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From the Department of Chemistry/Biochemistry and
Molecular/Cellular Biology Program, Arizona State University,
Tempe, Arizona 85287-1604
INTRODUCTION The GAL and PHO genes of
yeast provided some of the earliest evidence for specific nucleosome
changes on eukaryotic promoter regions, and they continue to contribute
unique insights to this emerging area. These nutrient-regulated systems
possess major advantages for chromatin studies. Gene activity is
tightly regulated and easily manipulated; firm genetic foundations
provide strong functional correspondence for biochemical analyses. The
promoter region nucleosome changes ("transactions") to be discussed
here include disruption, which refers to the loss of
nucleosome structure observed when transcription is activated, and
reorganization, which refers to the regeneration of promoter
region nucleosome structure and is associated with gene inactivation.
Results are from in vivo or nuclear chromatin analyses
unless otherwise stated. Other recent reviews also cover some of these
and related subjects (1-7).
Basic Relationships between Gene Expression and Promoter
Region Nucleosome Changes on PHO and GAL PHO
The incisive analysis of PHO5, mainly carried out by
Hörz and co-workers (1, 3), has made the PHO system a
major chromatin model. PHO5 encodes an acid phosphatase. It
is regulated by extracellular [phosphate], repressed by high
phosphate/induced to expression ("derepressed") by phosphate
deprivation (Table I). Induction depends
on the major, specific activator Pho4p1 and the subsidiary,
pleiotropic activator Pho2p (1, 3). Pho4p binds to the PHO5
upstream promoter elements UASp1 and UASp2 (Fig. 1A) via a domain in its
C terminus and activates transcription through its N-terminal domain.
Pho2p has multiple binding sites in the PHO5 upstream region
(Fig. 1A); binding of Pho2p to these sites in
vitro enhances Pho4p-UAS affinity
(8). Pho4p function is inhibited under repressing conditions by Pho80p,
probably through phosphorylation-dependent
effects on Pho4p subcellular localization (9).
Table I.
Regulators and gene activity
[View Larger Version of this Image (15K GIF file)]
The PHO5 upstream region is protected by an
array of positioned nucleosomes when the gene is inactive ( Nucleosome disruption constitutes a distinct process and is not merely
a subsidiary effect of PHO5 transcription because disruption occurs as usual in a TATA mutant in which PHO5 is not
transcribed at all (12). Both Pho2p and Pho4p are required for
disruption (13). However, Pho4p must play the major role because Pho4p overexpression can trigger disruption in a
pho2 1) The upstream nucleosomes help repress
PHO5 because their depletion, achieved by altering histone
stoichiometry, allows significant PHO5 expression under
repressed conditions (15). Moreover, the transition to the disrupted
(activated) state apparently depends on upstream nucleosome stability
because replacement of nucleosome Pho4p is predominantly
cytoplasmic in repressed cells but predominantly nuclear under induced
conditions (9). Higher nuclear [Pho4p] and an enhanced Pho4p-UAS
affinity in derepressed cells (11) may be sufficient to trigger
disruption. Pho4p probably initiates this process by binding to
UASp1 (11), possibly in concert, and cooperatively, with
Pho2p binding to its strong site that overlaps UASp1 in the
nucleosome-free region (Fig. 1A). This binding could serve
to anchor Pho4p while its activation domain mediates nucleosome
disruption and put Pho2p in a position to aid in the process. For
example, Pho2p may help expose the Pho4p activation domain by freeing
it from an intramolecular interaction with the Pho4p DNA binding domain
(17). However, neither Pho2p nor UASp1 is absolutely
required for disruption because in pho2 PHO5 disruption can be viewed as a multicomponent reaction
in which the Pho4p activation domain, Pho4p-UAS binding, Pho2p-DNA binding, and other contributions (see below) cooperate to provide enough energy to disrupt the upstream nucleosomes. This reaction must
be only modestly favorable because the presence of a hyperstable nucleosome in the array can prevent disruption (16). Mass action effects on this reaction might explain why overexpressed Pho4p can
itself produce disruption (Pho2 GAL
The GAL genes encode the enzymes and regulators needed
to utilize galactose as a carbon source (2, 18). The structural genes
(GAL1-10, The upstream chromatin regions on GAL genes
(GAL1-10, In the
inactive state (poised or repressed), positioned nucleosomes cover the
GAL10, During the initial steps of nuclear
isolation (cell harvest/spheroplast preparation) in our well defined
wild type strain, the disrupted structure of the induced
GAL1-10 and The GAL1-10 and GAL1-10 and The disruption/reorganization behavior described above is apparently
restricted to upstream nucleosomes because the induced pattern on the
GAL1 coding region (21, 31) is not affected at all during
these procedures that so radically alter the upstream regions (28).
