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* Biochip Technology Center, Argonne National Laboratory, Argonne, Illinois 60439-4833, USA;
Department of Cellular Biotechnologies and Hematology, University of Rome ‘La Sapienza’, 00161, Rome, Italy; and Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, USA
1Correspondence: Biochip Technology Center, Argonne National Laboratory, 9700 S. Cass Ave., Bldg. 202-A253, Argonne, IL 60439-4833, USA. E-mail: zlatanoj{at}everest.bim.anl.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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Key Words: DNA methylation • linker histone modifications • nucleosomal DNA • transcription regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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2
3
4
). Because the compact fiber should be refractory
to transcription, LHs have been thought of as nonspecific repressors.
Repression might occur at either of two levels in transcription:
initiation or elongation. Although compaction of chromatin structure
could have global effects at either of these levels, it is difficult to
imagine mechanisms by which compaction of chromatin fibers per
se would selectively repress specific genes. |
LINKER HISTONES AS REGULATORS OF INDIVIDUAL GENE TRANSCRIPTION |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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. That LHs are actually
involved in transcriptional regulation of individual genes was first
unambiguously shown in two systems: the selective transcription of
oocyte- and somatic-type 5S rRNA genes in early Xenopus
development (e.g., refs 6
, 7
), and the activation and
repression of transcription of certain genes in Tetrahymena
knockouts for histone H1 (8)
. Recent in vitro
experiments elucidated the mechanism of the differential expression of
the two types of 5S rRNA genes in somatic cells and concluded that the
differential effect of H1 on expression was due to the different manner
in which H1 influences the positions of nucleosomes over the coding
sequence of the oocyte and somatic genes (9
, 10
, reviewed
in ref 11
). Since in this case histone H1 is bound to both
the transcriptionally active and inactive chromatin template, we will
not include it in our ensuing discussion, which will focus mainly on
transcriptional activation through H1 displacement.
Interesting results on the selective effect of LHs on transcription of
individual genes were also obtained using an in vivo system
for inducible overexpression of different histone H1 subtypes in
cultured mouse cells (12
, 13)
. Overexpression of
H1o, the differentiation-specific variant
(14)
, led to reduced steady-state transcription levels for
all pol II genes studied; overexpression of another somatic H1 subtype,
H1c, affected different genes differentially, showing either
stimulation or no effect. Moreover, these subtype-specific effects were
shown to be due to differences in the structure of the subtype globular
domains (15)
. In these cases, H1 clearly does not have the
global effects on transcription that might be expected from its role in
chromatin condensation. The molecular mechanism(s) underlying these
subtype-specific effects on transcription of individual genes remain(s)
to be elucidated.
To understand how specific regulation by LHs might occur, it is helpful
to note that in many promoters or enhancers, the binding sites for
critical transcription factors are buried in nucleosomal structure.
Although some factors may be able to bind to DNA that is coiled onto
nucleosomes, this does not appear to be the general rule
(16)
. It is possible that the nucleosome covering a
binding site is actually removed to allow transcriptional initiation.
There remain, however, many examples in which the impeding nucleosome
seems to remain in place, but is somehow altered so as to allow access
by transcription factors.
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LINKER HISTONES ARE GATES TO NUCLEOSOMAL DNA |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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). This appears to be a
spontaneous process on LH-depleted chromatin (18)
or with
isolated core particles (19)
. However, the presence of LHs
bound to nucleosomes occupying gene regulatory regions would be
expected to act as a block against such unpeeling (Fig. 1
).
