12.1 THE NUCLEUS OF A EUKARYOTIC CELL 481

which binds to the complex and causes its disassembly as in-dicated in step 4, Figure 12.7a. This is the apparent functionof the high level of Ran-GTP in the nucleus: it promotes thedisassembly of complexes imported from the cytoplasm. Theimported cargo is released into the nucleoplasm, and one por-tion of the NLS receptor (the importin b subunit) is shuttledback to the cytoplasm together with the bound Ran-GTP(step 5). Once in the cytoplasm, the GTP molecule boundto Ran is hydrolyzed, releasing Ran-GDP from the importinb subunit. Ran-GDP is returned to the nucleus, where it isconverted back to the GTP-bound state for additional roundsof activity. Importin a is transported back to the cytoplasm byone of the exportins.

Ran-GTP plays a key role in the escort of macromole-cules from the nucleus, just as it does in their import from thecytoplasm. Recall that Ran-GTP is essentially confined to thenucleus. Whereas Ran-GTP induces the disassembly of im-ported complexes, as shown in step 4 of Figure 12.7a, Ran-GTP promotes the assembly of exported complexes. Proteinsexported from the nucleus contain amino acid sequences(called nuclear export signals, or NESs) that are recognized bytransport receptors that carry them through the nuclear enve-lope to the cytoplasm. Most of the traffic moving in this direc-tion consists of various types of RNA molecules—especiallymRNAs, rRNAs, and tRNAs—that are synthesized in thenucleus and function in the cytoplasm. In most cases, theseRNAs move through the NPC as ribonucleoproteins (RNPs).

Transport of an mRNP from the nucleus to cytoplasmis associated with extensive remodeling; certain proteins arestripped from the mRNA, while others are added to the com-plex. Transport of mRNPs does not appear to require Ran butdoes require the activity of an RNA helicase located on the cy-toplasmic filaments of the NPC. It is speculated that the heli-case provides the motive force to move the mRNA into thecytoplasm. Numerous studies have demonstrated a functionallink between pre-mRNA splicing and mRNA export; onlymature (i.e., fully processed) mRNAs are capable of nuclearexport. If an mRNA still contains an unspliced intron, thatRNA is retained in the nucleus.

Chromosomes and Chromatin

Chromosomes seem to appear out of nowhere at the begin-ning of mitosis and disappear once again when cell divisionhas ended. The appearance and disappearance of chromo-somes 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 tothis question.

Packaging the Genome An average human cell containsabout 6.4 billion base pairs of DNA divided among 46 chro-mosomes (the value for a diploid, unreplicated number ofchromosomes). Each unreplicated chromosome contains asingle, continuous DNA molecule; the larger the chromo-some, the longer the DNA it contains. Given that each basepair is about 0.34 nm in length, 6 billion base pairs would con-stitute a DNA molecule fully 2 m long. How is it possible to

fit 2 meters of DNA into a nucleus only 10 mm (1 3 1025 m)in diameter and, at the same time, maintain the DNA in astate that is accessible to enzymes and regulatory proteins?Just as important, how is the single DNA molecule of eachchromosome organized so that it does not become hopelesslytangled with the molecules of other chromosomes? The an-swers lie in the remarkable manner in which a DNA moleculeis packaged.

Nucleosomes: The Lowest Level of Chromosome OrganizationChromosomes are composed of DNA and associated protein,which together is called chromatin. The orderly packagingof eukaryotic DNA depends on histones, a remarkable groupof small proteins that possess an unusually high content ofthe basic amino acids arginine and lysine. Histones are di-vided into five classes, which can be distinguished by theirarginine/lysine ratio (Table 12.1). The amino acid sequencesof histones, particularly H3 and H4, have undergone verylittle change over long periods of evolutionary time. The H4histones of both peas and cows, for example, contain 102 aminoacids, and their sequences differ at only 2 amino acid residues.Why are histones so highly conserved? One reason is histonesinteract with the backbone of the DNA molecule, which isidentical in all organisms. In addition, nearly all of the aminoacids in a histone molecule are engaged in an interaction withanother molecule, either DNA or another histone. As a result,very few amino acids in a histone can be replaced with otheramino acids without severely affecting the function of theprotein.