This restriction is consistent with the likely localization of the
transaction-mediating factors Gal4p/Gal80p to the upstream regions.
Gal4p/Gal80p probably carry out these nucleosome transactions via the
constitutive, stoichiometric complex they are thought to form in
vivo (2).
Some More General Considerations The
specific GAL and PHO regulators may promote
transactions indirectly, by acting through other factors. For example,
Pho4p may simply recruit RNA polymerase II holoenzyme to carry out the PHO5 nucleosome disruption (32). One of the several
multiprotein complexes known to destabilize nucleosomes (4, 5, 7), e.g. RSC (33), may also help in disruption. Specific
regulators might also directly participate in transactions, acting
through their activation domains or other regions. For example, genetic analysis of the Gal4p C-terminal (activation) domain has defined a
specific activation "face" that contains Thr and Tyr (two each) as
the major residues (34). If these residues were to target the
hydrogen-bonding interactions that stabilize the octamer, between
H2A-H2B dimers and the H3-H4 tetramer (35), Gal4p could cooperate
directly with other factors in disrupting nucleosomes. It is also
important to consider that specific regulators probably carry out
nucleosome transactions and gene activation while in some kind of
organized three-dimensional superstructure (36), which will influence
their operation. For example, very little Gal4p can be isolated from
cells, presumably because it is present in an insoluble structure
in vivo (2).
On both PHO and GAL, the upstream nucleosome
transactions alter TATA exposure and thus can affect the ability of
TBP/PIC to access the TATA, a process crucial to transcription
initiation. These nucleosome transactions might therefore be closely
regulated; by regulating both (disruption/reorganization) the cell can
use nucleosome occupation of the TATA as a controllable switch, perhaps a fairly late one, in the activation pathway. Disruption permits activation. Reorganization may be part of the mechanism that
deactivates expression in response to the appropriate inactivating
signal(s). This switch could be controlled by regulator-sensitive
competition between nucleosomes and the TBP/PIC; activators facilitate
TBP/PIC occupation (and nucleosome removal); negative factors like
Gal80p promote nucleosome occupation. For example, the ability of Pho4p and Gal4p to activate transcription correlates directly with their ability to disrupt upstream nucleosomes (14, 27). Also, TBP mutants
that bind less well to DNA, and thus could compete less well with
nucleosomes, decrease the ability of Gal4p to activate transcription
(37). Some of these features might account for the uniqueness of
upstream transactions (compared with those on coding regions, see
above).
Histones play
specific roles in GAL and PHO expression through
their N-terminal tails. For example, removal of H4 tails decreases the
level of induced GAL1 and PHO5 expression ~20-
and ~4-fold, respectively (38). Removal of H3 tails has little effect
on PHO5 but causes GAL1 to be hyperexpressed
under induced conditions (39). These tails are not involved in the
histone-histone interactions that hold together the octamer (35) and
thus are free to engage in interactions with intranucleosomal DNA,
linker DNA, other nucleosomes, or non-histone proteins.
Acetylation of the lysines in H3 and H4 tails has long been linked to
transcriptionally active chromatin. Acetylation could destabilize
nucleosomes and thus facilitate disruption, because less positively
charged, acetylated histone tails should interact more weakly with
intranucleosomal DNA. However, removal of tails, e.g. of H4,
should also diminish these interactions and thus facilitate disruption
and therefore gene activation. Instead, PHO5 and
GAL1 transcription decreases (38). The inhibitory effects of
H4 tail loss might reflect the specific involvement of these tails in the nucleosome disruption that accompanies gene activation. For instance, they could be contacts for nucleosome-disrupting machinery. H4 tail loss results in increased protection at the TATA-proximal end
of GAL1-10 nucleosome B, suggesting that these tails
normally prevent the formation of a repressive (nucleosome) structure
and thus maintain transcription factor access around the TATA (40). These mechanisms might require acetylated H4 tails.
