Page 29 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN002G-104 May 17, 2001 20:53

Chromatin Structure and Modification 821

tion is mechanistically similar to that between heterochro-
matin and euchromatin). Within such a large domain of
less compacted chromatin, targeted chromatin remodeling
over short DNA segments then induces hypersensitivity,
i.e., a more dramatic disruption of histone–DNA contacts.

2. Modification

An additional important observation on the β-globin locus
related to a different type of chromatin structure alteration
observed in vivo: a change in the covalent modification
status of histone tails within a domain of transcription-
ally active chromatin. To better illuminate the significance
of this finding, we must briefly review the biochemistry
of such modifications. As mentioned earlier, in 1964 it
FIGURE 11 Binding of the thyroid hormone receptor (TR) to
chromatin generates a DNAse I hypersensitive site (arrowheads). was discovered that particular lysine residues in the NH 2 -
“TRE” represents the TR binding site. terminal tails of the core histones are reversibly covalently
modified by acetylation:
+
··· NH + Ac-CoA ··· NH 2 CO CH 3 .
It is important to note that the disruption of chromatin 3
structure that is described as a “hypersensitive” site is a This observation instantly suggested an electrostatic
highly localized event and only affects ca. 50–500 base mechanism for gene regulation: the histone tails carry a
pairs of DNA (i.e., only several nuclesomes’ worth)— wealth of positive charge on their basic amino acids and
thus, while the insect relative of GR, the ecdysone recep- are thus expected to bind tightly to the negatively charged
tor, functions via highly similar mechanisms, it should not phosphates in the DNA backbone. Acetylation eliminates
be inferred that polytene chromosome puffing observed the charge on the target amino acid side chain, and thus
some 25 years prior by Clever and Karlson in Chironomus a hyperacetylated histone tail would be expected to bind
after ecdysone injection (see Section II) is due to such less tightly to the DNA, and make it more visible to the
remodeling—puffing is a large-scale event which affects intranuclear world. Thus, there must be a correlation be-
stretches of the genome that are many thousands of bp tweenchromatinhyperacetylationandtranscriptionalacti-
long. A molecular correlate to “puffing” was discovered vation (and, conversely, between chromatin deacetylation
in the lab of C. Crane-Robinson in 1994 in studies of and transcriptional repression).
the globin gene cluster. A marvel of gene regulation, the This model makes a lot of sense from first principle a
globin genes in higher vertebrates have been investigated priori considerations, but is it supported by experimental
ingreatdetail,inpartbecausetheyrepresentoneofthebest evidence? The correlation between levels of acetylation
characterized instances of a “chromosomal domain”—a and transcription clearly exists in vivo. A very powerful
large continuous stretch of the genome in which several tool in making this experimental determination has been
genes reside whose expression is coordinately regulated antibodies that selectively recognize hyperacetylated or
(forinstance,inmammals,aprogressionofgeneactivation deacetylated histone tails. These have been used with great
occurs, with “fetal” globin genes active during embryonic success in two experimental strategies. The first, fluores-
development, and a switch to “adult” type globin genes cent in situ hybridization (FISH), is cytological: it allows
after birth). In the chicken genome, the β-globin locus one to examine histone acetylation status over entire chro-
encompasses ca. 33,000 base pairs; by using nucleases, mosomes. In the most general sense, it involves the prepa-
Crane-Robinson’s research group showed that in erythro- ration of nuclei from cells (or tissue) under biochemically
cytes (i.e., the cell type that transcribes globin genes) this mild conditions, the immobilization of their chromatin
entire portion of the chromosome was more sensitive to content on a suitable support, and the probing of the re-
nucleases than a transcriptionally inert one. sulting karyotype with an antibody of choice, followed by
We emphasize that the distinction between nuclease probing with a “secondary” antibody that reveals where
sensitivity and hypesensitivity is not merely a semantic the original antibody bound. The secondary antibody is
one. During the activation of gene expression in vivo,it chemically coupled to a fluorescent dye, and this allows
is quite likely that as a first step, a large stretch of chro- visualization under a microscope of entire chromosomes
matin undergoes some structural transition to a more ac- with brightly fluorescent segments that have bound to the
cessible conformation (it is possible that such a transi- antibody.