In the early 1970s, it was found that when chromatin wastreated with nonspecific nucleases, most of the DNA was con-verted to fragments of approximately 200 base pairs in length.In contrast, a similar treatment of naked DNA (i.e., DNA de-void of proteins) produced a randomly sized population offragments. This finding suggested that chromosomal DNAwas protected from enzymatic attack, except at certain peri-odic sites along its length. It was presumed that the proteinsassociated with the DNA were providing the protection. In1974, using the data from nuclease digestion and other typesof information, Roger Kornberg, then at Harvard University,proposed an entirely new structure for chromatin. Kornbergproposed that DNA and histones are organized into repeatingsubunits, called nucleosomes. We now know that each nucleo-

TABLE 12.1 Calf Thymus Histones

Number UEP*

of Mass (3 1026

Histone residues (kDa) %Arg %Lys year)

H1 215 23.0 1 29 8H2A 129 14.0 9 11 60H2B 125 13.8 6 16 60H3 135 15.3 13 10 330H4 102 11.3 14 11 600

*Unit evolutionary period: the time for a protein’s amino acid sequence tochange by 1 percent after two species have diverged.

482 Chapter 12 THE CELL NUCLEUS AND THE CONTROL OF GENE EXPRESSION

some contains a nucleosome core particle consisting of 146 basepairs of supercoiled DNA (page 390) wrapped almost twicearound a disk-shaped complex of eight histone molecules(Figure 12.8a). The histone core of each nucleosome consistsof two copies each of histones H2A, H2B, H3, and H4 as-sembled into an octamer, as discussed below. The remaininghistone—type H1—resides outside the nucleosome core par-ticle. The H1 histone is referred to as a linker histone becauseit binds to part of the linker DNA that connects one nucleo-some core particle to the next. Fluorescence studies indicatethat H1 molecules continuously dissociate and reassociatewith chromatin. Together the H1 protein and the histone oc-tamer interact with about 168 base pairs of DNA. H1 histonemolecules can be selectively removed from the chromatinfibers by subjecting the preparation to solutions of low ionicstrength. When H1-depleted chromatin is observed underthe electron microscope, the nucleosome core particles andnaked linker DNA can be seen as separate elements, which to-gether appear like “beads on a string” (Figure 12.8b).

Our understanding of DNA packaging has been greatlyadvanced in recent years by dramatic portraits of the nucleo-some core particle obtained by X-ray crystallography (Fig-ure 12.9). The eight histone molecules that comprise anucleosome core particle are organized into four heterodimers:two H2A-H2B dimers and two H3-H4 dimers (Figure12.9a,c). Dimerization of histone molecules is mediated bytheir C-terminal domains, which consist largely of a helices(represented by the cylinders in Figure 12.9c) folded into acompact 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, flexi-ble tail (represented by the dashed lines of Figure 12.9c) thatextends past the DNA helix and into the surroundings. These

tails are targets of a variety of covalent modifications whosekey functions will be explored later in the chapter.

Histone modification is not the only mechanism to alterthe histone character of nucleosomes. In addition to the four“conventional” core histones discussed above, several alternateversions of the H2A and H3 histones are also synthesized inmost cells. The importance of these histone variants, as theyare called, remains largely unexplored, but they are thought tohave specialized functions (Table 12.2). The localization andapparent function of one of these variants, CENP-A, is dis-cussed on page 496. Another variant, H2A.X, is distributedthroughout the chromatin, where it replaces conventionalH2A in a fraction of the nucleosomes. H2A.X becomes phos-phorylated at sites of DNA-strand breakage and may play arole in recruiting the enzymes that repair the DNA. Two othercore histone variants—H2A.Z and H3.3—can be incorpo-rated into nucleosomes of genes as they become activated andmay play a role in promoting the transcription of that geneticlocus.