The hyperexpression (GAL1) produced by H3 tail removal
suggests a different function for these tails. The level of
hyperexpression is roughly the same as the hyperexpression caused by
Gal80p loss, ~2-3-fold (2). H3 tails could thus play a role in
Gal80p-dependent nucleosome reorganization; their
acetylation might inhibit reorganization and thus favor the disrupted
state. Gal80p modulates the level of induced GAL1 expression
(2), and this is presumably why GAL80 is more highly
expressed (5-10-fold) in galactose, even though Gal80p inhibition of
Gal4p is relaxed and GAL genes are activated. This
modulation of GAL1 expression might be implemented by
enhancement of the potential for Gal80p-dependent
reorganization of upstream nucleosomes through increased Gal80p
levels.
Does disruption reflect complete
octamer loss, partial histone loss (most likely H2A-H2B (41)), or some
kind of conformational change? Complete loss of H2A-H2B, leaving only
the H3-H4 tetramer, should expose DNA near nucleosome ends and for ~ 20 bp around the dyad (42). This kind of change is observed for the
disrupted, TATA-containing nucleosome on the modestly induced
GAL80 gene (28). However, on GAL1-10 or
PHO5, disruption results in strong cleavage throughout
nucleosomal regions A-C, or A conformational change that exposes DNA to cleavage without core
histone loss has been suggested for the disruption of a nucleosome on
the mouse mammary tumor virus promoter (1, 7). Other changes that could
expose nucleosomal DNA without histone loss include a partial peeling
away of DNA from the octamer, as suggested to occur during RNA
polymerase transcription through a nucleosome (44) and recently
proposed as a model for factor binding to nucleosomal DNA (45) and
nucleosome sliding along DNA (46). Sliding may occur on the
GAL1 coding region during induction (21) but probably does
not explain the GAL1-10 upstream region changes (28).
Nucleosomes possess inherent instability. For example, in
vitro at physiological salt concentrations, there is spontaneous, low level octamer loss from purified polynucleosomal templates (47).
In vivo disruption mechanisms may depend on, and amplify, these inherent tendencies. Examples of such inherent pathways may be 1)
the peeling off of nucleosomal DNA triggered in vitro by DNA
binding proteins (45) and 2) sequential histone loss (H2A-H2B and then
H3-H4), which is the exact reversal of the nucleosome assembly pathway
(5, 35, 47, 48).
Nucleosome transactions probably involve bidirectional
reactions whose "equilibrium" position can be shifted, in either
direction, by factors or processes that affect the participating
species. For example, in disruptions that involve histone loss, the
presence of an acceptor for the dissociated histones should
thermodynamically favor H2A-H2B and octamer loss from DNA. The rapidity
and reversibility of PHO and GAL transactions
suggest that dissociated histones could remain nearby, perhaps bound to
histone acceptors. Potential histone acceptors include:
nucleosome-disrupting complexes (4, 5, 7, 49), which might provide at
least transient histone binding in addition to, or as a means of,
destabilizing histone-DNA interactions; reorganizing factors (Gal80p),
which might transiently accept dissociated histones and then redonate
them to DNA; intermediate filament-like proteins (nuclear lamins and
some matrix proteins), which may displace histones from DNA as well as
bind them (50).
In vitro, the DNA binding domains of transcription
activators can displace histones from DNA containing activator binding sites, solely by competitive binding (cf. Refs. 3 and 4). A
cooperative model was proposed to explain these results (51). However,
in vivo both Gal4p and Pho4p need their activation domains to disrupt nucleosomes; even at overexpressed levels, their DNA binding
domains alone cannot trigger disruption (14, 27). The role of the
activation domains in disruption is unknown. Possibly they recruit
other DNA binding proteins, e.g. TBP/PIC, RNA polymerase II
holoenzyme, etc. (52), which then cooperate together to displace histones, perhaps in concert with gene-specific factors like Pho4p or
Gal4p. The exposure of DNA binding sites, such as UASp2 for Pho4p or the TATA for TBP/PIC, should also promote the removal of
histones from those sequences. Disruption and reorganization may result
from the cooperation of a number of individual and energetically modest
effects like factor-DNA binding, other direct and indirect actions of
specific regulators, histone acceptors, and acetylation. Also, in
vitro disruption typically requires ATP (49); this may also be
true in vivo (28, 30).
The PHO and GAL systems
demonstrate that nucleosome transactions can be rapid, specifically
regulated by transcription factors, unique to promoter regions, and a
part of transcriptional control, regulating UAS and TATA accessibility.
Transactions may utilize subsidiary cellular factors, as well as
specific features (N-terminal tails) of the histones themselves. It
will be important to determine precisely what kind of change(s) occurs
in nucleosome disruption. This will provide insight on possible
mechanisms and what kind of subsidiary factors or processes are needed
to support the transactions. The location of nucleosome disruption
functions within activation domains will help distinguish the multiple
roles of activators and how these roles are implemented.