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To understand how LH binding could exert such a blocking action,
we must consider the way these histones interact with the nucleosomal
structure. Most LHs possess a well-defined three-dimensional structure:
a short amino-terminal random-coiled basic portion of the molecule is
followed by a structured globular domain and a long
carboxyl-terminal unstructured basic tail (see ref 1
). The
globular domain is believed to be situated at or near the
entry-exit of the DNA into the particle, although there are at least
three models for its exact location (reviewed in refs 4
, 20
21
22
23
). The binding of the globular domain at this position
allows the carboxyl-terminal tail to interact with both the incoming
and outgoing linker DNA helices, bringing them close together, with the
formation of a so-called ‘stem’ structure of 
30 bp in length
(24
, 25
, see schematic in Fig. 1
). Thus, the LH binding
site may be seen as a ‘gate’, which may either be closed when the
protein is bound or opened when the protein is released, so as to allow
invasion of the nucleosome by protein factors. In such a scenario, the
problem of gaining access to a DNA site covered by a nucleosome reduces
to the problem of removing a particular LH. Once a single protein
factor has bound to a portion of the DNA partially peeled from a
nucleosome, cooperative binding of other factors may be expected,
as proposed by Polach and Widom (19
, 26)
.
Alternatively, replacement of LH by other protein factors may repress transcription. A repressive protein factor would have to bind more strongly than LH to the nucleosome entry region to lock the DNA so tightly as to preclude nucleosome opening and hence transcription, even when appropriate transcription factors were present. It is our contention that LH replacements by both kinds of factors occur (see below) and constitute important, and subtly tunable, mechanisms for the regulation of transcriptional initiation.
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PROTEIN FACTORS THAT CAN DISPLACE LINKER HISTONES |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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lists a number of especially clear examples, where both LH displacement
and consequential regulation of transcription have been demonstrated.
LH replacement by protein factors may result in both transcriptional
activation (cases 1–3) and inactivation (case 4) (Table 1
; Fig. 1
).
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Although it cannot be ruled out that displacement of LHs is an
active process in which the incoming factor plays a direct role in the
histone removal, scenarios that do not demand this are also tenable. It
has long been known that LHs, in contrast to core histones, are labile
enough to undergo some redistribution in chromatin under physiological
conditions (27
, 28)
. The temporary spontaneous release of
LHs would allow recurrent ‘windows of opportunity’ for either
‘opening’ or ‘closing’ factors to bind (see Fig. 1
). We define an
opening factor as a transcription factor that by virtue of its binding
to the LH binding site on the nucleosome would keep the gate to
nucleosomal DNA open, i.e., would allow the unpeeling of the DNA end(s)
off the histone octamer as required for gene-specific transcription
factor binding. An opening factor may well be reversible in binding,
allowing reformation of the closed nucleosome if transcription factors
do not shortly enter to continue the unpeeling process. In the context
of this transcription initiation scenario, closing factors should be
defined as factors whose binding to the LH binding site is essentially
irreversible, unless subsequent modifications in DNA and/or proteins
promote their release. Once bound, they will preclude the dynamic
unpeeling of the nucleosomal DNA end(s) necessary for transcription
activation to occur.
A very simple example of a closing factor may be found in the LH
variant H5, present in large amounts in terminally differentiated
erythrocytes of birds and some fish. This histone has a much higher
(200 fold; ref 29
) affinity for DNA than do the H1
variants found in transcriptionally active cells, and it systematically
replaces H1 during the final stages of erythroid differentiation. As it
does so, transcription and replication activity essentially ceases.
Other examples of bone fide closing factors could be the
proteins that bind specifically to methylated DNA (30
, 31
;
see also below).
Finally, it may be relevant to note the existence of proteins that bind
directly to H1 in vitro and thus may fall into a category
distinct from either the opening or closing factors, which may not
require direct interaction with the LH. A long known example is the
nucleolar protein nucleolin (e.g., ref 32
), which induces
chromatin decondensation by binding to H1. A more recent example is
prothymosin 
(ProT), an abundant acidic nuclear protein thought
to be involved in cell proliferation. ProT shows high affinity for
H1 in vitro (33)
, and its complex with H1 can
be immunoisolated from crude cell extracts (34)
.