TABLE 12.2 Histone Variants

Type Variant Location Likely function

H2AH2A.X Throughout chromatin DNA repairH2A.Z Euchromatin TranscriptionmacroH2A Inactive X chromosome Transcriptional

silencingH3

CENP-A Centromeres Kinetochoreassembly

H3.3 Transcribed loci Transcription

Histone

octamer

DNA

(a)

H1

(b)

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

FIGURE 12.8 Nucleosomal organization of chromatin. (a) Schematicdiagram showing the structure of a nucleosome core particle and an as-sociated histone H1 molecule. The core particle itself consists of approx-imately 1.8 turns (146 base pairs) of negatively supercoiled DNAwrapped around eight core histone molecules (two each of H2A, H2B,H3, and H4). The H1 linker histone binds near the sites where DNA

12.1 THE NUCLEUS OF A EUKARYOTIC CELL 483

DNA and core histones are held together by severaltypes of noncovalent bonds, including ionic bonds betweennegatively charged phosphates of the DNA backbone andpositively charged residues of the histones. The two mole-cules make contact at sites where the minor groove of theDNA faces inward toward the histone core, which occurs atapproximately 10 base-pair intervals (the white hooks in Fig-ure 12.9c). In between these points of contact, the two mole-cules are seen to be separated by considerable space, whichmight provide access to the DNA for transcription factors andother DNA-binding proteins. For many years, histones werethought of as inert, structural molecules but, as we will see infollowing sections, these small proteins play critically impor-tant roles in determining the activity of the DNA with whichthey are associated. It has also become evident that chromatinis a dynamic cellular component in which histones, regulatoryproteins, and a myriad variety of enzymes move in and out ofthe nucleoprotein complex to facilitate the complex tasks ofDNA transcription, compaction, replication, recombination,and repair.

We began this section by wondering how a nucleus 10 mmin diameter can pack 200,000 times this length of DNA withinits boundaries. The assembly of nucleosomes is the first im-portant step in the compaction process. With a nucleotide–

nucleotide spacing of 0.34 nm, the 200 base pairs of a single10-nm nucleosome would stretch nearly 70 nm if fully ex-tended. Consequently, it is said that the packing ratio of theDNA of nucleosomes is approximately 7:1.

Higher Levels of Chromatin Structure A DNA moleculewrapped around nucleosome core particles of 10-nm diameteris the lowest level of chromatin organization. Chromatin doesnot, however, exist within the cell in this relatively extended,“beads-on-a-string” state. When chromatin is released fromnuclei and prepared at physiologic ionic strength, a fiber ofapproximately 30-nm thickness is observed (Figure 12.10a).Despite more than two decades of investigation, the structureof the 30-nm fiber remains a subject of debate. Two models inwhich the nucleosomal filament is coiled into the higher-order, thicker fiber are shown in Figure 12.10b,c. The modelsdiffer in the relative positioning of nucleosomes within thefiber. Recent research favors the “zig-zag” model depicted inFigure 12.10b, in which successive nucleosomes along theDNA are arranged in different stacks and alternating nucleo-somes become interacting neighbors. Regardless of how it isaccomplished, the assembly of the 30-nm fiber increases theDNA-packing ratio an additional 6-fold, or about 40-foldaltogether.

(a) (b)

H3

H4

H2A

H2B

N

C

C

N

N

H4

H2B

H3'H3

aNaN

aC

2

1

3

4

5

6

7

0

b

(c)

N

FIGURE 12.9 The three-dimensional structure of a nucleosome asrevealed by X-ray crystallography. (a) A nucleosome core particleviewed down the central axis of the DNA superhelix, showing the posi-tion of each of the eight histone molecules of the core octamer. Thehistones are seen to be organized into four dimeric complexes. Each hi-stone dimer binds 27 to 28 base pairs of DNA, with contacts occurringwhere the minor groove of the DNA faces the histone core. (b) The diskshape of the nucleosome core particle is evident in this view perpendicu-lar to the central axis. The two H3-H4 dimers are associated with oneanother in the center of the core particle to form a tetramer, whereas thetwo H2A-H2B dimers are positioned on each side of the (H3-H4)2

