Page 22 - 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

814 Chromatin Structure and Modification

As elaborated in the next section, a examination of the
nucleosome structure fails to explain these data—all nu-
cleosomes appear to be the same by available structural
criteria, and assemble the entire genome into what seems
to be a homogeneous fiber (see following). Remarkably,
thinning out that fiber has markedly idiosyncratic effects
on genome behavior—and some understanding as to pos-
sible mechanisms for enabling such nonuniform responses
is beginning to emerge.

C. Ontology of the Nucleosome
Evidence that DNA in the nucleus may be organized into
some sort of repetitive entity first came from analysis of
the way the genome inside the nucleus is seen by nucleases
(i.e., enzymes that degrade DNA by cleaving the phospho-
FIGURE 3 The linker histone—a ribbon representation of its
diester bond between two adjacent nucleotides), both en-
“winged-helix” structure.
dogenous to the cell and ectopic. Such experiments by R.
otherwise, however, and showed that histone depletion— Williamson in 1970, and by D. Hewish and L. Burgoyne in
and a concomitant decrease in the extent to which DNA is 1973, demonstrated that DNA released from the genome
assembled into chromatin—does not have nucleus-wide in this way assumes a nonrandom length distribution—
transcriptional consequences, although the upregulation which was unexpected, because nucleases were not known
of specific genes was indeed observed. to have a substrate preference within DNA. By way of ex-
Conclusiveevidencetothiseffectwasrecentlyprovided ample, Fig. 4 shows an agarose gel containing DNA sam-
in work from the lab of R. Young. This study made use ples after treatment with a nuclease. When the enzyme
of the fact that the entire genome of budding yeast has
been sequenced, and it was therefore possible to perform
genome-wide expression profiling analysis. This remark-
able experiment requires a custom-made “microarray”:a
silicon chip containing a grid, into each cell of which a dif-
ferent single-stranded nucleic acid probe corresponding to
a given yeast gene is placed and immobilized (a total of
5900 genes were thus analyzed in one experiment). Mes-
senger RNA is prepared from wild-type and mutant cells
at defined timepoints following inactivation of the histone
H4 gene, and the change in the levels of each message is
determined by hybridizing each mRNA sample to a sep-
arate chip (in actual fact, for detection purposes, a copy
of the mRNA labeled with a fluorescent dye is prepared)
and then measuring the difference—if any—in the levels
of the mRNA hybridizing to each cell in the array.
The major result from this study is that expression
of ca. 75% of the yeast genes is not significantly al-
tered by histone depletion. This indicates that in budding
yeast, chromatin does not have a genome-wide transcrip-
tional repression function. Remarkably, of the remain-
ing 25% of the genes, ca. 15% were upregulated more
than threefold, while 10% were downregulated more than
threefold. These data suggest that nucleosomes have gene-
specific roles in transcriptional control, and, surprisingly,
that the assembly into chromatin is not only required for
FIGURE 4 Chromatin treatment with micrococcal nuclease
the repression of some genes but also for the activation of (MNase) yields a nonrandom distribution of DNA fragments—
some others. evidence for the nucleosome’s existence.