How can transcription be repressed

Molecular cloning and analysis of these genes then indicated that they contain conserved sequences of base pairs called homeoboxes that encode the DNA-binding domains homeodomains of transcription factors. A wide variety of additional homeodomain proteins have since been identified in fungi, plants, and other animals, including humans. Vertebrate homeobox genes are strikingly similar to their Drosophila counterparts in both structure and function, demonstrating the highly conserved roles of these transcription factors in animal development.

The Antennapedia mutation. Antennapedia mutant flies have legs growing out of their heads in place of antennae. A Head of a normal fly. B Head of an Antennapedia mutant. Courtesy of F. Rudolf Turner, Indiana University. Two other families of DNA -binding proteins , leucine zipper and helix-loop-helix proteins, contain DNA-binding domains formed by dimerization of two polypeptide chains. The leucine zipper contains four or five leucine residues spaced at intervals of seven amino acids, resulting in their hydrophobic side chains being exposed at one side of a helical region.

This region serves as the dimerization domain for the two protein subunits, which are held together by hydrophobic interactions between the leucine side chains. Immediately following the leucine zipper is a region rich in positively charged amino acids lysine and arginine that binds DNA.

The helix-loop-helix proteins are similar in structure, except that their dimerization domains are each formed by two helical regions separated by a loop. An important feature of both leucine zipper and helix-loop-helix transcription factors is that different members of these families can dimerize with each other. Thus, the combination of distinct protein subunits can form an expanded array of factors that can differ both in DNA sequence recognition and in transcription-stimulating activities.

Both leucine zipper and helix-loop-helix proteins play important roles in regulating tissue-specific and inducible gene expression, and the formation of dimers between different members of these families is a critical aspect of the control of their function.

The activation domains of transcription factors are not as well characterized as their DNA -binding domains. Some, called acidic activation domains, are rich in negatively charged residues aspartate and glutamate ; others are rich in proline or glutamine residues.

These activation domains are thought to stimulate transcription by interacting with general transcription factors , such as TFIIB or TFIID, thereby facilitating the assembly of a transcription complex on the promoter. An important feature of these interactions is that different activators can bind to different general transcription factors or TAFs, providing a mechanism by which the combined action of multiple factors can synergistically stimulate transcription—a key feature of transcriptional regulation in eukaryotic cells.

Synergistic action of transcriptional activators. Gene expression in eukaryotic cells is regulated by repressors as well as by transcriptional activators. Like their prokaryotic counterparts, eukaryotic repressors bind to specific DNA sequences and inhibit transcription. In some cases, eukaryotic repressors simply interfere with the binding of other transcription factors to DNA Figure 6.

For example, the binding of a repressor near the transcription start site can block the interaction of RNA polymerase or general transcription factors with the promoter , which is similar to the action of repressors in bacteria.

Other repressors compete with activators for binding to specific regulatory sequences. Some such repressors contain the same DNA-binding domain as the activator but lack its activation domain. As a result, their binding to a promoter or enhancer blocks the binding of the activator, thereby inhibiting transcription. Action of eukaryotic repressors. A Some repressors block the binding of activators to regulatory sequences.

B Other repressors have active repression domains that inhibit transcription by interactions with general transcription factors.

In contrast to repressors that simply interfere with activator binding, many repressors called active repressors contain specific functional domains that inhibit transcription via protein-protein interactions Figure 6. Many active repressors have since been found to play key roles in the regulation of transcription in animal cells, in many cases serving as critical regulators of cell growth and differentiation.

As with transcriptional activators, several distinct types of repression domains have been identified. The functional targets of repressors are also diverse. Some repressors inhibit transcription by interacting with general transcription factors , such as TFIID; others are thought to interact with specific activator proteins. The regulation of transcription by repressors as well as by activators considerably extends the range of mechanisms that control the expression of eukaryotic genes.

One important role of repressors may be to inhibit the expression of tissue-specific genes in inappropriate cell types.

For example, as noted earlier, a repressor -binding site in the immunoglobulin enhancer is thought to contribute to its tissue-specific expression by suppressing transcription in nonlymphoid cell types. Other repressors play key roles in the control of cell proliferation and differentiation in response to hormones and growth factors see Chapters 13 and In the preceding discussion, the transcription of eukaryotic genes was considered as if they were present within the nucleus as naked DNA.