tetramer. (c) A simplified, schematic model of half of a nucleosome coreparticle, showing one turn (73 base pairs) of the DNA superhelix and

four core histone molecules. The four different histones are shown inseparate colors, as indicated by the key. Each core histone is seen to con-sist of (1) a globular region, called the “histone fold,” consisting of threea helices (represented by the cylinders) and (2) a flexible, extendedN-terminal tail (indicated by the letter N) that projects out of the his-tone disk and past the DNA double helix. The intermittent points ofinteraction between the histone molecules and the DNA are indicatedby white hooks. The dashed lines indicate the outermost portion of thehistone tails; these flexible tails lack a defined tertiary structure andtherefore 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.)

484 Chapter 12 THE CELL NUCLEUS AND THE CONTROL OF GENE EXPRESSION

Maintenance of the 30-nm fiber depends on the interac-tion between histone molecules of neighboring nucleosomes.Linker histones and core histones have both been implicatedin higher-order packaging of chromatin. If, for example, H1linker histones are selectively extracted from compacted chro-matin, the 30-nm fibers uncoil to form the thinner, more ex-tended beaded filament shown in Figure 12.8b. Adding backH1 histone leads to restoration of the higher-order structure.Core histones of adjacent nucleosomes may interact with oneanother by means of their long, flexible tails. Structural stud-ies indicate, for example, that the N-terminal tail of an H4histone from one nucleosome core particle can reach out andmake extensive contact with both the linker DNA betweennucleosome particles and the H2A/H2B dimer of adjacentparticles. These types of interactions are thought to mediatethe folding of the nucleosomal filament into a thicker fiber. Infact, chromatin fibers prepared with H4 histones that lacktheir tails are unable to fold into higher-order fibers.

The next stage in the hierarchy of DNA packaging isthought to occur as the 30-nm chromatin fiber is gatheredinto a series of large, supercoiled loops, or domains, thatmay be compacted into even thicker (80–100 nm) fibers.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 atype II topoisomerase that presumably regulates the degree ofDNA supercoiling. The topoisomerase would also be expectedto untangle the DNA molecules of different loops should theybecome intertwined. Normally, loops of chromatin fibers arespread out within the nucleus and cannot be visualized, buttheir presence can be revealed under certain circumstances.For instance, when isolated mitotic chromosomes are treatedwith solutions that extract histones, the histone-free DNAcan be seen to extend outward as loops from a protein scaffold(Figure 12.11).

The mitotic chromosome represents the ultimate in chro-matin compactness; 1 mm of mitotic chromosome length typ-ically contains approximately 1 cm of DNA, which representsa packing ratio of 10,000:1. This compaction occurs by apoorly understood process that is discussed in Section 14.2.An overview of the various levels of chromatin organization,from the nucleosomal filament to a mitotic chromosome, isdepicted in Figure 12.12.

Heterochromatin and Euchromatin After mitosis has beencompleted, most of the chromatin in highly compacted mi-

FIGURE 12.10 The 30-nm fiber: a higherlevel of chromatin structure. (a) Elec-tron micrograph of a 30-nm chromatinfiber released from a nucleus followinglysis of the cell in a hypotonic salt solu-tion. (b) In the “zig-zag” model, thelinker DNA is present in a straight,extended state that criss-crosses backand forth between consecutive core par-ticles, which are organized into twoseparate stacks of nucleosomes. Thelower portion of the figure shows howthe two stacks of nucleosomes are coiledinto a higher-order helical structure.(c) In the “solenoid” model, the linkerDNA gently curves as it connects con-secutive core particles, which are organ-ized into a single, continuous helicalarray containing about 6–8 nucleosomesper turn. In these models, the histoneoctamer is shown in blue, the DNA inmagenta, and the linker H1 histone inyellow. (A: COURTESY OF BARBARA

HAMKALO AND JEROME B. RATTNER; B,C:

FROM SEPIDEH KHORASANIZADEH, CELL

116:262, 2004; BY PERMISSION OF CELL

PRESS.)

