site-Specific Proteolysis

Many transcription factors undergo proteolytic cleavage at specific amino-acid residues, particularly in response to exogenous signals. Site-specific proteolysis often converts an inactive precursor protein into an active transcriptional regulator. One example is NF-κB. Although phosphorylation can regulate NF-κB by controlling its binding to IκB (see Fig. 4-12A), proteolysis can also regulate NF-κB (see Fig. 4-12B). A 105-kDa precursor of the 50-kDa subunit of NF-κB (p50), which we mentioned above, binds to and thereby retains the 65-kDa subunit of NF-κB (p65) in the cytoplasm. Proteolysis of this larger precursor yields the 50-kDa subunit that together with the 65-kDa subunit constitutes the active NF-κB transcription factor.

Another form of site-specific proteolysis, which creates active transcription factors from inactive membrane-tethered precursors, is called regulated intramembranous proteolysis (RIP). One example is the sterol regulatory element–binding protein (SREBP), a membrane protein that normally resides in the endoplasmic reticulum. In response to depletion of cellular cholesterol, SREBP undergoes RIP, which releases an N-terminal fragment containing a bHLH motif. The proteolytic fragment translocates to the nucleus, where it binds to DNA and activates transcription of genes that encode enzymes involved in cholesterol biosynthesis and the LDL (low-density lipoprotein) receptor (see p. 42). imageN4-11

N4-11

Examples of RIP

Contributed by Peter Igarashi

In addition to SREBP noted in the text, other proteins that undergo RIP are Notch and APP—all span the membrane at least once.

Notch is a plasma membrane receptor whose cytoplasmic domain is released in response to Delta, a membrane-bound ligand that regulates cell fate during development.

Amyloid precursor protein (APP) is a protein of unknown function that is cleaved in the membrane to produce the extracellular amyloid β peptide implicated in Alzheimer disease. For Notch and APP, the intramembrane cleavage does not take place until a primary cleavage event removes the bulk of the protein on the extracytoplasmic face. Although the cleaved sites differ in these proteins, the net effect of the first step is to shorten the extracytoplasmic domain to <30 amino acids, which allows the second cleavage to release a portion of the cytoplasmic domain.

The epithelial Na+ channel ENaC—a heterotrimer that consists of α, β, and γ subunits—also undergoes intramembrane proteolysis. While the ENaC is still in the vesicular subcompartment of the secretory pathway, the protease furin cleaves the extracellular domain of the α subunit twice (after consensus RXXR motifs), releasing a peptide of 26 amino acids. By itself, this modification increases the open probability of ENaC to ~0.30. Furin also cleaves the γ subunit, but only once. A variety of extracellular proteases—including prostasin, elastase, and plasmin—can make a second cut after the protein has reached the plasma membrane. The result of the γ-subunit cleavages is to increase the open probability to nearly 1.0. Thus, both examples of proteolysis greatly increase the activity of the channel.

Reference

Hughey RP, Carattino MD, Kleyman TR. Role of proteolysis in the activation of epithelial sodium channels. Curr Opin Nephrol Hypertens. 2007;16:444–450.

Other Post-Translational Modifications

In addition to phosphate groups, a variety of other covalent attachments can affect the activity of transcription factors (Table 4-3). These small molecules—such as acetyl groups, methyl groups, sugars or peptides, hydroxyl groups, or nitro groups—attach to specific amino-acid residues in the transcription factor. Post-translational modifications of transcription factors can affect their stability, intracellular localization, dimerization, DNA-binding properties, or interactions with coactivators. For example, acetylation of lysine residues in the p53 transcription factor increases binding to DNA and inhibits degradation. Methylation of an arginine residue in the coactivator CBP inhibits its interaction with the transcription factor CREB. O-linked glycosylation, a covalent modification in which sugar groups attach to serine or threonine residues, stimulates NF-κB. Ubiquitin is a small peptide that is covalently attached to lysine groups in proteins. Addition of multiple ubiquitin groups (polyubiquitination) frequently results in degradation of the protein via the proteasome (see pp. 33–34). However, addition of a single ubiquitin group (monoubiquitination) may stimulate the activity of a transcription factor, perhaps by increasing its affinity for transcriptional elongation factors. Conversely, sumoylation, covalent modification of lysine residues with small ubiquitin-like modifiers (SUMOs), may inhibit activity by altering the localization of a transcription factor within the nucleus. As we will see in the next section, extracellular signals often trigger post-translational modifications to regulate the activity of transcription factors.