However, this is not the case. The DNA of all eukaryotic cells is tightly bound to histones , forming chromatin. The basic structural unit of chromatin is the nucleosome , which consists of base pairs of DNA wrapped around two molecules each of histones H2A, H2B, H3, and H4, with one molecule of histone H1 bound to the DNA as it enters the nucleosome core particle see Figure 4. The chromatin is then further condensed by being coiled into higher-order structures organized into large loops of DNA.

This packaging of eukaryotic DNA in chromatin clearly has important consequences in terms of its availability as a template for transcription, so chromatin structure is a critical aspect of gene expression in eukaryotic cells.

Indeed, both activators and repressors regulate transcription in eukaryotes not only by interacting with general transcription factors and other components of the transcriptional machinery, but also by inducing changes in the structure of chromatin.

The relationship between chromatin structure and transcription is evident at several levels. First, actively transcribed genes are found in decondensed chromatin, corresponding to the extended nm chromatin fibers discussed in Chapter 4 see Figure 4.

For example, microscopic visualization of the polytene chromosomes of Drosophila indicates that regions of the genome that are actively engaged in RNA synthesis correspond to decondensed chromosome regions Figure 6.

Similarly, actively transcribed genes in vertebrate cells are present in a decondensed fraction of chromatin that is more accessible to transcription factors than is the rest of the genome.

Decondensed chromosome regions in Drosophila. A light micrograph showing decondensed regions of polytene chromosomes arrows , which are active in RNA synthesis. Courtesy of Joseph Gall, Carnegie Institute. Decondensation of chromatin , however, is not sufficient to make the DNA an accessible template for transcription.

Even in decondensed chromatin, actively transcribed genes remain bound to histones and packaged in nucleosomes, so transcription factors and RNA polymerase are still faced with the problem of interacting with chromatin rather than with naked DNA.

The tight winding of DNA around the nucleosome core particle is a major obstacle to transcription, affecting both the ability of transcription factors to bind DNA and the ability of RNA polymerase to transcribe through a chromatin template. This inhibitory effect of nucleosomes is relieved by acetylation of histones and by the binding of two nonhistone chromosomal proteins called HMG and HMG to nucleosomes of actively transcribed genes.

HMG stands for high-mobility group proteins; these proteins migrate rapidly during gel electrophoresis. Additional proteins called nucleosome remodeling factors facilitate the binding of transcription factors to chromatin by altering nucleosome structure. Acetylation of histones has been correlated with transcriptionally active chromatin in a wide variety of cell types Figure 6.

The core histones H2A, H2B, H3 and H4 have two domains : a histone fold domain, which is involved in interactions with other histones and in wrapping DNA around the nucleosome core particle, and an amino-terminal tail domain, which extends outside of the nucleosome. The amino-terminal tail domains are rich in lysine and can be modified by acetylation at specific lysine residues. Acetylation reduces the net positive charge of the histones, and may weaken their binding to DNA as well as altering their interactions with other proteins.

Importantly, recent experiments have provided direct evidence that histone acetylation facilitates the binding of transcription factors to nucleosomal DNA, indicating that histone acetylation increases the accessibility of chromatin to DNA-binding proteins. In addition, direct links between histone acetylation and transcriptional regulation have come from experiments showing that transcriptional activators and repressors are associated with histone acetyltransferases and deacetylases, respectively.

This association was first revealed by cloning a gene encoding a histone acetyltransferase from Tetrahymena. Unexpectedly, the sequence of this histone acetyltransferase revealed that it was closely related to a previously known yeast transcriptional coactivator called Gcn5p, which stimulates transcription in association with several different sequence-specific transcriptional activators.

Further experiments revealed that Gcn5p itself has histone acetyltransferase activity, suggesting that transcriptional activation results directly from histone acetylation. These results have been extended by the finding that several mammalian transcriptional coactivators are also histone acetyltransferases, as is a general transcription factor TAFII, a component of TFIID. Conversely, histone deacetylases which remove the acetyl groups from histone tails are associated with transcriptional repressors in both yeast and mammalian cells.

Histone acetylation is thus regulated by both transcriptional activators and repressors, indicating that it plays a key role in eukaryotic gene expression.

Histone acetylation. A The core histones have histone-fold domains, which interact with other histones and with DNA in the nucleosome, and N-terminal tails, which extend outside of the nucleosome. The N-terminal tails of the core histones e. Nucleosome remodeling factors are protein complexes that facilitate the binding of transcription factors by altering nucleosome structure Figure 6.

