Structural Organization of Eukaryotic Chromosomes

We turn now to the question of how DNA molecules are organized within eukaryotic cells. Because the total length of cellular DNA is up to a hundred thousand times a cell's length, the packing of DNA is crucial to cell architecture. During interphase, when cells are not dividing, the genetic material exists as a nucleoprotein complex called chromatin, which is dispersed through much of the nucleus. Further folding and compaction of chromatin during mitosis produces the visible metaphase chromosomes, whose morphology and staining characteristics were detailed by early cytogeneticists. In this section, we consider the properties of chromatin and its organization into chromosomes. Important features of chromosomes in their entirety are covered in the next section.

Eukaryotic Nuclear DNA Associates with Histone Proteins to Form Chromatin

When the DNA from eukaryotic nuclei is isolated in isotonic buffers (i.e., buffers with the same salt concentration found in cells, «0.15 M KCl), it is associated with an equal mass of protein as chromatin. The general structure of chromatin has been found to be remarkably similar in the cells of all eukaryotes, including fungi, plants, and animals.

The most abundant proteins associated with eukaryotic DNA are histones, a family of small, basic proteins present in all eukaryotic nuclei. The five major types of histone proteins—termed H1, H2A, H2B, H3, and H4—are rich in positively charged basic amino acids, which interact with the negatively charged phosphate groups in DNA.

The amino acid sequences of four histones (H2A, H2B, H3, and H4) are remarkably similar among distantly related species. For example, the sequences of histone H3 from sea urchin tissue and calf thymus differ by only a single amino acid, and H3 from the garden pea and calf thymus differ only in four amino acids. Minor histone variants encoded by genes that differ from the highly conserved major types also exist, particularly in vertebrates.

The amino acid sequence of H1 varies more from organism to organism than do the sequences of the other major histones. In certain tissues, H1 is replaced by special his-tones. For example, in the nucleated red blood cells of birds, a histone termed H5 is present in place of H1. The similarity in sequence among histones from all eukaryotes suggests that they fold into very similar three-dimensional conformations, which were optimized for histone function early in evolution in a common ancestor of all modern eukaryotes.

Chromatin Exists in Extended and Condensed Forms

When chromatin is extracted from nuclei and examined in the electron microscope, its appearance depends on the salt concentration to which it is exposed. At low salt concentration in the absence of divalent cations such as Mg+2, isolated chromatin resembles "beads on a string" (Figure 10-19a). In this extended form, the string is composed of free DNA called "linker" DNA connecting the beadlike structures termed nucleosomes. Composed of DNA and histones, nu-cleosomes are about 10 nm in diameter and are the primary structural units of chromatin. If chromatin is isolated at physiological salt concentration («0.15 M KCl, 0.004 M Mg+2), it assumes a more condensed fiberlike form that is 30 nm in diameter (Figure 10-19b).

Structure of Nucleosomes The DNA component of nu-cleosomes is much less susceptible to nuclease digestion than is the linker DNA between them. If nuclease treatment is carefully controlled, all the linker DNA can be digested, releasing individual nucleosomes with their DNA component. A nucleosome consists of a protein core with DNA wound around its surface like thread around a spool. The core is an octamer containing two copies each of his-tones H2A, H2B, H3, and H4. X-ray crystallography has shown that the octameric histone core is a roughly disk-shaped molecule made of interlocking histone subunits

▲ EXPERIMENTAL FIGURE 10-19 The extended and condensed forms of extracted chromatin have very different appearances in electron micrographs. (a) Chromatin isolated in low-ionic-strength buffer has an extended "beads-on-a-string" appearance. The "beads" are nucleosomes (10-nm diameter) and

(Figure 10-20). Nucleosomes from all eukaryotes contain 147 base pairs of DNA wrapped slightly less than two turns around the protein core. The length of the linker DNA is more variable among species, ranging from about 15 to 55 base pairs.

In cells, newly replicated DNA is assembled into nucleo-somes shortly after the replication fork passes, but when isolated histones are added to DNA in vitro at physiological salt concentration, nucleosomes do not spontaneously form.

▲ FIGURE 10-20 Structure of the nucleosome based on x-ray crystallography. (a) Space-filling model shown from the front (left) and from the side (right, rotated clockwise 90°). H2A is yellow; H2B is red; H3 is blue; H4 is green. The sugar-phosphate backbone of the DNA strands is shown as white tubes. The N-terminal tails of the eight histones and the two H2A C-terminal tails, involved in the "string" is connecting (linker) DNA. (b) Chromatin isolated in buffer with a physiological ionic strength (0.15 M KCl) appears as a condensed fiber 30 nm in diameter. [Part (a) courtesy of S. McKnight and O. Miller, Jr.; part (b) courtesy of B. Hamkalo and J. B. Rattner.]

However, nuclear proteins that bind histones and assemble them with DNA into nucleosomes in vitro have been characterized. Proteins of this type are thought to assemble his-tones and newly replicated DNA into nucleosomes in vivo as well.

