Heterochromatin Causes Difficulty for Access to DNA in Eukaryotes

In bacteria, the DNA is freely accessible to RNA polymerase and regulatory proteins. However, in eukaryotes, the DNA is coiled around the histones, forming nucleosomes, as discussed in Ch. 4. The nucleosomes are wound into a helix and held close together largely by interactions due to the histone proteins. Densely packaged DNA is referred to as heterochromatin and cannot be transcribed, because the RNA polymerase cannot gain access to the promoters. Generally, visible DNA in electron micrographs is heterochromatin, whereas DNA that is not dense is called euchromatin (Fig. 10.09).

The linker histone, H1, has two arms extending from its central spherical domain. The central part of H1 binds to its own nucleosome and the two arms are thought to bind to the nucleosomes on either side, however the exact arrangement is uncertain. The histones of the nucleosome core (H2A, H2B, H3 and H4) have a body of about 80 amino acids and a tail of 20 amino acids at the N-terminal end that faces outwards from the core. Interactions due to these tails are believed to be important in nucleo-some aggregation and higher level folding of the chromatin.

The histone tails contain several lysine residues that may have acetyl groups added or removed. All four of the core histones may be acetylated, although H3 and H4 are heterochromatin A highly condensed form of chromatin that cannot be transcribed because it cannot be accessed by RNA polymerase

FIGURE 10.08 MyoD and Its Alternative Partners

A) MyoD and E12 both possess basic DNA binding domains. When MyoD dimerizes with E12 the dimer therefore binds to DNA. B) In contrast, Id protein lacks a basic region. When MyoD dimerizes with Id, this dimer cannot bind DNA.

Acetylation of histones controls access of regulatory proteins to the DNA.

Heterochromatin Causes Difficulty for Access to DNA in Eukaryotes 271

FIGURE 10.09 Heterochromatin Versus Euchromatin

The nucleus shown contains regions of densely packed heterochromatin and less densely packed euchromatin regions. This electron micrograph is of a neuroglial cell nucleus from an insect nervous system, magnified by 3980. Note the nucleolus (brown) and condensed DNA (dark red) in the nucleus. Mitochondria (pink) can be seen in the surrounding cytoplasm. Copyright Dennis Kunkel.

FIGURE 10.09 Heterochromatin Versus Euchromatin

The nucleus shown contains regions of densely packed heterochromatin and less densely packed euchromatin regions. This electron micrograph is of a neuroglial cell nucleus from an insect nervous system, magnified by 3980. Note the nucleolus (brown) and condensed DNA (dark red) in the nucleus. Mitochondria (pink) can be seen in the surrounding cytoplasm. Copyright Dennis Kunkel.

FIGURE 10.10 Acetylation of Histone Tails Disaggregates Nucleosomes

A) Closely packed nucleosomes are stabilized by binding of histone tails to histones in the next nucleosome. B) When the tail of H4 is acetylated, it no longer binds to histones in an adjacent nucleosome. This promotes disaggregation of neighboring nucleosomes. [Histone H1 binds to the linker DNA between the nucleosomes but is not shown in this figure for the sake of clarity.]

most often modified. The degree of acetylation affects the state of nucleosome aggregation and therefore of gene expression. Non-acetylated histones form highly condensed heterochromatin, whereas acetylated histones form less condensed chromatin. Note that the nucleosomes themselves are not disassembled by acetylation, but their clustering is loosened up (Fig. 10.10).

Enzymes known as histone acetyl transferases (HATs) add acetyl groups and histone deacetylases (HDACs) remove them. Several proteins previously known as co-activators are actually HATs. Examples include the human CBP and p300 proteins acetylation Addition of an acetyl (CH3CO) group histone acetyl transferase (HAT) Enzyme that adds acetyl groups to histones histone deacetylase (HDAC) Enzyme that removes acetyl groups from histones

FIGURE 10.11 Acetylation and Deacetylation of Histones

A) Acetylation of histone tails is performed by co-activators known as histone acetyl transferases (HATs). B) Deacetylation of histone tails is due to a repressor complex containing both a DNA-binding subunit and a deacetylase.

