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1 THE NUCLEUS OF A EUKARYOTIC CELL

481

which binds to the complex and causes its disassembly as indicated in step 4, Figure 12.7a. This is the apparent function of the high level of Ran-GTP in the nucleus: it promotes the disassembly of complexes imported from the cytoplasm. The imported cargo is released into the nucleoplasm, and one portion of the NLS receptor (the importin subunit) is shuttled back to the cytoplasm together with the bound Ran-GTP (step 5). Once in the cytoplasm, the GTP molecule bound to Ran is hydrolyzed, releasing Ran-GDP from the importin subunit. Ran-GDP is returned to the nucleus, where it is converted back to the GTP-bound state for additional rounds of activity. Importin is transported back to the cytoplasm by one of the exportins. Ran-GTP plays a key role in the escort of macromolecules from the nucleus, just as it does in their import from the cytoplasm. Recall that Ran-GTP is essentially conned to the nucleus. Whereas Ran-GTP induces the disassembly of imported complexes, as shown in step 4 of Figure 12.7a, RanGTP promotes the assembly of exported complexes. Proteins exported from the nucleus contain amino acid sequences (called nuclear export signals, or NESs) that are recognized by transport receptors that carry them through the nuclear envelope to the cytoplasm. Most of the trafc moving in this direction consists of various types of RNA moleculesespecially mRNAs, rRNAs, and tRNAsthat are synthesized in the nucleus and function in the cytoplasm. In most cases, these RNAs move through the NPC as ribonucleoproteins (RNPs). Transport of an mRNP from the nucleus to cytoplasm is associated with extensive remodeling; certain proteins are stripped from the mRNA, while others are added to the complex. Transport of mRNPs does not appear to require Ran but does require the activity of an RNA helicase located on the cytoplasmic laments of the NPC. It is speculated that the helicase provides the motive force to move the mRNA into the cytoplasm. Numerous studies have demonstrated a functional link between pre-mRNA splicing and mRNA export; only mature (i.e., fully processed) mRNAs are capable of nuclear export. If an mRNA still contains an unspliced intron, that RNA is retained in the nucleus.

t 2 meters of DNA into a nucleus only 10 m (1 10 5 m) in diameter and, at the same time, maintain the DNA in a state that is accessible to enzymes and regulatory proteins? Just as important, how is the single DNA molecule of each chromosome organized so that it does not become hopelessly tangled with the molecules of other chromosomes? The answers lie in the remarkable manner in which a DNA molecule is packaged. Nucleosomes: The Lowest Level of Chromosome Organization Chromosomes are composed of DNA and associated protein, which together is called chromatin. The orderly packaging of eukaryotic DNA depends on histones, a remarkable group of small proteins that possess an unusually high content of the basic amino acids arginine and lysine. Histones are divided into ve classes, which can be distinguished by their arginine/lysine ratio (Table 12.1). The amino acid sequences of histones, particularly H3 and H4, have undergone very little change over long periods of evolutionary time. The H4 histones of both peas and cows, for example, contain 102 amino acids, and their sequences differ at only 2 amino acid residues. Why are histones so highly conserved? One reason is histones interact with the backbone of the DNA molecule, which is identical in all organisms. In addition, nearly all of the amino acids in a histone molecule are engaged in an interaction with another molecule, either DNA or another histone. As a result, very few amino acids in a histone can be replaced with other amino acids without severely affecting the function of the protein. In the early 1970s, it was found that when chromatin was treated with nonspecic nucleases, most of the DNA was converted to fragments of approximately 200 base pairs in length. In contrast, a similar treatment of naked DNA (i.e., DNA devoid of proteins) produced a randomly sized population of fragments. This nding suggested that chromosomal DNA was protected from enzymatic attack, except at certain periodic sites along its length. It was presumed that the proteins associated with the DNA were providing the protection. In 1974, using the data from nuclease digestion and other types of information, Roger Kornberg, then at Harvard University, proposed an entirely new structure for chromatin. Kornberg proposed that DNA and histones are organized into repeating subunits, called nucleosomes. We now know that each nucleo-