Structure of Condensed Chromatin When extracted from cells in isotonic buffers, most chromatin appears as fibers «30 nm in diameter (see Figure 10-19b). In these condensed condensation of the chromatin, are not visible because they are disordered in the crystal. (b) Ribbon diagram of the histones showing the lengths of the histone tails (dotted lines) not visible in the crystal structure. H2A N-terminal tails at the bottom, C-terminal tails at the top. [Part (a) after K. Luger et al., 1997, Nature 389:251; part (b) K. Luger and T J. Richmond, 1998, Curr. Opin. Gen. Dev. 8:140.]

▲ FIGURE 10-21 Solenoid model of the 30-nm condensed chromatin fiber in a side view. The octameric histone core (see Figure 10-20) is shown as an orange disk. Each nucleosome associates with one H1 molecule, and the fiber coils into a solenoid structure with a diameter of 30 nm. [Adapted from M. Grunstein, 1992, Sci. Am. 267:68.]

fibers, nucleosomes are thought to be packed into an irregular spiral or solenoid arrangement, with approximately six nucleosomes per turn (Figure 10-21). H1, the fifth major histone, is bound to the DNA on the inside of the solenoid, with one H1 molecule associated with each nucleosome. Recent electron microscopic studies suggest that the 30-nm fiber is less uniform than a perfect solenoid. Condensed chro-matin may in fact be quite dynamic, with regions occasionally partially unfolding and then refolding into a solenoid structure.

The chromatin in chromosomal regions that are not being transcribed exists predominantly in the condensed, 30-nm fiber form and in higher-order folded structures whose detailed conformation is not currently understood. The regions of chromatin actively being transcribed are thought to assume the extended beads-on-a-string form.

Modification of Histone Tails Controls Chromatin Condensation

Each of the histone proteins making up the nucleosome core contains a flexible amino terminus of 11-37 residues extending from the fixed structure of the nucleosome; these termini are called histone tails. Each H2A also contains a flexible C-terminal tail (see Figure 10-20b). The histone tails are required for chromatin to condense from the beads-on-a-string conformation into the 30-nm fiber. Several positively charged lysine side chains in the histone tails may interact with linker DNA, and the tails of one nucleosome likely interact with neighboring nucleosomes. The histone tail lysines, especially those in H3 and H4, undergo reversible acetylation and deacetylation by enzymes that act on specific lysines in the N-termini. In the acetylated form, the positive charge of the lysine e-amino group is neutralized, thereby eliminating its interaction with a DNA phosphate group. Thus the greater the acetylation of histone N-termini, the less likely chromatin is to form condensed 30-nm fibers and possibly higher-order folded structures.

The histone tails can also bind to other proteins associated with chromatin that influence chromatin structure and processes such as transcription and DNA replication. The interaction of histone tails with these proteins can be regulated by a variety of covalent modifications of histone tail amino acid side chains. These include acetylation of lysine e-amino groups, as mentioned earlier, as well as methylation of these groups, a process that prevents acetylation, thus maintaining their positive charge. Arginine side chains can also be methylated. Serine and threonine side chains can be phosphorylated, introducing a negative charge. Finally, a single 76-amino-acid ubiquitin molecule can be added to some lysines. Recall that addition of multiple linked ubiquitin molecules to a protein can mark it for degradation by the proteasome (Chapter 3). In this case, the addition of a single ubiquitin does not affect the stability of a histone, but influences chro-matin structure. In summary, multiple types of covalent modifications of histone tails can influence chromatin structure by altering histone-DNA interactions and interactions between nucleosomes and by controlling interactions with additional proteins that participate in the regulation of transcription, as discussed in the next chapter.

The extent of histone acetylation is correlated with the relative resistance of chromatin DNA to digestion by nucle-ases. This phenomenon can be demonstrated by digesting isolated nuclei with DNase I. Following digestion, the DNA is completely separated from chromatin protein, digested to completion with a restriction enzyme, and analyzed by Southern blotting (see Figure 9-26). An intact gene treated with a restriction enzyme yields characteristic fragments. When a gene is exposed first to DNase, it is cleaved at random sites within the boundaries of the restriction enzyme cut sites. Consequently, any Southern blot bands normally seen with that gene will be lost. This method has been used to show that the transcriptionally inactive ^-globin gene in non-erythroid cells, where it is associated with relatively unacety-lated histones, is much more resistant to DNase I than is the active, transcribed ^-globin gene in erythroid precursor cells, where it is associated with acetylated histones (Figure 10-22). These results indicate that the chromatin structure of nontranscribed DNA is more condensed, and therefore more protected from DNase digestion, than that of transcribed DNA. In condensed chromatin, the DNA is largely inaccessible to DNase I because of its close association with histones and other less abundant chromatin proteins. In contrast, actively transcribed DNA is much more accessible to DNase I digestion because it is present in the extended, beads-on-a-string form of chromatin.

Genetic studies in yeast indicate that specific histone acetylases are required for the full activation of transcription of a number of genes. Consequently, as discussed in


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