FOOTNOTES ACKNOWLEDGEMENTS I thank Dr. Neal Woodbury for critical
reading of the manuscript and Rena Klingenberg for patiently typing the
revisions.
REFERENCES
Volume 272, Number 43,
Issue of October 24, 1997
pp. 26795-26798
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
MINIREVIEW:
Nucleosome Transactions on the Promoters of the Yeast
GAL and PHO Genes*
INTRODUCTION
Basic Relationships between Gene Expression and Promoter
Region Nucleosome Changes on PHO and GAL
Some More General Considerations
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Gene
Major promoter
Major
activator
Major repressor
Induction conditions
Inactive in
PHO5
UASp1
Pho4p
Pho80p
Low Pi
High
Pi
UASp2
GAL1-10
4
UASG
Gal4p
Gal80p
Galactose
Glu (repressed)
gly/lac (poised)
Fig. 1.
Organization of the PHO5 and
GAL1-10 upstream regions. Along the lines are located
the UAS, TATA (boxed T), and transcription start sites
(thin arrows). Major tick marks on the
lines lie at 100-bp intervals. Below the lines,
nucleosome positions are located to scale by rectangles.
Stippled rectangles are the nucleosomes that are disrupted
by gene activation; open rectangles are other positioned
nucleosomes. A, PHO5. The strong Pho2p binding
sites are located by thick vertical arrows, and nucleosomes
that are subject to disruption/reorganization are labeled
1 to 4. B, GAL1-10.
Disrupted/reorganized nucleosomes are labeled A-C.
1 to 4,
Fig. 1A). These nucleosomes cover UASp2 (but not
UASp1), some Pho2p binding sites, and the TATA. Induction
of PHO5 expression causes strong exposure of the DNA within
these nucleosomal regions to restriction enzyme, micrococcal nuclease,
and DNase I cleavage, indicating that the normal structure of these
four upstream nucleosomes is disrupted under induced conditions (10).
This should expose upstream binding sites to transcription factors.
Indeed, Pho4p occupies both of its UAS binding sites in derepressed
chromatin; neither is occupied in repressed chromatin (11). Note that
disruption is only operationally defined; the exact nature of the
change that causes DNA exposure (partial or complete histone loss or nucleosome unfolding) remains unclear on this and other genes (see
below).
mutant, but Pho2p overexpression in
a pho4 mutant cannot. More specifically,
disruption requires the Pho4p N-terminal activation domain (14); even
overexpression of derivatives that lack this domain cannot produce
disruption. Nucleosome disruption is suggested to be a dedicated
function of the Pho4p activation domain (14).
2 DNA with a sequence that can form
a hyperstable nucleosome results in PHO5 inhibition and
persistence of the inactive nucleosome array structure under induced
conditions (16). 2) Overexpression of truncated Pho4p derivatives can
force Pho4p binding to a UAS located in the non-nucleosomal
(UASp1) site but not to a UAS located in nucleosome 2
(11, 14). Thus, nucleosome 2 very strongly restricts
UASp2 access.
(13) or UASp1 (11) strains, overexpressed
Pho4p can itself produce disruption. Disruption in the
UASp1 strain does require a functional
UASp2 (11). This result and the observation that
Pho4p-UASp2 binding is only observed in disrupted chromatin
(11) suggests that Pho4p-UASp2 binding and nucleosome disruption are tightly linked.
/UASp1
strains). The four-nucleosome array disrupts as a unit (11); these
nucleosomes may be structurally linked and their disruption cooperative. Chromosomal context does not play a major role because the
array and its disruption occur as usual when PHO5 is in a CEN plasmid (12). We can look forward to further analysis of this
intriguing chromatin transition.
7) are strongly induced by galactose
through the specific activator Gal4p (Table I). Gal4p activates
transcription through a domain in its C terminus while bound, via an
N-terminal DNA binding domain, to upstream GAL-specific
promoter elements, the UASG. In non-galactose carbon
sources, the structural genes are either repressed (glucose) or in a
poised state (gly/lac), inactive but very rapidly inducible if
galactose becomes available. In both types of carbon source, Gal80p
inhibits Gal4p by directly interacting with its C-terminal activation
domain (18). The presence of galactose relaxes this inhibition,
allowing Gal4p to activate expression.