Chromosomes and Chromatin

Chromosomes seem to appear out of nowhere at the beginning of mitosis and disappear once again when cell division has ended. The appearance and disappearance of chromosomes provided early cytologists with a challenging question: What is the nature of the chromosome in the nonmitotic cell? We are now able to provide a fairly comprehensive answer to this question.
Packaging the Genome An average human cell contains about 6.4 billion base pairs of DNA divided among 46 chromosomes (the value for a diploid, unreplicated number of chromosomes). Each unreplicated chromosome contains a single, continuous DNA molecule; the larger the chromosome, the longer the DNA it contains. Given that each base pair is about 0.34 nm in length, 6 billion base pairs would constitute a DNA molecule fully 2 m long. How is it possible to

TABLE 12.1 Calf Thymus Histones

Number of residues Mass (kDa) ( %Arg %Lys UEP* 10 6 year)

Histone

H1 H2A H2B H3 H4

215 129 125 135 102

23.0 14.0 13.8 15.3 11.3

1 9 6 13 14

29 11 16 10 11

8 60 60 330 600

*Unit evolutionary period: the time for a proteins amino acid sequence to change by 1 percent after two species have diverged.

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Histone octamer

H1

DNA (a) (b)

FIGURE 12.8 Nucleosomal organization of chromatin. (a) Schematic diagram showing the structure of a nucleosome core particle and an associated histone H1 molecule. The core particle itself consists of approximately 1.8 turns (146 base pairs) of negatively supercoiled DNA wrapped around eight core histone molecules (two each of H2A, H2B, H3, and H4). The H1 linker histone binds near the sites where DNA

enters and exits the nucleosome. Two alternate positions of the H1 molecule are shown. (b) Electron micrograph of chromatin bers released from the nucleus of a Drosophila cell in a buffer of low ionic strength. The nucleosome core particles are approximately 10 nm in diameter and are connected by short strands of naked linker DNA, which are approximately 2 nm in diameter. (B: COURTESY OF OSCAR L. MILLER, JR.)

some contains a nucleosome core particle consisting of 146 base pairs of supercoiled DNA (page 390) wrapped almost twice around a disk-shaped complex of eight histone molecules (Figure 12.8a). The histone core of each nucleosome consists of two copies each of histones H2A, H2B, H3, and H4 assembled into an octamer, as discussed below. The remaining histonetype H1resides outside the nucleosome core particle. The H1 histone is referred to as a linker histone because it binds to part of the linker DNA that connects one nucleosome core particle to the next. Fluorescence studies indicate that H1 molecules continuously dissociate and reassociate with chromatin. Together the H1 protein and the histone octamer interact with about 168 base pairs of DNA. H1 histone molecules can be selectively removed from the chromatin bers by subjecting the preparation to solutions of low ionic strength. When H1-depleted chromatin is observed under the electron microscope, the nucleosome core particles and naked linker DNA can be seen as separate elements, which together appear like beads on a string (Figure 12.8b). Our understanding of DNA packaging has been greatly advanced in recent years by dramatic portraits of the nucleosome core particle obtained by X-ray crystallography (Figure 12.9). The eight histone molecules that comprise a nucleosome core particle are organized into four heterodimers: two H2A-H2B dimers and two H3-H4 dimers (Figure 12.9a,c). Dimerization of histone molecules is mediated by their C-terminal domains, which consist largely of helices (represented by the cylinders in Figure 12.9c) folded into a compact mass in the core of the nucleosome. In contrast, the N-terminal segment of each core histone (and also the C-terminal segment of H2A) takes the form of a long, exible tail (represented by the dashed lines of Figure 12.9c) that extends past the DNA helix and into the surroundings. These

