Page 28 - 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

820 Chromatin Structure and Modification

how anything pertinent to gene-specific regulation could 1. Remodeling
occur on such a homogeneous, monotonously reiterative
As was the case with the initial identification of subunit
substrate as the “beads-on-a-string”fiber. Thus, all tran-
composition of chromatin, the first clues to chromatin re-
scriptional and other regulatory phenomena were thought
modelingphenomenaconcomitantwithalterationsingene
to occur on nucleosome-free stretches of “naked” DNA,
activity came from the use of nucleases. In the late 1970s,
although it remained unclear, whether such stretches can
C. Wu and S. Elgin, and, independently, S. Nedospasov
be found in vivo.
and G. Georgiev used nucleases such as DNAse I and mi-
This notion was also supported by experimental ob-
crococcal nuclease (MNase) to probe chromatin in vivo.
servations on nonhistone factor interactions with chro-
The notion behind this approach was the use of low quan-
matin templates in vitro. Such analysis indicated that many
tities of enzyme—the prediction being that mature chro-
proteins—for example, the TATA-box binding protein,
matin, i.e., DNA that is tightly complexed with histones,
TBP—cannot bind to their DNA sites when they are as-
would not be accessible to cleavage by nuclease, and thus
sembled into nucleosomes. From these, and other obser-
only DNA stretches that were not assembled into conven-
vations, emerged a model according to which inactive re-
tionalchromatinwouldbe“visible”tothenuclease.These
gions of the genome were packaged into nucleosomes;
investigators used an elegant technical trick—“indirect
chromatin, thus, was viewed as a general (nonspecific),
end-labeling”—to reveal such DNA stretches. Wu and
repressive entity. The elimination of histones from active
Nedospasov made the same observation—that regulatory
regions of the genome was then thought to set the stage
DNA (for example, promoters of active genes, or of genes
for binding by nonhistone factors and transcriptional ac-
that are poised for upregulation) is preferentially accessi-
tivation via the recruitment of RNA polymerase.
ble to such nucleases and forms a “nuclease hypersensitive
Over the past years, however, several lines of evidence
site” in vivo. At the time, the structural basis of this en-
emerged that suggested this model was an oversimplifi-
tity was unclear; remarkably, it continues to be not entirely
cation. It became apparent that chromatin structure is not
certain to this day, although we have a much deeper under-
monotonous or homogeneously isomorphous; instead, ex-
standing of the molecular machines responsible for effect-
tensive localized alterations in its nucleoprotein fiber were
ing this chromatin structure alteration and of the properties
observed concomitant with changes in genomic activity
these machines exhibit in vitro.
at specific loci. In an important complementary develop-
An appreciation for the role of such hypersensitive sites
ment, a host of macromolecular complexes were discov-
to gene regulation grew in the early 1980s, when it was
ered that populate the nucleus and effect these alterations.
discovered that they are a relatively ubiquitous feature
In addition, specific gene loci were shown to have proper
of promoters and enhancers in the eukaryotic genome.
transcriptional control depend on their assembly into chro-
A seminal observation was made in 1984 using a model
matin. Finally, whole-genome expression profiling experi-
system that has provided many insights into the role of
mentsdescribedaboveprovidedstrongevidencethatchro-
chromatin in gene control: the regulation of transcription
matin is not a generalized repressor, and that many genes
of the mouse mammary tumor virus (MMTV) genome by
require chromatin for proper regulation.
theglucocorticoidreceptor(GR)anditsligands,thegluco-
corticoids (cortisol, corticosterone, and aldosterone). GR
is a small-molecule-regulated transcription factor: inac-
A. Chromatin Structure Alterations
That Occur in vivo tive in the absence of hormone, it translocates to the nu-
cleus in its presence, binds to target genes, and activates
While structural studies of the nucleosome provided very transcription. In 1974, it was discovered that transcription
important information for scholars of transcription, a ma- driven by MMTV—the etiologic agent of breast cancer
jor issue for understanding how the genome behaves in in mice—is rapidly upregulated by treatment with a syn-
chromatin form was to learn what—if anything—happens thetic GR ligand, dexamethasone. Ten years later, K. Zaret
to chromatin during gene activation and repression in vivo. and K. Yamamoto discovered that hormonal treatment in-
The cytological studies described earlier in this article duces a strong DNAse I hypersensitive site in the MMTV
(Section II) indicated that localized alterations do occur, promoter (called the “long terminal repeat,” or LTR), and
but the resolution limitations inherent in such methods that this hypersensitive site vanished rapidly upon removal
prevented an interpretation of the data in molecular terms: of hormone. This established a correlation between the
for example, puffs that form on polytene chromosomes extent of such remodeling and the level of transcription
are very conspicuous, but what exactly happens to the at this locus. An example of a DNAse I hypersensitive
chromatin fiber during puffing remained unclear (and, in- site induced by a nuclear hormone receptor is shown in
cidentally, still does). Fig. 11.