Page 34 - 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

826 Chromatin Structure and Modification

FIGURE 14 The chemical equation of ε-acetyllysine synthesis and degradation by HATs and HDACs.

nucleosomal arrays—but what these complexes actually in the mid-1980s in a screen for strains that would have
do to the chromatin fiber in vivo remains to be investigated. deficiencies in regulating enzymes involved in amino acid
biosynthesis; nothing was known about the mechanism
of its action until the lab of L. Guarente demonstrated
2. The Power of Amendments: HATs and HDACs
in 1992–1994 that it is involved in transcriptional control
The extensive evidence connecting histone tail acetylation by such transcription factors as Gal4p and Gcn4p. The
with transcription (Section IV.A.2) suffered from its inher- genetic approach that led to this discovery is very infor-
ently correlative nature—it was not clear, whether changes mative: the chimeric transcriptional activator Gal4-VP16
in acetylation status were causative to, or, instead, an ef- carries the DNA binding domain of the yeast transcription
fect of, changes in gene activity. This situation changed factor Gal4p, and the transcription activation domain of
quite dramatically in 1996. a protein from herpes simplex virus (HSV)—virally de-
As shown in Fig. 14, the reaction of acetylation is cat- rived proteins and regulatory DNA stretches have evolved
alyzed by histone acetyltransferases (HATs) and reversed toevokelarge-scaleresponsesfromthetranscriptionalma-
by histone deacetylases (HDACs). In the cases of both chinery, and thus are very commonly used in biology. An
classes of proteins, their enzymatic activity was used as a unexpected property of Gal4-VP16 was its toxicity to the
tool to purify them from cells. The laboratory of C. D. Allis yeast cell; work from M. Ptashne’s lab gave rise to the no-
developed a very unusual method for the detection of HAT tion of “squelching”—competition by a given transcrip-
activity: the in-gel assay. In this technique, a conventional tion factor for some limiting general component of the
polyacrylamide gel is prepared with a large quantity of basal transcriptional machinery. Thus, it was thought that
histone protein polymerized directly into the gel matrix! thte VP16 domain “squelches” important regulatory pro-
A crude protein mixture is then resolved on the gel, and teins away from other yeast promoters. Thus, reasoned
then an in loco reaction is performed by bathing the gel Guarente and his colleagues, whatever protein—when
3
in H-acetyl-CoA: the assumption is that the presumptive overexpressed—suppresses Gal4-VP16 toxicity must be
HAT will, after having migrated to a defined position in relevant to transcriptional control. Indeed, such a screen
the gel, utilize the histone substrate all around it in the gel, yielded the GCN5 locus; it was suggested that Gcn5p,
and label it. The position of the HAT activity, therefore, therefore, is a “transcriptional adaptor”—an entity that
can be visualized by fluorography. Allis’s lab identified transduces the regulatory signal being sent by the DNA-
a major source of HAT activity in nuclei of the ciliated bound regulator to the basal transcription machinery.
protozoan Tetrahymena that migrated at 55 kDA; its pu- Thus, the discovery of Allis and coworkers shone in a
rification and primary sequence characterization yielded new light: a protein found in ciliates enzymatically capa-
the striking observation of its high homology to a known ble of hyperacetylating histones has a very close relative
yeast protein, Gcn5p. in budding yeast—a protein that is required for transcrip-
To appreciate the significance of this discovery, it is tional activation and is thought to be an adaptor! This was
helpful to briefly review what was then known about the first instance of a direct connection between a spe-
GCN5 (in budding yeast, gene loci are designated by up- cific HAT and transcriptional control. In subsequent years,
percase italics, i.e., ABC1, while the protein product of Gcn5p in yeast and other organisms has been subjected to
that locus is written as “Abc1p”). This gene was isolated an extraordinarily comprehensive set of studies that firmly