tails are targets of a variety of covalent modications whose key functions will be explored later in the chapter. Histone modication is not the only mechanism to alter the histone character of nucleosomes. In addition to the four conventional core histones discussed above, several alternate versions of the H2A and H3 histones are also synthesized in most cells. The importance of these histone variants, as they are called, remains largely unexplored, but they are thought to have specialized functions (Table 12.2). The localization and apparent function of one of these variants, CENP-A, is discussed on page 496. Another variant, H2A.X, is distributed throughout the chromatin, where it replaces conventional H2A in a fraction of the nucleosomes. H2A.X becomes phosphorylated at sites of DNA-strand breakage and may play a role in recruiting the enzymes that repair the DNA. Two other core histone variantsH2A.Z and H3.3can be incorporated into nucleosomes of genes as they become activated and may play a role in promoting the transcription of that genetic locus.

TABLE 12.2 Histone Variants

Type Variant Location Likely function

H2A H2A.X H2A.Z macroH2A H3 CENP-A H3.3 Centromeres Transcribed loci Kinetochore assembly Transcription Throughout chromatin Euchromatin Inactive X chromosome DNA repair Transcription Transcriptional silencing

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483

7
N C N H3'H3

0 1

6
C

N H4 H2B

5
C

3
N N

H2A H2B

H3 H4

(a)

(b)

(c)

FIGURE 12.9 The three-dimensional structure of a nucleosome as

revealed by X-ray crystallography. (a) A nucleosome core particle viewed down the central axis of the DNA superhelix, showing the position of each of the eight histone molecules of the core octamer. The histones are seen to be organized into four dimeric complexes. Each histone dimer binds 27 to 28 base pairs of DNA, with contacts occurring where the minor groove of the DNA faces the histone core. (b) The disk shape of the nucleosome core particle is evident in this view perpendicular to the central axis. The two H3-H4 dimers are associated with one another in the center of the core particle to form a tetramer, whereas the two H2A-H2B dimers are positioned on each side of the (H3-H4)2 tetramer. (c) A simplied, schematic model of half of a nucleosome core particle, showing one turn (73 base pairs) of the DNA superhelix and

four core histone molecules. The four different histones are shown in separate colors, as indicated by the key. Each core histone is seen to consist of (1) a globular region, called the histone fold, consisting of three helices (represented by the cylinders) and (2) a exible, extended N-terminal tail (indicated by the letter N) that projects out of the histone disk and past the DNA double helix. The intermittent points of interaction between the histone molecules and the DNA are indicated by white hooks. The dashed lines indicate the outermost portion of the histone tails; these exible tails lack a dened tertiary structure and therefore do not appear in the X-ray structures shown in a and b.
(A,B: REPRINTED WITH PERMISSION FROM KAROLIN LUGER ET AL., NATURE 389:251, 1997; COURTESY OF TIMOTHY J. RICHMOND. C: AFTER DRAWING BY D. RHODES; COPYRIGHT 1997, BY MACMILLAN MAGAZINES LIMITED.)

DNA and core histones are held together by several types of noncovalent bonds, including ionic bonds between negatively charged phosphates of the DNA backbone and positively charged residues of the histones. The two molecules make contact at sites where the minor groove of the DNA faces inward toward the histone core, which occurs at approximately 10 base-pair intervals (the white hooks in Figure 12.9c). In between these points of contact, the two molecules are seen to be separated by considerable space, which might provide access to the DNA for transcription factors and other DNA-binding proteins. For many years, histones were thought of as inert, structural molecules but, as we will see in following sections, these small proteins play critically important roles in determining the activity of the DNA with which they are associated. It has also become evident that chromatin is a dynamic cellular component in which histones, regulatory proteins, and a myriad variety of enzymes move in and out of the nucleoprotein complex to facilitate the complex tasks of DNA transcription, compaction, replication, recombination, and repair. We began this section by wondering how a nucleus 10 m in diameter can pack 200,000 times this length of DNA within its boundaries. The assembly of nucleosomes is the rst important step in the compaction process. With a nucleotide