30nm

Fiber

30nm

Fiber

(a) (b) (c)

12.1 THE NUCLEUS OF A EUKARYOTIC CELL 485

totic chromosomes returns to its diffuse interphase condition.Approximately 10 percent of the chromatin, however, gener-ally remains in a condensed, compacted form throughout in-terphase. This compacted, densely stained chromatin is seenat the periphery of the nucleus in Figure 12.1a. Chromatinthat remains compacted during interphase is called hete-rochromatin to distinguish it from euchromatin, which re-turns to a dispersed state. When a radioactively labeled RNAprecursor such as [3H]uridine is given to cells that are subse-quently fixed, sectioned, and autoradiographed, the clumps ofheterochromatin remain largely unlabeled, indicating thatthey have relatively little transcriptional activity. The state of aparticular region of the genome, whether it is euchromatic orheterochromatic, is stably inherited from one cell generationto the next.

Heterochromatin is divided into two classes. Constitu-tive heterochromatin remains in the compacted state in allcells at all times and, thus, represents DNA that is perma-nently silenced. In mammalian cells, the bulk of the constitu-tive heterochromatin is found in the region that flanks thetelomeres and centromere of each chromosome and in a fewother sites, such as the distal arm of the Y chromosome inmale mammals. The DNA of constitutive heterochromatinconsists primarily of repeated sequences (page 394) and con-

tains relatively few genes. In fact, when genes that are nor-mally active move into a position adjacent to heterochromatin(having changed position as the result of transposition ortranslocation), they tend to become transcriptionally silenced,a phenomenon known as position effect. It is thought that het-erochromatin contains components whose influence canspread outward a certain distance, affecting nearby genes. Thespread of heterochromatin along the chromosome is appar-ently blocked by specialized barrier sequences (boundary ele-ments) in the genome. Constitutive heterochromatin alsoserves to inhibit genetic recombination between homologousrepetitive sequences. This type of recombination can lead toDNA duplications and deletions (Figure 10.22).

ScaffoldScaffold

DNA double helix(2 nm in diameter)

Nucleosomecore particle

DNA H1 histone

Core histones(8 subunits)

Nucleosome filament(10 nm in diameter)

30 nm fiber

Looped domains

Protein scaffoldMetaphasechromosome

FIGURE 12.11 Chromatin loops: a higher level of chromatin structure.Electron micrograph of a mitotic chromosome that had been treatedwith a solution of dextran sulfate to remove histones. The histone-depleted chromosome displays loops of DNA that are attached at theirbases to a residual protein scaffold. (FROM JAMES R. PAULSON AND U. K.

LAEMMLI, CELL 12:823, 1977; BY PERMISSION OF CELL PRESS.)

FIGURE 12.12 Levels of organization of chromatin. Naked DNA mol-ecules are wrapped around histones to form nucleosomes, which repre-sent the lowest level of chromatin organization. Nucleosomes areorganized into 30-nm fibers, which in turn are organized into loopeddomains. When cells prepare for mitosis, the loops become further com-pacted into mitotic chromosomes (see Figure 14.13).

486 Chapter 12 THE CELL NUCLEUS AND THE CONTROL OF GENE EXPRESSION

Unlike the constitutive variety, facultative heterochro-matin is chromatin that has been specifically inactivated dur-ing certain phases of an organism’s life or in certain types ofdifferentiated cells (as in Figure 17.9b). An example of facul-tative heterochromatin can be seen by comparing cells of afemale mammal to those of a male. The cells of males have atiny Y chromosome and a much larger X chromosome. Be-cause the X and Y chromosomes have only a few genes incommon, males have a single copy of most genes that are car-ried on the sex chromosomes. Although cells of females con-tain two X chromosomes, only one of them is transcriptionallyactive. The other X chromosome remains condensed as aheterochromatic clump (Figure 12.13a) called a Barr bodyafter the researcher who discovered it in 1949. Formation ofa Barr body ensures that the cells of both males and femaleshave the same number of active X chromosomes and thussynthesize equivalent amounts of the products encoded by X-linked genes.

