A second mechanism for influencing gene transcription in eukaryotes modifies the local chromatin structure around gene regulatory sequences. To fully understand how this mechanism works, we need to first understand chromatin structure and then consider how it can change and how these changes affect gene expression.

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The recruitment of transcriptional machinery by activators may appear to be somewhat similar in eukaryotes and bacteria, with the major difference being the number of interacting proteins in the transcriptional machinery. Indeed, two decades ago, many biologists pictured eukaryotic regulation simply as a biochemically more complicated version of what had been discovered in bacteria. However, this view has changed dramatically as biologists have considered the effect of the organization of genomic DNA in eukaryotes.

Compared with eukaryotic DNA, bacterial DNA is relatively “naked,” making it readily accessible to RNA polymerase. In contrast, eukaryotic chromosomes are packaged into chromatin, which is composed of DNA and proteins (mostly histones). The basic unit of chromatin is the nucleosome, which contains ~150 bp of DNA wrapped 1.7 times around a core of histone proteins (Figure 12-11). The nucleosome core contains eight histones, two subunits of each of the four histones: histones 2A, 2B, 3, and 4 (called H2A, H2B, H3, and H4) organized as two dimers of H2A and H2B and a tetramer of H3 and H4. Surrounding the nucleosome core is a linker histone, H1, which can compact the nucleosomes into higher-order structures that further condense the DNA.

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The packaging of eukaryotic DNA into chromatin means that much of the DNA is not readily accessible to regulatory proteins and the transcriptional apparatus. Thus, whereas prokaryotic genes are generally accessible and “on” unless repressed, eukaryotic genes are inaccessible and “off” unless activated. Therefore, the modification of chromatin structure is a distinctive feature of many eukaryotic processes including gene regulation (discussed in this chapter), DNA replication (Chapter 7), and DNA repair (Chapter 16). There are three major mechanisms to alter chromatin structure that will be discussed in depth in this section:

One way to alter chromatin structure might be simply to move the histone octamer along the DNA. In the 1980s, biochemical techniques were developed that allowed researchers to determine the position of nucleosomes in and around specific genes. In these studies, chromatin was isolated from tissues or cells in which a gene was on and compared with chromatin from tissue where the same gene was off. The result for most genes analyzed was that nucleosome positions changed, especially in a gene’s regulatory regions. Thus, which DNA regions are wrapped up in nucleosomes can change: nucleosome positions can shift on the DNA from cell to cell and over the life cycle of an organism. Transcription is repressed when the promoter and flanking sequences are wound up in a nucleosome, which prevents the initiation of transcription by RNA pol II. Activation of transcription would thus require nudging the nucleosomes away from the promoter. Conversely, when gene repression is necessary, nucleosomes shift into a position that prevents transcription. The changing of nucleosome position is referred to as chromatin remodeling. Chromatin remodeling is known to be an integral part of eukaryotic gene expression, and great advances are being made in determining the underlying mechanism(s) and the regulatory proteins taking part. Here, again, genetic studies in yeast have been pivotal.

Two genetic screens in yeast for mutants in seemingly unrelated processes led to the discovery of the same gene whose product plays a key role in chromatin remodeling. In both cases, yeast cells were treated with agents that would cause mutations. In one screen, these mutagenized yeast cells were screened for cells that could not grow well on sucrose (sugar nonfermenting mutants, snf). In another screen, mutagenized yeast cells were screened for mutants that were defective in switching their mating type (switch mutants, swi; see Section 12.5). Many mutants for different loci were recovered in each screen, but one mutant gene was found to cause both phenotypes. Mutants at the so-called swi2/snf2 locus (“switch–sniff”) could neither utilize sucrose effectively nor switch mating type.

What was the connection between the ability to utilize sugar and the ability to switch mating types? The Swi2-Snf2 protein was purified and discovered to be part of a large, multisubunit complex called the SWI–SNF complex that can reposition nucleosomes in a test-tube assay if ATP is provided as an energy source (Figure 12-12). In some situations, the multisubunit SWI–SNF complex activates transcription by moving nucleosomes that are covering the TATA sequences. In this way, the complex facilitates the binding of RNA polymerase II. The SWI–SNF complex is thus a co-activator.

Gal4 also binds to the SWI–SNF chromatin-remodeling complex and recruits it to activated promoters. Yeast strains containing a defective SWI–SNF complex show a reduced level of Gal4 activity. Why might an activator use multiple activation mechanisms? There are at least two reasons understood at present. The first is that target promoters may become less accessible at certain stages of the cell cycle or in certain cell types (in multicellular eukaryotes). For example, genes are less accessible during mitosis, when chromatin is more condensed. At that stage, Gal4 must recruit the chromatin-remodeling complex, whereas at other times, it might not be necessary to use the complex.