TABLE 4-3

Examples of Transcription Factors That Regulate Gene Expression in Response to Physiological Stimuli

PHYSIOLOGICAL STIMULUS

TRANSCRIPTION FACTOR

EXAMPLES OF PROTEINS ENCODED BY TARGETED GENE

Hypoxia

HIF-1α

VEGF, erythropoietin, glycolytic enzymes

DNA damage

p53

CIP1, GADD45, PCNA, MDM2

Cholesterol depletion

SREBP-1

HMG-CoA reductase, fatty-acid synthase, LDLl receptor

Viruses, oxidants

NF-κB

Tumor necrosis factor-alpha, interleukin-1β, interleukin-2, granulocyte colony-stimulating factor, inducible nitric oxide synthase, intercellular cell adhesion molecule

Heat stress

HSF1

Heat shock proteins, αB-crystallin

Fatty acids

PPARα

Lipoprotein lipase, fatty-acid transport protein, acyl-CoA synthetase, carnitine palmitoyltransferase I

CIP1, CDK (cyclin-dependent kinase)-interacting protein 1; GADD45, growth arrest and DNA damage 5; HSF1, heat shock factor protein 1; MDM2, mouse double minute 2 homolog (an E3 ubiquitin-protein ligase); PCNA, proliferating cell nuclear antigen.

The expression of some transcription factors is tissue specific

Some transcription factors are ubiquitous, either because these transcription factors regulate the transcription of genes that are expressed in many different tissues or because they are required for the transcription of many different genes. Examples of ubiquitous transcription factors are the DNA-binding transcription factors Sp1 and NF-Y, which bind to regulatory elements (i.e., GC boxes and CCAAT boxes, respectively—see p. 79) that are present in many gene promoters. Other transcription factors are present only in certain tissues or cell types; these transcription factors are involved in the regulation of tissue-specific gene expression.

Tissue-specific activators bind to enhancers present in the promoters and regulatory regions of genes that are expressed in a tissue-specific manner. Conversely, tissue-specific repressors bind to silencers that prevent transcription of a gene in nonexpressing tissues. Each tissue-specific transcription factor can regulate the expression of multiple genes. Because the short sequences of enhancers and silencers may occur by chance, the combined effect of multiple transcription factors—each binding to distinct regulatory elements near the gene—prevents illegitimate transcription in nonexpressing tissues. In addition to being activated by transcriptional activators, tissue-specific gene expression may also be regulated by transcriptional repression. In this case, transcriptional repressors prevent transcription of a gene in nonexpressing tissues. Tissue-specific expression probably also involves permanent silencing of nonexpressed genes through epigenetic modifications, such as DNA methylation (see pp. 95–96).

Pit-1 is an HTH-type tissue-specific transcription factor that regulates the pituitary-specific expression of genes encoding growth hormone, thyroid-stimulating hormone, and prolactin. MyoD and myogenin are bHLH-type transcription factors that bind to the E-box sequence CANNTG of promoters and enhancers of many genes expressed in skeletal muscle, such as myosin heavy chain and muscle creatine kinase. EKLF as well as GATA-1 and NF-E2 mediate the erythroid-specific expression of β-globin genes. The combined effects of HNF-1, HNF-3, HNF-4, C/EBP, and other transcription factors—each of which may individually be present in several tissues—mediate the liver-specific expression of genes such as albumin and α1-antitrypsin. Many tissue-specific transcription factors play important roles in embryonic development. For example, myogenin is required for skeletal muscle differentiation, and GATA-1 is required for the development of erythroid cells.