X Chromosome Inactivation Based on her studies of the in-heritance of coat color in mice, the British geneticist MaryLyon proposed the following in 1961:

1. Heterochromatinization of the X chromosome in femalemammals occurs during early embryonic development andleads to the inactivation of the genes on that chromosome.

2. Heterochromatinization in the embryo is a randomprocess in the sense that the paternally derived X chro-mosome and the maternally derived X chromosome standan 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 ma-ternal X can be inactivated in a neighboring cell. Once anX chromosome has been inactivated, its heterochromaticstate is transmitted through many cell divisions, so thatthe same X chromosome is inactive in all the descendantsof that particular cell.

3. Reactivation of the heterochromatinized X chromosomeoccurs in germ cells prior to the onset of meiosis.Consequently, both X chromosomes are active duringoogenesis, and all of the gametes receive a euchromaticX chromosome.

The Lyon hypothesis was soon confirmed.1 Because ma-ternally and paternally derived X chromosomes may containdifferent alleles for the same trait, adult females are in a sensegenetic mosaics, where different alleles function in differentcells. X-chromosome mosaicism is reflected in the patchworkcoloration of the fur of some mammals, including calico cats(Figure 12.13b,c). Pigmentation genes in humans are notlocated on the X chromosome, hence the absence of “calicowomen.” Mosaicism due to X inactivation can be demon-strated in women, nonetheless. For example, if a narrow beamof red or green light is shone into the eyes of a woman who is

(a) (b) (c)

FIGURE 12.13 The inactive X chromosome: an example of facultativeheterochromatin. (a) The inactivated X chromosome in the nucleus of awoman’s cells appears as a darkly staining heterochromatic structure, calleda Barr body (arrows). (b) A calico cat. Random inactivation of either Xchromosome in different cells during early embryonic development cre-ates a mosaic of tissue patches. Each patch comprises the descendants ofone cell that was present in the embryo at the time of inactivation. Thesepatches are visually evident in calico cats, which are heterozygotes with anallele 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 catsare virtually nonexistent: because all cells in the male have either theblack or orange coat color allele. (The white spots on this cat are due toa different, autosomal coat color gene.) (c) This kitten was cloned fromthe cat shown in b. The two animals are genetically identical but have dif-ferent coat patterns, a reflection of the random nature of the X inactivationprocess (and likely other random developmental events). (A: COURTESY OF

MURRAY L. BARR; B,C: COURTESY OF COLLEGE OF VETERINARY MEDICINE

AND BIOMEDICAL SCIENCES, TEXAS A&M UNIVERSITY.)

1The random inactivation of X chromosomes discussed here, which occursafter the embryo implants in the uterus, is actually the second wave of X chro-mosome inactivation to occur in the embryo. The first wave, which occurs veryearly in development, is not random but rather leads only to the inactivationof X chromosomes that had been donated by the father. This early inactivationof paternal X chromosomes is maintained in the cells that give rise to extra-embryonic 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 andrandom X inactivation subsequently occurs.

12.1 THE NUCLEUS OF A EUKARYOTIC CELL 487

a heterozygous carrier for red-green color blindness, patchesof retinal cells with defective color vision can be found inter-spersed among patches with normal vision.

The mechanism responsible for X inactivation has been afocus of attention since a 1992 report suggesting that inactiva-tion is initiated by a noncoding RNA molecule—rather than aprotein—that is transcribed from one of the genes (calledXIST in humans) on the X chromosome that becomes inacti-vated. The XIST RNA is a large transcript (over 17 kb long),which distinguishes it from many other noncoding RNAsthat tend to be quite small. The XIST RNA does not diffuseinto the nucleoplasm, but accumulates along the length of thechromosome just before it is inactivated.2 The XIST gene is re-quired to initiate inactivation, but not to maintain it from onecell generation to the next. This conclusion is based on thediscovery of tumor cells in certain women that contain an in-activated X chromosome whose XIST gene is deleted. X inac-tivation is thought to be maintained by DNA methylation(page 520) and repressive histone modifications, as discussedin the next section.