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A second reason is that many transcription factors act in combinations to control gene expression synergistically. We will see shortly that this combinatorial synergy is a result of the fact that chromatin-remodeling complexes and the transcriptional machinery are recruited more efficiently when multiple transcription factors act together.

Let’s look at the nucleosome more closely to see if any part of this structure could carry the information necessary to influence nucleosome position, nucleosome density, or both.

As already stated, most nucleosomes are composed of a histone octamer made up of two dimers of H2A and H2B and a tetramer of H3 and H4. Histones are known to be the most conserved proteins in nature; that is, histones are almost identical in all eukaryotic organisms from yeast to plants to animals. In the past, this conservation contributed to the view that histones could not take part in anything more complicated than the packaging of DNA to fit in the nucleus. However, recall that DNA with just its four bases was once considered too simple a molecule to carry the blueprint for all organisms on Earth.

Figure 12-13a shows a model of nucleosome structure that represents contributions from many studies. Of note is that the histone proteins are organized into the core octamer with some of their amino-terminal ends making electrostatic contacts with the phosphate backbone of the surrounding DNA. These protruding ends are called histone tails. Since the early 1960s, it has been known that specific basic amino acid residues (lysine and arginine) in the histone tails can be covalently modified by the attachment of acetyl and methyl groups (Figure 12-13b). These reactions, which take place after the histone protein has been translated and even after the histone has been incorporated into a nucleosome, are called post-translational modifications (PTMs).

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There are now known to be at least 150 different histone modifications that utilize a wide variety of molecules in addition to the acetyl and methyl groups already mentioned, including phosphorylation, ubiquitination, and ADP ribosylation. The covalent modification of histone tails is said to contribute to a histone code. Scientists coined the expression “histone code” because the covalent modification of histone tails is reminiscent of the genetic code. For the histone code, information is stored in the patterns of histone modification rather than in the sequence of nucleotides. With more than 150 known histone modifications, there are a huge number of possible patterns, and scientists are just beginning to decipher their effects on chromatin structure and transcriptional regulation. To add to this complexity, the code is likely not interpreted in precisely the same way in all organisms. The role of histone acetylation and methylation in gene expression is described below.

Histone acetylation, deacetylation, and gene expression The acetylation reaction is one of the best-characterized histone modifications:

Note that the reaction is reversible, which means that acetyl groups can be added by the enzyme histone acetyltransferase (HAT) and removed by the enzyme histone deacetylase (HDAC) from the same histone residue. For now, let’s see how the acetylation and deacetylation of histone amino acids influences chromatin structure and gene expression.

Evidence had been accumulating for years that the histones associated with the nucleosomes of active genes are rich in acetyl groups (said to be hyperacetylated), whereas inactive genes are underacetylated (hypoacetylated). The HAT enzyme proved very difficult to isolate. When it was finally isolated and its protein sequence deduced, it was found to be an ortholog of a yeast transcriptional activator called GCN5 (meaning that it was encoded by the same gene in a different organism). Thus, the conclusion was that GCN5 is a histone acetyltransferase. It binds to the DNA in the regulatory regions of some genes and activates transcription by acetylating nearby histones. Various protein complexes that are recruited by transcriptional activators are now understood to possess HAT activity.

How does histone acetylation alter chromatin structure and, in the process, facilitate changes in gene expression? The addition of acetyl groups to histone residues neutralizes the positive charge of lysine residues and reduces the interaction of the histone tails with the negatively charged DNA backbone. This results in more open chromatin as the electrostatic interactions between adjacent nucleosomes and between nucleosomes and adjacent DNA are reduced (Figure 12-14). In addition, histone acetylation, in conjunction with other histone modifications, influences the binding of regulatory proteins to the DNA. A bound regulatory protein may take part in one of several functions that either directly or indirectly increase the frequency of transcription initiation.

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Like other histone modifications, acetylation is reversible, and HDACs play key roles in gene repression. For example, in the presence of galactose and glucose, the activation of GAL genes is prevented by the Mig1 protein. Mig1 is a sequence-specific DNA-binding repressor that binds to a site between the UAS element and the promoter of the GAL1 gene (Figure 12-15). Mig1 recruits a protein complex called Tup1 that contains a histone deacetylase and that represses gene transcription. The Tup1 complex is an example of a corepressor, which facilitates gene repression but is not itself a DNA-binding repressor. The Tup1 complex is also recruited by other yeast repressors, such as MATα2, and counterparts of this complex are found in all eukaryotes.