The mechanism of action of nucleosome remodeling factors is not yet clear, but they appear to increase the accessibility of nucleosomal DNA to other proteins such as transcription factors without removing the histones. One possibility is that they catalyze the sliding of histone octamers along the DNA molecule, thereby repositioning nucleosomes to facilitate transcription factor binding.

The mechanisms by which nucleosome remodeling factors are targeted to actively transcribed genes also remain to be established, although some studies suggest that they can be brought to enhancer or promoter sites in association with transcriptional activators or as components of the RNA polymerase II holoenzyme see Figure 6. Furthermore, in addition to being required for HM silencing, most Sir proteins are required for the formation of telomeric heterochromatin and for the silencing of genes by telomeres.

One of the most remarkable components of the Sir repressosome is Sir2. Not only is this protein required for HM and telomeric silencing, but it is also required for transcriptional and recombinational silencing of the rDNA repeats in the nucleolus Lustig Through its function in the nucleolus, it suppresses nucleolar fragmentation and thereby serves as a longevity factor.

Recent biochemical studies of Sir2, which is the only Sir protein that is conserved in multicellular eukaryotes, have revealed that it is the prototype for a novel family of histone deacetylases Imai et al. A surprising aspect of Sir2 enzymology is that NAD is required as a cofactor. In the deacetylation reaction, the acetyl group is apparently transferred to the nicotinamide-linked ribose residue in NAD, displacing nicotinamide and producing acetyl-ADP-ribose Tanner et al.

The finding that Sir2 is a histone deacetylase provides the first known mechanistic link between the Sir repressosome and the hypoacetylated state of the loci silenced by the Sir repressosome. Unlike the local deacetylation brought about by the Ume6-recruited Sin3 complex, deacetylation by the Sir repressosome is apparently a long-range phenomenon. Some of the other Sir proteins, particularly Sir3 and Sir4, may provide the explanation for this difference.

Sir3 and Sir4 are able to bind the hypoacetylated N-terminal tails of histones H3 and H4, perhaps allowing Sir3 and Sir4 to spread along the chromatin fiber from the silencer Grunstein The recruitment of Sir2 by Sir3 and Sir4 may then result in the spread of the deacetylated domain.

The spreading of a repressed chromosomal state is not by any means limited to Sir-dependent repression in yeast.

A classic example of this kind of spreading is provided by the phenomenon of position effect variegation PEV in Drosophila Reuter and Spierer In this process, genes that become mislocalized to regions close to centromeric heterochromatin are silenced by the spreading of heterochromatin. Extensive genetic analysis of PEV has shown that the likelihood of spreading can be altered by changes in the concentration of chromatin components or in the concentration of enzymes that covalently modify histones Wallrath Just as the Sir repressosome generates a transcriptionally silent chromatin structure that is able to spread along the chromatin fiber, it is possible that the Groucho repressosome nucleates a silent chromosomal state.

This may allow these corepressors to spread along the chromatin fiber. Spreading of corepressors along chromatin may be a key to long-range repression. The corepressors may then spread along chromatin by virtue of their ability to bind hypoacetylated histones. The corepressor polymer may then recruit additional histone deacetylase HDAC.

Through such a repetitive process, a large chromosomal locus may be organized into a repressed state. B Short-range corepressors such as CtBP can also recruit histone deacetylase. This may result in the local deacetylation of nucleosomes, forming an altered chromatin structure that may displace neighboring activators.

As a result of the hypothesized inability of short-range repressors to polymerize, the effect may be strictly local. Although studies that directly address the possibility of Groucho spreading have not yet been reported, studies looking at the possibility of Tup1 spreading at the yeast STE6 locus have recently been carried out by two labs, and the results are contradictory. Furthermore, nucleosome positioning and repression are both dependent on the histone tails Roth et al.

In one report examining the question of Tup1 spreading, chromatin immunoprecipitation ChIP assays with Tup1 antibodies suggest a high density of Tup1 along the entire STE6 locus under repressive conditions Ducker and Simpson Furthermore, these experiments showed a sharp drop-off in Tup1 density upstream and downstream of the gene, suggesting the existence of boundary elements that somehow limit the spread of Tup1.

However, similar ChIP experiments from another laboratory failed to reproduce these findings Wu et al. The reason for the discrepancy is unclear and therefore the question of Tup1 spreading remains unresolved. For example, by recruiting histone deacetylases, Tup1 might generate a change in the local histone acetylation pattern. This altered acetylation state could serve as a signal for the recruitment of factors that are able to cooperatively spread along the template, organizing a repressed chromosomal domain.