The Histone Code and Formation of Heterochromatin

Figure 12.9c shows a schematic model of the nucleosome coreparticle with its histone tails projecting outward. But this isonly a general portrait that obscures important differencesamong nucleosomes. Cells contain a remarkable array of en-zymes that are able to add chemical groups to or remove themfrom specific amino acid residues in the histone tails. Thoseresidues that are subject to modification, most notably bymethylation, acetylation, or phosphorylation, are indicatedby the colored bars in Figure 12.14. The past few years hasseen the emergence of a hypothesis known as the histone

code, which postulates that the state and activity of a particu-lar region of chromatin depend on the specific modifications,or combinations of modifications, to the histone tails in thatregion. In other words, the pattern of modifications adorningthe tails of the core histones contains encoded informationgoverning the properties of those nucleosomes. Studies sug-gest that histone tail modifications act in two ways to influ-ence chromatin structure and function.

1. The modified residues serve as docking sites to recruit aspecific array of nonhistone proteins, which then deter-mine the properties and activities of that segment ofchromatin. A sampling of some of the specific proteinsthat bind selectively to modified histone residues is de-picted in Figure 12.15. Each of the proteins bound to thehistones in Figure 12.15 is capable of modulating someaspect of chromatin activity or structure.

2. The modified residues alter the manner in which the his-tone tails of neighboring nucleosomes interact with oneanother or with the DNA to which the nucleosomes arebound. Changes in these types of interactions can leadto changes in the higher order structure of chromatin.

Acetylation of the lysine residue at position 16 on histoneH4, for example, interferes with the formation of thecompact 30-nm chromatin fiber.

For the moment, we will restrict the discussion to the for-mation of heterochromatin as it occurs, for example, during

2Approximately 15 percent of genes on the chromosome escape inactivation byan unknown mechanism. The “escapees” include genes that are also present onthe Y chromosome, which ensures that they are expressed equally in both sexes.

Me-Lys

P

Ac

Ac

Me-Arg

Methyl

Phosphoryl

Acetyl

Me-Lys

P

Me-Lys

Me-Lys

Me-Arg

Ac

Ac

4

10

14

18

2628

36

9

17

23

Me-Arg

Ac

Ac

Ac

Ac

Ac

Ac

Ac

P

P

Ac

Ac

Ac

8

12

16

20 Me-Lys

1

1

5

5

9

20

12

5

15

3

27A

A

A

R

R

R

H3

H2B

H2AH4

FIGURE 12.14 Histone modifications and the “histone code.” Histonescan be enzymatically modified by the covalent addition of methyl, acetyl,and phosphate groups (and others not discussed). This illustration indi-cates the positions in the N-terminal tails of the four core histones atwhich each of these three groups can be added. Methyl groups are addedto either lysine or arginine residues, acetyl groups to lysine residues, andphosphate groups to serine residues. The matter is even more complex,because each lysine residue can have either one, two, or three addedmethyl groups, and each arginine residue can have either one or twoadded methyl groups. The number of added methyl groups can affect theaffinity 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 modifications are associated withparticular chromatin activities, which has led to the concept of a histonecode. The present discussion is restricted to the lysine residues, whichare best understood. The red letters A and R represent transcriptionalactivation and repression, respectively. Acetylation of lysines on bothhistones H3 and H4 is closely correlated with transcriptional activation.The effects of methylation of H3 and H4 lysines depends strongly onwhich of these residues is modified. For example, methylation of lysine 9of histone H3 (i.e., H3K9) is typically present in heterochromatin andassociated with transcriptional repression, as discussed in the text.Methylation of H3K27 and H4K20 is also strongly associated withtranscriptional repression, whereas methylation of H3K4 and H3K36 isassociated with activation. Just as there are enzymes that add each ofthese groups, there are also enzymes (deacetylases, demethylases, andphosphatases) that specifically remove them. (C: AFTER G. FELSENFELD

& M. GROUDINE, REPRINTED WITH PERMISSION FROM NATURE 421:450,

2003; © COPYRIGHT 2003, MACMILLAN MAGAZINES LIMITED.)