nucleotide spacing of 0.34 nm, the 200 base pairs of a single 10-nm nucleosome would stretch nearly 70 nm if fully extended. Consequently, it is said that the packing ratio of the DNA of nucleosomes is approximately 7:1. Higher Levels of Chromatin Structure A DNA molecule wrapped around nucleosome core particles of 10-nm diameter is the lowest level of chromatin organization. Chromatin does not, however, exist within the cell in this relatively extended, beads-on-a-string state. When chromatin is released from nuclei and prepared at physiologic ionic strength, a ber of approximately 30-nm thickness is observed (Figure 12.10a). Despite more than two decades of investigation, the structure of the 30-nm ber remains a subject of debate. Two models in which the nucleosomal lament is coiled into the higherorder, thicker ber are shown in Figure 12.10b,c. The models differ in the relative positioning of nucleosomes within the ber. Recent research favors the zig-zag model depicted in Figure 12.10b, in which successive nucleosomes along the DNA are arranged in different stacks and alternating nucleosomes become interacting neighbors. Regardless of how it is accomplished, the assembly of the 30-nm ber increases the DNA-packing ratio an additional 6-fold, or about 40-fold altogether.

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30nm Fiber

FIGURE 12.10 The 30-nm ber: a higher level of chromatin structure. (a) Electron micrograph of a 30-nm chromatin ber released from a nucleus following lysis of the cell in a hypotonic salt solution. (b) In the zig-zag model, the linker DNA is present in a straight, extended state that criss-crosses back and forth between consecutive core particles, which are organized into two separate stacks of nucleosomes. The lower portion of the gure shows how the two stacks of nucleosomes are coiled into a higher-order helical structure. (c) In the solenoid model, the linker DNA gently curves as it connects consecutive core particles, which are organized into a single, continuous helical array containing about 68 nucleosomes per turn. In these models, the histone octamer is shown in blue, the DNA in magenta, and the linker H1 histone in yellow. (A: COURTESY OF BARBARA
HAMKALO AND JEROME B. RATTNER; B,C: FROM SEPIDEH KHORASANIZADEH, CELL 116:262, 2004; BY PERMISSION OF CELL PRESS.)

(a)

(b)

(c)

Maintenance of the 30-nm ber depends on the interaction between histone molecules of neighboring nucleosomes. Linker histones and core histones have both been implicated in higher-order packaging of chromatin. If, for example, H1 linker histones are selectively extracted from compacted chromatin, the 30-nm bers uncoil to form the thinner, more extended beaded lament shown in Figure 12.8b. Adding back H1 histone leads to restoration of the higher-order structure. Core histones of adjacent nucleosomes may interact with one another by means of their long, exible tails. Structural studies indicate, for example, that the N-terminal tail of an H4 histone from one nucleosome core particle can reach out and make extensive contact with both the linker DNA between nucleosome particles and the H2A/H2B dimer of adjacent particles. These types of interactions are thought to mediate the folding of the nucleosomal lament into a thicker ber. In fact, chromatin bers prepared with H4 histones that lack their tails are unable to fold into higher-order bers. The next stage in the hierarchy of DNA packaging is thought to occur as the 30-nm chromatin ber is gathered into a series of large, supercoiled loops, or domains, that may be compacted into even thicker (80100 nm) bers. The DNA loops are apparently tethered at their bases to pro-

teins that are part of an organized nuclear scaffold or matrix (discussed on page 499). Included among these proteins is a type II topoisomerase that presumably regulates the degree of DNA supercoiling. The topoisomerase would also be expected to untangle the DNA molecules of different loops should they become intertwined. Normally, loops of chromatin bers are spread out within the nucleus and cannot be visualized, but their presence can be revealed under certain circumstances. For instance, when isolated mitotic chromosomes are treated with solutions that extract histones, the histone-free DNA can be seen to extend outward as loops from a protein scaffold (Figure 12.11). The mitotic chromosome represents the ultimate in chromatin compactness; 1 m of mitotic chromosome length typically contains approximately 1 cm of DNA, which represents a packing ratio of 10,000:1. This compaction occurs by a poorly understood process that is discussed in Section 14.2. An overview of the various levels of chromatin organization, from the nucleosomal lament to a mitotic chromosome, is depicted in Figure 12.12.
Heterochromatin and Euchromatin After mitosis has been completed, most of the chromatin in highly compacted mi-

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DNA double helix (2 nm in diameter) H1 histone

DNA Nucleosome core particle Core histones (8 subunits)

Nucleosome filament (10 nm in diameter)

Scaffold
30 nm fiber

FIGURE 12.11 Chromatin loops: a higher level of chromatin structure.

