Page 31 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

P1: GNH Revised Pages
Encyclopedia of Physical Science and Technology EN002G-104 May 17, 2001 20:53

Chromatin Structure and Modification 823

while those of histones H2A and H2B were seen to as- that show these enzymes to be directly involved in tran-
sume a random coil conformation. The working model scriptional regulation in vivo. These are reviewed in the
as to the local effects of acetylation on tail structure and next section.
interaction with DNA, then, suggests that it may disrupt
the (currently unknown) secondary structure the tails as- B. Chromatin Disruption and Modification:
sume, and lessen the extent to which the tails interact with The Enzymatic Machinery
DNA.
In interesting testimony to the powerful influence of
What is the relevance of these observations to transcrip-
methodology over the course scientific inquiry, investiga-
tional control? If, indeed, the tails become more loosely
tions of the molecular agents that effect chromatin struc-
associated with DNA upon hyperacetylation, it is possi-
ture alterations in vivo have followed a remarkably uni-
ble that the underlying DNA becomes more accessible to
form scheme, somewhat reminiscent of a Bildungsroman.
nonhistone regulators. In vitro experiments with purified
It begins with a genetic screen in budding yeast for strains
chromatin components and particular transcriptional reg-
that exhibit particular phenotypes related to gene control
ulators (A. Wolffe, J. Workman, and their colleagues) have
(e.g., the ability to activate a particular metabolical path-
found that histone hyperacetylation potentiates binding to
way), the molecular cloning of the underlying loci, and
nucleosomal substrates by such proteins as TFIIIA, Gal4,
their subsequent putative identification as transcriptional
and USF. Whether such potentiation of binding occurs in
regulators. Next, analysis in metazoa reveals the existence
vivo is unknown.
of homologs, and an enzymatic assay is developed that
As mentioned earlier, a segment of the histone H4 tail
demonstrates that these proteins have the capacity to alter
was seen making an internucleosomal contact in the crys-
chromatin structure. Mutational analysis reveals that there
tal structure. These data, and other observations, lent sup-
is a correlation between the enzymatic activity of the pro-
port to the hypothesis that tail acetylation affects higher-
tein and its ability to act in transcriptional control. Bio-
order folding of chromatin. Strong evidence to this effect
chemical analysis of whole-cell extracts then finds these
was obtained by J. Hansen and his colleagues, who used
proteins within large, multisubunit complexes, only a few
analytical ultracentrifugation to demonstrate that the hy-
polypeptides in which have enzymatic activity, while the
drodynamic properties of chromatin fibers can be mod-
rest are of uncertain function. Finally, in vitro experiments
ulated in vitro. An alteration in the ionic strength of the
are done that show these complexes can be targeted by
solution dramatically altered the shape of the nucleosomal
particular transcriptional activators or repressors, and that
fiber: as the concentration of Mg 2+ increased, the fiber be-
mutations in these transcription factors that alter the abil-
came much more compact. Importantly, the deacetylation
ity to interact with chromatin modifying and remodelling
of this fiber was then shown to have an identical effect:
complexes impair their properties as regulators.
thus, hyperacetylated chromatin adopts an extended con-
formation, and deacetylation promotes folding. It is possi-
1. Disrupt and Conquer: ATP-Dependent
ble, therefore, that the increased accessibility to nucleases
Chromatin Remodeling Engines
seen in transcriptionally active, hyperacetylated chromo-
somal domains in vivo is causally linked to a change in In the 1980s, genetic studies in laboratories of M. Carlson
the extent of chromatin compaction that can be observed and F. Winston identified a number of mutant yeast strains
in vitro. Additional support for such a connection comes that failed to metabolize sucrose; following convention,
from the work of A. Belmont and coworkers, who used the many loci revealed in the screen were called SNF
in vivo imaging techniques to demonstrate that transcrip- (sucrose nonfermenter) and given a number (i.e., SNF2,
tional activation is accompanied by the hyperacetylation SNF5, etc.). At approximately the same time, the lab of K.
and unfolding of a chromosomal domain spanning several Nasmyth discovered a number of loci in the yeast genome
hundred thousand base pairs. Most remarkably, this pro- that when mutated, incapacitated the switching of mat-
cess occurred even in the absence of transcription—this ing type; these were dubbed SWI (for “switch”) and also
important control experiment indicated that the unfolding numbered (SWI5, SWI2, etc.). The capacity to metabolize
is not a consequence of the passage by the RNA poly- sucrose is dependent on the activation of specific genes
merase II complex (an imposing entity) through the chro- that belong to the cognate metabolic pathway (in partic-
mosome, and is an independently regulated phenomenon. ular, an invertase); the switching of mating type requires
Whatever the structural consequences of acetylation on the upregulation of a gene that encodes an endonucle-
chromatin, some of the strongest evidence in favor of its ase required for initiating a DNA recombination reaction.
direct role in controlling the genome comes from data il- Thus, it appeared that a common feature of the two other-
luminating the abundance inside the nucleus of enzymes wise quite unrelated phenotypes in the mutant strains (fail-
effecting histone tail modification, and from experiments ure to metabolize sugar or the incapacity to mate) was a