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Methylation is another post-translational modification of arginine and lysine residues in the histone tails that can result in altered chromatin and gene expression. The enzyme histone methyltransferase (HMTase) adds one, two, or three methyl groups to a specific amino acid in the tail of histone H3. One of the reactions catalyzed by HMTase is shown here:

The amino acid lysine is abbreviated with a “K.” As such, these post-translational modifications of lysine 9 of histone H3 are referred to as H3K9me1, H3K9me2, and H3K9me3, respectively.

Unlike acetylation, the addition of methyl groups can either activate or repress gene expression. Recall that acetylation of lysine acts to neutralize the positive histone charge and, in this direct way, activates gene expression by reducing interactions between nucleosomes and DNA, opening chromatin. In contrast, methylation of specific lysine residues, which does not affect charge, creates binding sites for other proteins that either activate or repress gene expression depending on the residues modified. For example, methylation of H3 lysine residue 4 [H3K4(me)] is associated with the activation of gene expression and is enriched in nucleosomes near the start of transcription. There is a very different outcome when H3K9 or H3K27 are methylated. These modifications, which are associated with gene repression and tightly packed chromatin, will be discussed in greater detail later in this chapter.

An important feature of chromatin structure is that it can be inherited. This form of inheritance is given a name—epigenetic inheritance—and defined operationally as the inheritance of chromatin states from one cell generation to the next. What this inheritance means is that, in DNA replication, both the DNA sequence and the chromatin structure are faithfully passed on to the next cell generation. However, unlike the sequence of DNA, chromatin structure can change in the course of the cell cycle and during successive generations of cell division.

Recall that prokaryotic replication is orchestrated at the replication fork by the replisome, a molecular machine that includes two DNA pol III holoenzymes and accessory proteins (see Figure 7-20). In eukaryotes, replication of chromatin means that the replisome not only has to copy the nucleotide sequence of the parental strands but also has to disassemble the nucleosomes in the parental strands and reassemble them in the daughter molecules. During this process, the old histones from existing nucleosomes are randomly distributed to the daughter molecules and new histones are delivered to the replisome. The randomly distributed old histones serve as templates to guide the modification of new histones. In this way, the old histones with their modified tails and the new histones with unmodified tails are assembled into nucleosomes that become associated with both daughter strands (Figure 12-16). The modifications carried by the old histones are responsible in part for epigenetic inheritance. As such, these old modifications are called epigenetic marks because they guide the modification of the new histones.

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Unlike the common (also called consensus) histones that are added during DNA replication, eukaryotes also have other histones, called histone variants, that can replace the consensus histones that have already been assembled into nucleosomes. For example, two variants for histone H2 are called H2A.Z and H2A.X, and one H3 variant is called CENP-A. Given that histones can be modified in so many ways, why might it be necessary to replace one histone with a variant? While scientists are just beginning to understand the different roles of histone variants, a common theme is that they provide a quick way to change chromatin by replacing one histone code with another. CENP-A, for example, replaces H3 in centromeric DNA, and its presence is thought to define centromere function. The role of histone variant H2A.Z in identifying damaged DNA for rapid repair is discussed in detail in Chapter 16.

There is another important epigenetic mark in most (but not all) eukaryotes. This mark is not a histone modification; rather, it is the addition of methyl groups to DNA residues after replication. An enzyme usually attaches these methyl groups to the carbon-5 position of a specific cytosine residue.

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In mammals, the methyl group is usually added to the cytosine in a CG dinucleotide. The pattern of methylation is called symmetric methylation because the methyl groups are present on both strands in the same context:

A remarkable number of C residues are methylated in mammals: 70 to 80 percent of all CG dinucleotides are methylated genome-wide. Interestingly, most of the unmethylated CG dinucleotides are found in clusters near gene promoters. These regions are called CpG islands, where the “p” represents the phosphodiester bond. Hence, C methylation is associated with inactive regions of the genome.

Like histone modifications, DNA methylation marks can be stably inherited from one cell generation to the next. The inheritance of DNA methylation is better understood than the inheritance of histone modifications. Semiconservative replication generates daughter helices that are methylated on only the parental strand. DNA molecules methylated on only one strand are termed hemimethylated. Methyl groups are added to unmethylated strands by DNA methyltransferases that have a high affinity for these hemimethylated substrates. These enzymes are guided by the methylation pattern on the parental strand (Figure 12-17). As you will see later in the chapter, because DNA methylation is more stable than histone modifications, it is often associated with regions of the genome that are maintained in an inactive state for the entire lifetime of an organism. Such regions will be discussed later in this chapter.