Electron micrograph of a mitotic chromosome that had been treated with a solution of dextran sulfate to remove histones. The histonedepleted chromosome displays loops of DNA that are attached at their bases to a residual protein scaffold. (FROM JAMES R. PAULSON AND U. K.
LAEMMLI, CELL 12:823, 1977; BY PERMISSION OF CELL PRESS.)

Looped domains

Metaphase chromosome

Protein scaffold

totic chromosomes returns to its diffuse interphase condition. Approximately 10 percent of the chromatin, however, generally remains in a condensed, compacted form throughout interphase. This compacted, densely stained chromatin is seen at the periphery of the nucleus in Figure 12.1a. Chromatin that remains compacted during interphase is called heterochromatin to distinguish it from euchromatin, which returns to a dispersed state. When a radioactively labeled RNA precursor such as [3H]uridine is given to cells that are subsequently xed, sectioned, and autoradiographed, the clumps of heterochromatin remain largely unlabeled, indicating that they have relatively little transcriptional activity. The state of a particular region of the genome, whether it is euchromatic or heterochromatic, is stably inherited from one cell generation to the next. Heterochromatin is divided into two classes. Constitutive heterochromatin remains in the compacted state in all cells at all times and, thus, represents DNA that is permanently silenced. In mammalian cells, the bulk of the constitutive heterochromatin is found in the region that anks the telomeres and centromere of each chromosome and in a few other sites, such as the distal arm of the Y chromosome in male mammals. The DNA of constitutive heterochromatin consists primarily of repeated sequences (page 394) and con-

FIGURE 12.12 Levels of organization of chromatin. Naked DNA molecules are wrapped around histones to form nucleosomes, which represent the lowest level of chromatin organization. Nucleosomes are organized into 30-nm bers, which in turn are organized into looped domains. When cells prepare for mitosis, the loops become further compacted into mitotic chromosomes (see Figure 14.13).

tains relatively few genes. In fact, when genes that are normally active move into a position adjacent to heterochromatin (having changed position as the result of transposition or translocation), they tend to become transcriptionally silenced, a phenomenon known as position effect. It is thought that heterochromatin contains components whose inuence can spread outward a certain distance, affecting nearby genes. The spread of heterochromatin along the chromosome is apparently blocked by specialized barrier sequences (boundary elements) in the genome. Constitutive heterochromatin also serves to inhibit genetic recombination between homologous repetitive sequences. This type of recombination can lead to DNA duplications and deletions (Figure 10.22).

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(a)

(b)

(c)

FIGURE 12.13 The inactive X chromosome: an example of facultative heterochromatin. (a) The inactivated X chromosome in the nucleus of a womans cells appears as a darkly staining heterochromatic structure, called a Barr body (arrows). (b) A calico cat. Random inactivation of either X chromosome in different cells during early embryonic development creates a mosaic of tissue patches. Each patch comprises the descendants of one cell that was present in the embryo at the time of inactivation. These patches are visually evident in calico cats, which are heterozygotes with an allele for black coat color residing on one X chromosome and an allele

for orange coat color on the other X. This explains why male calico cats are virtually nonexistent: because all cells in the male have either the black or orange coat color allele. (The white spots on this cat are due to a different, autosomal coat color gene.) (c) This kitten was cloned from the cat shown in b. The two animals are genetically identical but have different coat patterns, a reection of the random nature of the X inactivation process (and likely other random developmental events). (A: COURTESY OF
AND

MURRAY L. BARR; B,C: COURTESY OF COLLEGE OF VETERINARY MEDICINE BIOMEDICAL SCIENCES, TEXAS A&M UNIVERSITY.)