488 Chapter 12 THE CELL NUCLEUS AND THE CONTROL OF GENE EXPRESSION

a striking difference. The lysine residue at the #9 position(Lys9 or K9) of the H3 histone in heterochromatic domains islargely methylated, whereas this same residue in euchromaticdomains tends to be unmethylated, although it is often acety-lated. Removal of the acetyl groups from H3 and H4 histonesis among the initial steps in conversion of euchromatin intoheterochromatin. The correlation between transcriptional re-pression and histone deacetylation can be seen by comparingthe inactive, heterochromatic X chromosome of female cells,which contains deacetylated histones, to the active, euchro-matic X chromosome, whose histones exhibit a normal levelof acetylation (Figure 12.16). Histone deacetylation is accom-panied by methylation of H3K9, which is catalyzed by anenzyme (a histone methyltransferase) that appears to be dedi-cated to this, and only this, particular function. This enzyme,called SUV39H1 in humans, can be found localized withinheterochromatin, where it may stabilize the heterochromaticnature of the region through its methylation activity.

The formation of a methylated lysine at the #9 positionendows the histone H3 tail with an important property: itbecomes capable of binding with high affinity to proteinsthat contain a particular domain, called a chromodomain. Thehuman genome contains at least 30 proteins with chromo-domains, the best studied of which is heterochromatic protein 1(or HP1). HP1 has been implicated in the formation andmaintenance of heterochromatin. Once bound to an H3tail, HP1 is thought to interact with other proteins, including(1) SUV39H1, the enzyme responsible for methylating theH3K9 residue and (2) other HP1 molecules on nearby nu-cleosomes. These binding properties of the HP1 moleculepromote the formation of an interconnecting network ofmethylated nucleosomes, leading to a compacted, higher-order chromatin domain. Most importantly, this state is trans-mitted through cell divisions from one cell generation to thenext (discussed on page 497).

Studies in a number of organisms indicate that smallRNAs, similar in nature to those involved in RNA interfer-ence (page 449), play an important role in targeting a particu-lar region of the genome to undergo H3K9 methylation andsubsequent heterochromatinization. If, for example, compo-

X chromosome inactivation. For the sake of simplicity, wewill focus on modification of a single residue—lysine 9 ofH3—which will illustrate the general principles by which cellsutilize the histone code. The actions of several other histonemodifications are indicated in Figure 12.14 and discussed inthe accompanying legend, and another example is describedon page 517. As we will see throughout this chapter, tech-niques have been developed in recent years to analyze changesthat affect genome transcription, such as histone modifica-tions, on a genome-wide level, rather than simply looking atthese changes one gene at a time. This has given us a muchbroader view of the general importance of each of these phe-nomena than was possible only a few years ago.

Comparison of the nucleosomes present within hete-rochromatic versus euchromatic chromatin domains revealed

BPTF

CHD1

ING2

HP1 14-3-3 Rsc4 PC EAF3 CRB2

JMJD2

Brd2

Me Me

Ac

Ac Ac

MeAcPMeMe

H3K K S K K K4 9 10 14 27 36

K

20K K

16 8K

12 H4

Taf1 Bdf1

FIGURE 12.15 Examples of proteins that bind selectively to modifiedH3 or H4 residues. Each of the bound proteins possesses an activitythat alters the structure and/or function of the chromatin. There is anadded complexity that is not shown in this drawing in that modifica-tions at one histone residue can influence events at other residues, a

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

CELL PRESS.)

FIGURE 12.16 Experimental demonstration of a correlation betweentranscriptional activity and histone acetylation. This metaphase chro-mosome spread has been labeled with fluorescent antibodies to acety-lated histone H4, which fluoresce green. It is evident that all of thechromosomes except the inactivated X stain brightly with the antibodyagainst the acetylated histone. (FROM P. JEPPESEN AND B. M. TURNER,

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