7, 80) contain a sizable (~170
bp) stretch of DNA that is permanently nucleosome-free (19-22), in
every carbon source, plus or minus Gal4p/Gal80p (23). The UASG
on GAL1-10 (Fig. 1B), GAL7 (21), and
GAL80 (22) all lie completely within the non-nucleosomal regions. Thus, Gal4p can bind to all of these UASG without
disrupting nucleosomes. The ability of Gal4p to access and bind to the
UASG in gly/lac helps poise cells for rapid inducibility,
thus enabling a quick switch to the better carbon source galactose (2).
On PHO5, conditional UASp2 accessibility helps implement
repression (see above). Restricted UAS accessibility and Pho4p
subcellular location probably control Pho4p-UASp binding; Pho4p levels
are the same under activating or repressing conditions (24).
Gal4p-UASG binding appears to be determined mainly by Gal4p
levels (2).
7, and 80
7, and 80 TATA and the
GAL1 and 80 transcription start sites and
surround the GAL1 TATA (19-22) (Fig. 1B). These
nucleosomes help repress gene activity because nucleosome depletion in
non-galactose carbon sources allows some TATA-dependent GAL1 expression (25). Galactose induction triggers the
Gal4p-dependent disruption of all these upstream
nucleosomes (19-22, 26-28). For GAL1-10 nucleosome B, it
is known that disruption depends on the transcription activation domain
of Gal4p (27); this is likely to be true for the other upstream
nucleosomes, for example A and C (Fig. 1B), which are
disrupted simultaneously with B (28). Nucleosome disruption exposes the
TATA to various exogenous probes (19-22, 26-28) and thus should also
enhance its exposure to TBP/PIC, thereby facilitating transcription
initiation. Also, the first DNA melting for GAL10 and
GAL1 transcription occurs within disrupted nucleosome A and
C regions (29). Release of the negative supercoiling restrained by
those nucleosomes might aid this initial strand separation. Note that
the DNA binding and nucleosome disruption functions of Gal4p act at
sites that are distinct and almost certainly quite spatially distant in
the chromatin structure. In Pho4p, these functions act, at least in
part, on the same chromosomal region (UASp2). In Gal4p, DNA binding and
disruption are independent, i.e. one can occur without the
other (gly/lac); in Pho4p these functions seem to be linked.
80 upstream regions is completely
reorganized. However, in isogenic gal80D mutants
under the same conditions, this upstream nucleosome reorganization does
not occur (28). Thus, Gal80p must be required for the reorganization observed in wild type. This reorganization produces the typical inactive (present in non-galactose carbon sources) upstream nucleosome structure and probably involves the same process that normally reorganizes these regions in response to galactose absence (28).
80 upstream
nucleosomes that are reorganized during spheroplast preparation can be
disrupted by simply incubating the prepared spheroplasts in galactose.
This disruption resembles in vivo disruption in several ways
(28) and is probably carried out by the same process. To observe
disruption, spheroplasts must be prepared from induced cells. This
probably indicates that other steps, such as recruitment of
transcription factors/disruption machinery, are required to set up the
readily disrupted state.
80 upstream nucleosomes are
completely disrupted within 10-15 min in spheroplast treatments and
completely reorganized sometime within a 1-2-h protocol (28).
PHO5 upstream nucleosome disruption is well under way within
15 min after shifting pho80ts cells from
24 °C (permissive for Pho80p function/PHO5 repressed) to
37 °C (restrictive for Pho80p function/PHO5 induced). The
reverse temperature shift in 80ts mutants or phosphate
incubation of spheroplasts from induced wild type cells triggers
reorganization to the inactive array structure within 15 min (30).
Thus, nucleosome transactions of both types can occur rapidly, without
the need for DNA replication or cell growth (30).
1 to 4, and the chromatin digest
pattern resembles a naked DNA pattern. This suggests octamer loss. On
the weakly regulated PHO8, derepression causes only
instability (unfolding?), and partial accessibility increases in the
upstream nucleosomes (43). Upstream nucleosome disruption may thus
involve different types of changes on different genes, depending
perhaps on expression level or tightness of regulation. This variation
might reflect the sequential nature of disruption, i.e. loss
of H2A-H2B dimers first and then the H3-H4 tetramer, and/or the
presence of multiple disruption pathways (4, 5, 7).
1
The abbreviations used are: Pho4p, Pho4 protein;
UAS, upstream activation sequence; gly/lac, glycerol/lactate; PIC,
preinitiation complex; TBP, TATA binding protein; bp, base
pair(s).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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