Unlike the constitutive variety, facultative heterochromatin is chromatin that has been specically inactivated during certain phases of an organisms life or in certain types of differentiated cells (as in Figure 17.9b). An example of facultative heterochromatin can be seen by comparing cells of a female mammal to those of a male. The cells of males have a tiny Y chromosome and a much larger X chromosome. Because the X and Y chromosomes have only a few genes in common, males have a single copy of most genes that are carried on the sex chromosomes. Although cells of females contain two X chromosomes, only one of them is transcriptionally active. The other X chromosome remains condensed as a heterochromatic clump (Figure 12.13a) called a Barr body after the researcher who discovered it in 1949. Formation of a Barr body ensures that the cells of both males and females have the same number of active X chromosomes and thus synthesize equivalent amounts of the products encoded by X-linked genes. X Chromosome Inactivation Based on her studies of the inheritance of coat color in mice, the British geneticist Mary Lyon proposed the following in 1961:
1. Heterochromatinization of the X chromosome in female mammals occurs during early embryonic development and leads to the inactivation of the genes on that chromosome. 2. Heterochromatinization in the embryo is a random process in the sense that the paternally derived X chromosome and the maternally derived X chromosome stand an equal chance of becoming inactivated in any given cell. Consequently, at the time of inactivation, the paternal X

can be inactivated in one cell of the embryo, and the maternal X can be inactivated in a neighboring cell. Once an X chromosome has been inactivated, its heterochromatic state is transmitted through many cell divisions, so that the same X chromosome is inactive in all the descendants of that particular cell. 3. Reactivation of the heterochromatinized X chromosome occurs in germ cells prior to the onset of meiosis. Consequently, both X chromosomes are active during oogenesis, and all of the gametes receive a euchromatic X chromosome. The Lyon hypothesis was soon conrmed.1 Because maternally and paternally derived X chromosomes may contain different alleles for the same trait, adult females are in a sense genetic mosaics, where different alleles function in different cells. X-chromosome mosaicism is reected in the patchwork coloration of the fur of some mammals, including calico cats (Figure 12.13b,c). Pigmentation genes in humans are not located on the X chromosome, hence the absence of calico women. Mosaicism due to X inactivation can be demonstrated in women, nonetheless. For example, if a narrow beam of red or green light is shone into the eyes of a woman who is
1

The random inactivation of X chromosomes discussed here, which occurs after the embryo implants in the uterus, is actually the second wave of X chromosome inactivation to occur in the embryo. The rst wave, which occurs very early in development, is not random but rather leads only to the inactivation of X chromosomes that had been donated by the father. This early inactivation of paternal X chromosomes is maintained in the cells that give rise to extraembryonic tissues (e.g., the placenta) and is not discussed in the text. Early paternal X inactivation is erased in cells that give rise to embryonic tissue and random X inactivation subsequently occurs.