Indeed, the idea that the covalent modification state of histone tails might serve as a code that is read by various effector proteins to generate changes in gene expression has been the subject of much recent interest Jenuwein and Allis Although histone deacetylation probably accounts for part of the ability of Groucho to repress transcription, it is unlikely to represent the whole story.

Just as activation by enhancers has long been thought to involve stimulatory interactions between enhancer-bound activators and the basal machinery, it is likely that repression involves inhibitory interactions between silencer-bound repressors and the basal machinery. Long-range repression could therefore require the formation of a DNA loop that brings a silencer, with its interacting repressors and corepressors, into the vicinity of the core promoter, with its interacting basal transcriptional machinery.

Evidence that basal machinery interactions might mediate repression by Groucho family repressors comes from studies of the androgen receptor AR; Yu et al. Furthermore, in an in vitro transcription system reconstituted from highly purified components presumably devoid of histones, AES abolished transcriptional activation by AR, although basal transcription was unaffected. A possible interpretation of these findings is that, after recruitment of AES by a regulatory factor, loop formation allows an interaction between AES and TFIIE that serves to block preinitiation complex function.

Although this is an attractive model, it does not account for the inability of AES to interfere with basal transcription. If AES truly inhibits the basal machinery, one might expect to observe repression of basal transcription. A number of studies have suggested that Tup1 might function by basal machinery interactions, and, in particular, by interactions with the mediator complex Gromoller and Lehming ; Papamichos-Chronakis et al.

It was first characterized in yeast, and analogous complexes have been identified in metazoans. In both yeast and metazoans, the mediator complex is believed to interact functionally with a wide variety of activators. In an elegant study showing a functional interaction between Tup1 and the basal machinery, the Srb7 subunit of the mediator complex was found to bind Tup1 Gromoller and Lehming A mutant allele of srb7 was created encoding a protein that was unable to bind Tup1, but that was able to rescue the lethality caused by an srb7 deletion.

This srb7 allele was found to result in phenotypes reminiscent of those associated with tup1 mutations, such as cell clumping and decreased mating efficiency.

In addition, several Tup1 target promoters displayed severely compromised Tup1-mediated repression. These experiments strongly suggest that an interaction between Tup1 and the holoenzyme interferes with the function of the basal machinery. Further experiments included in the study described above suggest that the same region of Srb7 that contacts Tup1 also contacts Med6, a component of the mediator complex that is required for the stimulation of transcription by a number of activator proteins.

This intriguing model has a very interesting implication. It suggests that factors that work through the basal machinery to mediate long-range repression may work in an activator-selective manner. For example, if the model is correct, it would suggest that Tup1 should preferentially interfere with activation by factors that work through Med6, whereas factors that work by other mechanisms should be relatively resistant to repression by Tup1.

Interactions between Tup1 and the mediator. For simplicity, the general transcription factors have been omitted. A number of activators Act require Med6 to activate transcription. These activators may stimulate an interaction between Med6 and Srb7, leading to activation. B After recruitment by a repressor Rep , Tup1 as a component of the Ssn6—Tup1 complex may block activation by competing with Med6 for binding to Srb7.

In some cases of gene regulation, mechanisms are required that will allow repressors to block activation of a given promoter by activators bound close to the repressor binding site, while still allowing activation by more distantly bound activators.

Although, as discussed above, long-range repression may sometimes allow for activator-specific repression, short-range repression may be a more flexible way to achieve this kind of control. For example, the distance over which a short-range repressor is able to work appears to be dependent on repressor concentration.

Thus, short-range repressors may provide a sensitive means of responding to a transcription factor concentration gradient Hewitt et al. Repressors that regulate the expression of the Drosophila pair-rule genes such as eve and hairy provide an excellent example of short-range repression.

Pair-rule genes are generally expressed in seven transverse stripes along the anteroposterior axis of the early embryo Ingham The spatial control of pair-rule gene expression is largely dependent on the transcription factors encoded by the gap genes e. These factors work via multiple autonomous enhancers in the pair-rule genes Akam An individual enhancer often directs a single stripe of expression.

Because the enhancers function independently of one another to direct stripes of expression at different positions along the anteroposterior axis, the characteristic seven-stripe expression pattern can be generated by an appropriate combination of enhancers within a single locus. The ability of these multiple enhancers to function autonomously is critically dependent on the ability of the repressor proteins that interact with these enhancers to function in a short-range manner.

