8.5: Genes and Chromatin in Eukaryotes

Chromosomes and chromatin are a uniquely eukaryotic association of DNA with proteins of different types and amounts. Bacterial DNA (as well as prokaryotic DNA generally) is relatively “naked”—that is, not visibly associated with protein.

CHALLENGE

Why do you think prokaryotic DNA is mostly “naked”?

The electron micrograph of an interphase cell (Figure 8.12) reveals that the chromatin can itself exist in various states of condensation.

Figure 8.12: Transmission micrograph of euchromatin and heterochromatin in the nucleus.

Chromatin is maximally condensed during mitosis to form chromosomes. During interphase, chromatin exists in more- or less-condensed forms, called heterochromatin and euchromatin, respectively.

Transition between these chromatin forms involves changes in the amounts and types of proteins that can bind to the chromatin during gene regulation (i.e., when genes are turned on or off). Experiments (to be described later) showed that active genes tend to be in the more dispersed euchromatin, where enzymes of replication and transcription have easier access to the DNA. Transcriptionally inactive genes are heterochromatic, obscured by additional chromatin proteins present in heterochromatin.

CHALLENGE

What likely effects of the chemical modification of chromatin proteins could explain their behavior?

We can define three levels of chromatin organization in general terms:

1. DNA is wrapped around histone proteins (nucleosomes) like “beads on a string.”
2. Multiple nucleosomes are coiled (condensed) into 30 nm fiber (solenoid) structures.
3. The 30 nm fibers are packed in higher order to form the familiar metaphase chromosome

CHALLENGE

Describe the difference(s) between anaphase and metaphase chromosomes, using appropriate terminology.

These aspects of chromatin structure were determined by gentle disruption of the nuclear envelope of nuclei, followed by salt extraction of extracted chromatin. Salt extraction dissociates most of the proteins from the chromatin. The results of a low-salt extraction are shown in Figure 8.13.

Figure 8.13: Low-salt fractionation of interphase nuclei yields 10 nm nucleosome beads on a string.

When the low-salt extract is centrifuged and the pellet is resuspended, the remaining chromatin looks like a 10 nm filament of attached nucleosomes, or beads on a string. DNA wrapped nucleosomes are the beads, which are in turn linked by uniform lengths of the metaphorical DNA “string.”

After a high-salt chromatin extraction, the structure visible in the electron microscope is the 30 nm solenoid, the coil of nucleosomes modeled in Figure 8.14. As shown in the illustration, increasing the salt concentration of an already-extracted nucleosome preparation will cause the “necklace” to fold into the 30 nm solenoid structure.

Figure 8.14: High-salt chromatin extraction from nuclei, or high-salt treatment of 10 nm filaments selectively removes chromatin proteins, yielding 30 nm solenoid structures, essentially coils of 10 nm filaments

In fact, there are at least five levels (orders) of chromatin structure. These are illustrated below in Figure 8.15. The first two orders, as we have discussed, are the string of beads (#1) and the solenoid (#2). Other extraction protocols have revealed other aspects of chromatin structure (#3 and #4). Chromosomes in metaphase (#5) of mitosis are the “highest order,” or most condensed form of chromatin.

Figure 8.15: Five different levels (orders) of chromatin structure.

The results of deoxyribonuclease (DNAse I) digestion of a beads-on-a-string extract are shown in Figure 8.16. DNase I briefly digests nucleosome “necklaces,” degrading DNA between the “beads” and leaving behind shortened, different length 10 nm filaments. After a longer digestion, only single beads (nucleosomes) remain, bound to small amounts of DNA.

Figure 8.16: Short digestion times with DNase I leave behind shortened 10 nm filaments (or beads on a string). Longer digestion releases single nucleosomes associated with some DNA.

Roger Kornberg, one son of Nobel laureate Arthur Kornberg (discoverer of the first DNA polymerase enzyme of replication—see the next chapter), participated in the discovery and characterization of nucleosomes while still a postdoc! He found that each nucleosome is associated with about two hundred base pairs of DNA. Electrophoresis of DNA that had been extracted from digests of nucleosome beads-on-a-string showed that nucleosomes are separated by a DNA “linker” of about eighty base pairs. DNA extracted from nucleosomes was about 147 base pairs long, a closer estimate of the DNA wrapped around the proteins of the nucleosome. It is appropriate to note here that Roger Kornberg earned the 2006 Nobel Prize for Chemistry for his work on eukaryotic transcription and the structure of RNA polymerase (among other things), topics we cover in a later chapter! Five histone proteins could be isolated from nucleosomes and separated by electrophoresis; the results are illustrated in Figure 8.17.

Figure 8.17: Electrophoretic separation of five histones extracted from 10 nm filaments (left) and the role of histones in the nucleosome.

Histones are basic proteins containing many lysine and arginine amino acids. Their positively charged side chains enable these amino acids to bind to the acidic negatively charged phosphodiester backbone of double helical DNA. The DNA wraps around octamers, each of which is composed of two out of four histones (H3, H2A, H3B, H4), to form the nucleosome (Figure 8.17, right). Histone H1 serves as a linker between nucleosomes. About a gram of histones is associated with each gram of DNA.

CHALLENGE

Histones are among the most highly conserved proteins. Very few amino-acid differences distinguish a human histone from histones in a mouse, sea urchin, or yeast cell. Why do you think that is?

172-2 Nucleosomes: DNA and Protein

173-2 Chromatin Structure: Dissecting Chromatin

As you might guess, an acidic extraction of chromatin should selectively remove the basic histone proteins, leaving behind an association of DNA with nonhistone proteins. This proves to be the case. An electron micrograph of the chromatin remnant after an acid extraction of metaphase chromosomes is shown in Figure 8.18. DNA freed of the regularly spaced histone-based nucleosomes, loops out and away from the long axis of the chromatin. The dark material along this axis is a protein scaffold, which makes up what’s left after histone extraction. Much of this protein is topoisomerase, an enzyme that prevents DNA from breaking apart under the strain of replication (to be detailed in a later chapter).

Figure 8.18: Acid extraction of chromatin removes histones, leaving nonhistone proteins behind. (See the original micrograph at Acid Extract of Chromatin.)