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a heterozygous carrier for red-green color blindness, patches of retinal cells with defective color vision can be found interspersed among patches with normal vision. The mechanism responsible for X inactivation has been a focus of attention since a 1992 report suggesting that inactivation is initiated by a noncoding RNA moleculerather than a proteinthat is transcribed from one of the genes (called XIST in humans) on the X chromosome that becomes inactivated. The XIST RNA is a large transcript (over 17 kb long), which distinguishes it from many other noncoding RNAs that tend to be quite small. The XIST RNA does not diffuse into the nucleoplasm, but accumulates along the length of the chromosome just before it is inactivated.2 The XIST gene is required to initiate inactivation, but not to maintain it from one cell generation to the next. This conclusion is based on the discovery of tumor cells in certain women that contain an inactivated X chromosome whose XIST gene is deleted. X inactivation is thought to be maintained by DNA methylation (page 520) and repressive histone modications, as discussed in the next section.
The Histone Code and Formation of Heterochromatin Figure 12.9c shows a schematic model of the nucleosome core particle with its histone tails projecting outward. But this is only a general portrait that obscures important differences among nucleosomes. Cells contain a remarkable array of enzymes that are able to add chemical groups to or remove them from specic amino acid residues in the histone tails. Those residues that are subject to modication, most notably by methylation, acetylation, or phosphorylation, are indicated by the colored bars in Figure 12.14. The past few years has seen the emergence of a hypothesis known as the histone code, which postulates that the state and activity of a particular region of chromatin depend on the specic modications, or combinations of modications, to the histone tails in that region. In other words, the pattern of modications adorning the tails of the core histones contains encoded information governing the properties of those nucleosomes. Studies suggest that histone tail modications act in two ways to inuence chromatin structure and function. 1. The modied residues serve as docking sites to recruit a specic array of nonhistone proteins, which then determine the properties and activities of that segment of chromatin. A sampling of some of the specic proteins that bind selectively to modied histone residues is depicted in Figure 12.15. Each of the proteins bound to the histones in Figure 12.15 is capable of modulating some aspect of chromatin activity or structure. 2. The modied residues alter the manner in which the histone tails of neighboring nucleosomes interact with one another or with the DNA to which the nucleosomes are bound. Changes in these types of interactions can lead to changes in the higher order structure of chromatin.
2 Approximately 15 percent of genes on the chromosome escape inactivation by an unknown mechanism. The escapees include genes that are also present on the Y chromosome, which ensures that they are expressed equally in both sexes.

H2B

Acetyl Methyl Phosphoryl

Me-Lys P Ac 14 17 Me-Arg 23 Ac 10 9

H3
4

Ac

12 Ac 15 Ac
Me-Arg 26 P 28 27 36

Ac

18

Me-Lys Ac

20 Ac

Me-Lys

Me-Lys

R
Me-Lys 16

20 Ac Ac P 1
H2A

R
Ac 8 Ac

Ac 12

5 Ac 3 Me-Arg 1 P

H4

FIGURE 12.14 Histone modications and the histone code. Histones can be enzymatically modied by the covalent addition of methyl, acetyl, and phosphate groups (and others not discussed). This illustration indicates the positions in the N-terminal tails of the four core histones at which each of these three groups can be added. Methyl groups are added to either lysine or arginine residues, acetyl groups to lysine residues, and phosphate groups to serine residues. The matter is even more complex, because each lysine residue can have either one, two, or three added methyl groups, and each arginine residue can have either one or two added methyl groups. The number of added methyl groups can affect the afnity of the residue for an interacting protein. Unless otherwise noted, we will restrict the discussion to tri-methylated lysine residues (e.g., H3K9me3 or H3K36me3). Certain modications are associated with particular chromatin activities, which has led to the concept of a histone code. The present discussion is restricted to the lysine residues, which are best understood. The red letters A and R represent transcriptional activation and repression, respectively. Acetylation of lysines on both histones H3 and H4 is closely correlated with transcriptional activation. The effects of methylation of H3 and H4 lysines depends strongly on which of these residues is modied. For example, methylation of lysine 9 of histone H3 (i.e., H3K9) is typically present in heterochromatin and associated with transcriptional repression, as discussed in the text. Methylation of H3K27 and H4K20 is also strongly associated with transcriptional repression, whereas methylation of H3K4 and H3K36 is associated with activation. Just as there are enzymes that add each of these groups, there are also enzymes (deacetylases, demethylases, and phosphatases) that specically remove them. (C: AFTER G. FELSENFELD
& M. GROUDINE, REPRINTED WITH PERMISSION FROM NATURE 421:450, 2003; COPYRIGHT 2003, MACMILLAN MAGAZINES LIMITED.)