At the same time, these repressors are unable to interfere with activation by other stripe enhancers in the eve locus because the activator binding sites within the other enhancers are hundreds to thousands of base pairs away, thus ensuring enhancer autonomy.

Therefore, an understanding of how CtBP represses transcription may go a long way toward explaining short-range repression. It should be noted, however, that Drosophila CtBP was first isolated in a yeast two-hybrid screen for proteins that interact with Hairy, which, as discussed above, is a Groucho-interacting long-range repressor Poortinga et al.

It should also be noted that the short-range repressors at work in the early embryo can, at least in some cases, function by CtBP-independent mechanisms La Rosee-Borggreve et al. Whereas the Drosophila version of CtBP interacts with the short-range repressors mentioned above, mammalian CtBP has been found to interact with a number of mammalian factors, including E1A Boyd et al.

Therefore, the theme of employing short peptide motifs to recruit corepressors seems to extend to CtBP as well as Groucho. A number of studies suggest that CtBP may function, at least in part, by recruiting histone deacetylases Sundqvist et al. In addition, repression by a Gal4—CtBP fusion protein was found, at least in some cases, to be sensitive to TSA, a specific inhibitor of histone deacetylases Criqui-Filipe et al.

How do we explain the apparent contradiction that arises from the possibility that both long- and short-range corepressors may function through histone deacetylation? There are a number of possibilities. Alternatively, the differences between long- and short-range corepressors could relate to the different properties of different histone deacetylases. Groucho has thus far only been found to bind class I histone deacetylases, whereas CtBP appears to bind both class I and class II histone deacetylases Bertos et al.

Perhaps the different repertoires of histone deacetylases recruited by different corepressors result in different histone acetylation patterns in the surrounding chromatin. As discussed previously, certain histone acetylation patterns could result in the recruitment of chromatin-remodeling enzymes that organize large, transcriptionally repressed domains.

In contrast, other histone acetylation patterns might only generate short-range changes in chromatin structure that result in the ejection of activators from nearby binding sites Fig.

As with Groucho, it is likely that histone deacetylase interactions do not fully account for the ability of CtBP to repress transcription. Another possible mechanism for short-range repression, which is sometimes referred to as quenching Gray and Levine b , involves interactions of repressors and the corepressors they recruit with activators bound to nearby sites.

Thus, once a short-range corepressor is recruited to a gene by an interaction with a repressor, it could be transferred to a nearby DNA-bound activator protein. The transferred corepressor could then serve to block activation, perhaps by obstructing an interaction between the activation domain and the general machinery. One aspect of CtBP-mediated repression would seem to make the quenching model unattractive.

Given the great diversity in activation domains, the interactions between CtBP and the activators would therefore need to be quite promiscuous. Such promiscuity might be expected to result in a myriad of nonproductive interactions with irrelevant nuclear components. A possible solution to this problem comes from the idea that, in the absence of highly evolved complementary interaction surfaces, it may nonetheless be possible to impose specificity by localization Ptashne and Gann The ability of repressors and corepressors to function by multiple mechanisms, including chromatin interactions and transcriptional machinery interactions, appears to be widespread in eukaryotic gene regulation.

This multifunctionality may allow corepressors to shut off gene expression in ways that are tailored to the goal of the repression. For example, if the goal is to transiently repress transcription in response to a temporary change in the environment, then repression via transcriptional machinery interactions, which should be rapidly reversible, might be the best option.

In contrast, if the goal is to generate a repressed epigenetic state, then repression via covalent changes in histone structure might be the preferred option. This is because the semiconservative redistribution of histones during S phase might allow such changes to be maintained from one cell generation to the next. Indeed, a number of repressed states that have been linked to changes in chromatin including HM silencing and PEV are known to be heritable Jenuwein and Allis The availability of both short- and long-range repressors adds yet another layer of flexibility to gene regulation.

Long-range repression provides the possibility of shutting down an entire locus regardless of how many separate regulatory modules control the activity of that locus. On the other hand, short-range repression provides a way to control the activity of one enhancer without interfering with the activity of others. This enhancer autonomy appears to be especially important at complex loci containing multiple enhancers, each required for a distinct portion of an intricate pattern of expression.

View all Transcriptional repression: the long and the short of it Albert J. Figure 1. Figure 2. Figure 3. Previous Section Next Section.

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