Page 30 - 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

822 Chromatin Structure and Modification

A model system best suited for such analysis is one ChIP has been applied extensively to analyze the hi-
where an entire chromosome has an altered expression stone tail acetylation status over particular stretches of
state; the best characterized example comes from stud- various genomes (from budding yeast to humans). The
ies of dosage compensation: this evolutionarily con- general conclusion from these experiments is that, indeed,
served device for coping with a different autosome to sex transcriptional repression is accompanied by localized hi-
chromosome ratio between genders in metazoa changes stone deacetylation, while transcriptional activation oc-
expression levels of the X chromosome depending on gen- curs within loci that are associated with hyperacetylated
der. For example, in mammals, one of the two X chromo- histones: for example, theDNAse I sensitive domain of
somes in females is inactivated (thus, identical expression the active chicken β-globin locus that was described ear-
levels are achieved for X-linked loci between males and lier was found to be hyperacetylated by ChIP analysis (C.
females). In insects, on the other hand, the single X in Crane-Robinson and colleagues), while transcriptionally
males is transcriptionally upregulated twofold, thus ad- silent stretches of yeast chromatin, such as the mating type
justing its expression level to that of two X chromosomes loci, are deacetylated (J. Broach and colleagues).
in females. FISH analysis by B. Turner and colleagues It is quite striking that our current biochemical insight
demonstrated that the inactive X in mammalian females into the enzymatic reaction of histone tail acetylation,
is hypoacetylated; this provides an important correlate to the causative agents of this modification (Section IV.C),
Ohno’s and Lyon’s observations (v.s.) from the 1960s that and their involvement in transcriptional control in vivo is
this chromosome is condensed into heterochromatin and not paralleled by a similar understanding of the structural
transcriptionally silenced. On the other hand, FISH anal- effects of histone tail hyperacetylation on chromatin, or of
ysis in Drosophila revealed that the transcriptionally “hy- the mechanistic underpinnings of the general stimulatory
peractive” X chromosome in males is hyperacetylated rel- effect that this modification has on the transcriptional
ative to X chromosomes in females. machinery.
The second experimental approach that revealed a The structural puzzles come in part from the fact that
correlation between states of acetylation and levels of the tails fail to appear in X-ray crystallographic analysis.
transcriptional activity is chromatin immunoprecipitation It is clear that in the case of histones H3 and H2B, the
(ChIP). This method was developed by M. Solomon and tails emerge into solution by passing through the two
A. Varshavsky, and further by D. Allis and M. Gorovsky. adjacent DNA double helices that lie on the surface of the
This technique allows one to detemine if a protein of in- octamer, but their subsequent path—assuming a defined
terest interacts in vivo with a DNA stretch of interest; the one exists, which is not at all clear—is unknown. In
reagents required for such analysis are an antibody against addition, a short segment of the histone H4 tail—seven
a particular protein and knowledge of the primary DNA se- amino acids—can be seen making contact with a histone
quence of the locus of interest. This ingenious and power- H2A/H2B dimer in an adjacent nucleosomal particle.
ful method begins by taking an in vivo snapshot of protein– How—or whether—the tails engage the DNA of the
DNA and protein–protein interactions in the nucleus via nucleosome they belong to is unknown.
a brief incubation of living cells or tissue in formalde- Faced with a void of structural understanding from crys-
hyde; this small molecule rapidly penetrates into the cell tallographic analysis, scientists turned to other biophysi-
and introduces covalent crosslinks between proteins and cal methods to investigate what happens to the tails upon
DNA that they are bound to, as well as between proteins acetylation. In 1982, E. M. Bradbury and colleagues used
that are in sufficient physical proximity (i.e., are in a com- NMR analysis of peptides corresponding to the histone
plex). Chromatin is then isolated from cells and sheared H4 tail, and found that it bound only weakly to DNA, and
(by acoustical means, i.e., sonication) into small—ca. 500 that hyperacetylation abolished this binding. By thermal
base pairs—fragments. An immunoprecipitation is then denaturation analysis these scientists derived a quantita-
performed to isolate from this complex mixture the pro- tive estimate of the effect of acetylation: the intact pep-
tein of interest (and whatever happens to be covalently at- tide was seen binding to DNA with an affinity of 50 pM,
tached to it): the antibody is immobilized on a suspension while acetylation reduced it to 10 µM! The magnitude
of agarose beads, and the beads are then mixed extensively of this effect was subsequently shown to depend on the
with the sonicated chromatin to allow the antibody to bind number of lysine residues acetylated, and in the context
its antigen. The beads are then isolated by centrifugation, of the nucleosome—rather than as isolated peptides—the
whichredistributesthetargetproteinfromsolutionintothe histone tails continued to make some contacts with the
pellet (together with the beads). The cross links between DNA even when hyperacetylated. In studies using circular
the protein and DNA are then eliminated, and the DNA dichroism spectra (J. Parello and colleagues), DNA-bound
isolated. Finally, the presence of a given DNA sequence stretches of both histone H4 and H3 tails were found to
in this isolate is assayed by PCR or by Southern blotting. be highly structured and adopt an α-helical conformation,