Acetylation of the lysine residue at position 16 on histone H4, for example, interferes with the formation of the compact 30-nm chromatin ber. For the moment, we will restrict the discussion to the formation of heterochromatin as it occurs, for example, during

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Chapter 12 THE CELL NUCLEUS AND THE CONTROL OF GENE EXPRESSION

BPTF CHD1 ING2

Brd2 HP1 14-3-3 Rsc4 PC EAF3 CRB2 JMJD2 Ac K 16 12 8 K K Ac Ac Taf1 Bdf1

Me H3 K 4

Me K 9

P S 10

Ac K 14

Me K 27

Me K 36

Me K 20

H4

FIGURE 12.15 Examples of proteins that bind selectively to modied

H3 or H4 residues. Each of the bound proteins possesses an activity that alters the structure and/or function of the chromatin. There is an added complexity that is not shown in this drawing in that modications at one histone residue can inuence events at other residues, a

phenomenon known as cross-talk. For example, the binding of the heterochromatin protein HP1 to H3K9 is blocked by phosphorylation of the adjacent serine residue (H3S10), which typically occurs during mitosis. (FROM T. KOUZARIDES, CELL 128:696, 2007, BY PERMISSION OF
CELL PRESS.)

X chromosome inactivation. For the sake of simplicity, we will focus on modication of a single residuelysine 9 of H3which will illustrate the general principles by which cells utilize the histone code. The actions of several other histone modications are indicated in Figure 12.14 and discussed in the accompanying legend, and another example is described on page 517. As we will see throughout this chapter, techniques have been developed in recent years to analyze changes that affect genome transcription, such as histone modications, on a genome-wide level, rather than simply looking at these changes one gene at a time. This has given us a much broader view of the general importance of each of these phenomena than was possible only a few years ago. Comparison of the nucleosomes present within heterochromatic versus euchromatic chromatin domains revealed

FIGURE 12.16 Experimental demonstration of a correlation between

transcriptional activity and histone acetylation. This metaphase chromosome spread has been labeled with uorescent antibodies to acetylated histone H4, which uoresce green. It is evident that all of the chromosomes except the inactivated X stain brightly with the antibody against the acetylated histone. (FROM P. JEPPESEN AND B. M. TURNER,
COVER OF

CELL VOL. 74, NO. 2, 1993; BY PERMISSION OF CELL PRESS.)

a striking difference. The lysine residue at the #9 position (Lys9 or K9) of the H3 histone in heterochromatic domains is largely methylated, whereas this same residue in euchromatic domains tends to be unmethylated, although it is often acetylated. Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin. The correlation between transcriptional repression and histone deacetylation can be seen by comparing the inactive, heterochromatic X chromosome of female cells, which contains deacetylated histones, to the active, euchromatic X chromosome, whose histones exhibit a normal level of acetylation (Figure 12.16). Histone deacetylation is accompanied by methylation of H3K9, which is catalyzed by an enzyme (a histone methyltransferase) that appears to be dedicated to this, and only this, particular function. This enzyme, called SUV39H1 in humans, can be found localized within heterochromatin, where it may stabilize the heterochromatic nature of the region through its methylation activity. The formation of a methylated lysine at the #9 position endows the histone H3 tail with an important property: it becomes capable of binding with high afnity to proteins that contain a particular domain, called a chromodomain. The human genome contains at least 30 proteins with chromodomains, the best studied of which is heterochromatic protein 1 (or HP1). HP1 has been implicated in the formation and maintenance of heterochromatin. Once bound to an H3 tail, HP1 is thought to interact with other proteins, including (1) SUV39H1, the enzyme responsible for methylating the H3K9 residue and (2) other HP1 molecules on nearby nucleosomes. These binding properties of the HP1 molecule promote the formation of an interconnecting network of methylated nucleosomes, leading to a compacted, higherorder chromatin domain. Most importantly, this state is transmitted through cell divisions from one cell generation to the next (discussed on page 497). Studies in a number of organisms indicate that small RNAs, similar in nature to those involved in RNA interference (page 449), play an important role in targeting a particular region of the genome to undergo H3K9 methylation and subsequent heterochromatinization. If, for example, compo-