FIGURE 10.11 Acetylation and Deacetylation of Histones

A) Acetylation of histone tails is performed by co-activators known as histone acetyl transferases (HATs). B) Deacetylation of histone tails is due to a repressor complex containing both a DNA-binding subunit and a deacetylase.

Access to eukaryotic DNA involves moving or restructuring the nucleosomes.

involved in cell cycle control and differentiation. Similarly, several so-called co-repressor proteins are histone deacetylases. Co-activators and co-repressors do not bind to the DNA, itself, but bind to transcription factors that have already bound to the DNA (Fig. 10.11).

In addition to disaggregating the nucleosomes by acetylation, a further step is needed to provide access to the DNA itself. This is performed by chromatin remodeling complexes. These carry out two main types of remodeling. Firstly, they can slide nucleosomes along a DNA molecule, so exposing sequences for transcription. Secondly, they are able to rearrange the histones, so remodeling nucleosomes into a looser structure that allows access to the DNA. ATP is used to provide energy for this remodeling.

There are two families of chromatin remodeling complexes. The larger Swi/Snf ("switch sniff") complexes consist of eight to 12 proteins and bind to DNA strongly (Fig. 10.12). Swi/Snf can both slide and remodel nucleosomes. Apparently, Swi/Snf merges two nucleosomes into a new, looser structure. The smaller ISWI ("imitation switch") complexes contain two to six polypeptides and can slide nucleosomes but cannot rearrange them. They bind to histones rather than to DNA (Fig. 10.12). (The Swi factors were named after the switching of mating type; Snf factors refer to sucrose nonfermenting mutants. Both were found first in yeast.)

Binding of the chromatin remodeling complexes by transcription factors targets them to the stretch of DNA that needs opening up. The precise order in which transcription factors, histone acetyl transferases and chromatin remodeling complexes bind appears to vary from promoter to promoter. A generalized overall sequence of events for the activation of a eukaryotic gene is as follows:

chromatin remodeling complex A protein assembly that rearranges the histones of chromatin in order to allow transcription co-repressor In prokaryotes—a small signal molecule needed for some repressor proteins to bind to DNA;in eukaryotes—an accessory protein, often a histone deacetylase, involved in gene repression ISWI ("imitation switch") complex Smaller type of chromatin remodeling complex Swi/Snf ("switch sniff") complex Larger type of chromatin remodeling complex

Methylation of DNA in Eukaryotes Controls Gene Expression 273

A) Sliding

B) Remodeling

A) Sliding

B) Remodeling

FIGURE 10.12 Sliding and Remodeling of Nucleosomes

A) Sliding of the nucleosome relative to the DNA exposes a previously inaccessible promoter. B) A remodeling complex such as Swi/Snf can merge two nucleosomes and loosen the winding of DNA, making more of the DNA accessible.

FIGURE 10.12 Sliding and Remodeling of Nucleosomes

A) Sliding of the nucleosome relative to the DNA exposes a previously inaccessible promoter. B) A remodeling complex such as Swi/Snf can merge two nucleosomes and loosen the winding of DNA, making more of the DNA accessible.

1. A transcription factor binds to the DNA.

2. A histone acetyl transferase binds to the transcription factor.

3. The HAT acetylates the histones in the vicinity and the association of the nucle-osomes is loosened.

4. The chromatin remodeling complex slides or rearranges the nucleosomes, allowing binding access to the DNA.

5. Further transcription factors bind.

6. RNA polymerase binds to the DNA.

7. Initiation requires a positive signal to be transmitted via the mediator complex from one or more specific transcription factors.

Consider, for example, the yeast HO gene, which encodes an endonuclease required for the switching of mating type in yeast. First, the Swi5p transcription factor binds. Then the Swi/Snf complex binds to Swi5p. Next to arrive is SAGA, a histone acetyl transferase, which depends for its binding on the presence of Swi/Snf. The his-tones in the promoter region are then acetylated. This allows another transcription factor, SBF, to bind. This then allows the general transcription factors to bind, followed by the RNA polymerase (Fig. 10.13).

Methylation of DNA is often used to control gene expression during development of higher organisms.

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