Furthermore, a fraction of H1 is released when chromatin is challenged
with ProT (34)
. Chromatin from cells overexpressing
ProT reveal biochemical characteristics of H1-depleted chromatin,
again suggesting active removal of H1 by the protein (35)
.
These studies involving bulk chromatin should be extended to
specific-gene chromatin to see whether the observed H1-mediated effect
is a general consequence of H1 depletion on chromatin higher order
structure or whether gene-specific effects could also be involved.
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Further levels of regulation will bias linker histone displacement |
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Linker histone phosphorylation |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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, 37
). There are several sites for phosphorylation on each linker
histone, and part of the confusion may result from the differential
effects phosphorylation at different sites may have (see, for example,
ref 38
). Although there has also been evidence that
phosphorylation is closely correlated with transcription, it has been
unclear as to how this could function at the level of particular genes.
A clue may be found in recent studies by Lee and Archer
(39)
, who demonstrate that only phosphorylated H1 can be
displaced from the MMTV promoter by the action of glucocorticoid
receptor. This observation, coupled with the discovery that long
exposure to glucocorticoids leads to H1 dephosphorylation, explains the
hitherto puzzling ‘refractory’ state of the promoter obtained on long
exposure. Thus, there is now at least one clear example in which H1
phosphorylation is connected to the expression of a particular gene.
It may be relevant to note that phosphorylation/dephosphorylation of
other, histone H1-like proteins may also be used as a tool for
transcriptional regulation. There also appears to be at least one
example in which dephosphorylation of an H1-like protein leads to gene
activation. The protein MDBP-2-H1 is an H1-like protein that in
roosters binds to the vitellogenin III gene promoter, inhibiting
transcription (40)
. Treatment with estradiol leads to
dephosphorylation of this protein, similar to the observations with H1
in the MMTV system (see above). This in turn leads to release of the
histone, accompanied by transcriptional activation.
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Linker histone poly(ADP-ribosyl)ation |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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H1 histone is one of the best substrates (41)
for
poly(ADP-ribose) polymerase, which builds or transfers ADP-ribose
polymers onto H1 and other proteins both in a covalent and/or
noncovalent manner. Covalent binding leads to the presence of short
ADP-ribose chains on the modified protein, e.g., 8–10 units of ADPR on
H1 (42)
, whereas noncovalent binding leads to attachment
of long and branched polymers (100–200 ADPR units) to specific
domains on the protein (43)
. Noncovalent interactions are
far stronger than would be expected from simple electrostatic
interactions, so much so that this binding is resistant to strong
acids, detergents, chaotropes, and high ionic-strength conditions
(43)
. Poly(ADP-ribose) polymerase is involved in the
noncovalent modification through an unusual mechanism: to perform this
transfer step, termed heteromodification, it is necessary for the two
monomers in the catalytically active dimer to first modify each other,
building long and branched polymers on numerous sites in their
automodification domains (44)
. It is clear that the
presence of ADP-ribose polymers on the linker histone molecule changes
its charge dramatically and is expected to affect both H1-DNA and H1-H1
interactions, which are important in chromatin organization. Electron
micrographs have shown that in vitro
poly(ADP-ribosyl)ation of polynucleosomes leads to significant
relaxation of chromatin structure (45)
.
Poly(ADP-ribosyl)ation does not detach H1 from the internucleosomal
regions, an observation subsequently confirmed by a different
experimental approach (42)
.
If poly(ADP-ribosyl)ation does not cause or help LH displacement,
what is the possible mechanism whereby this modification modulates the
expression of specific genes? Recent evidence suggests that in
vivo this modification plays a regulatory role in protecting
genomic DNA methylation pattern (46
, 47)
, particularly in
maintaining the unmethylated state of CpG islands in the promoters of
constitutively expressed housekeeping genes (48)
(see also
below). The molecular mechanism involved in the interplay between H1
poly(ADP-ribosyl)ation and gene-specific methylation pattern is still
enigmatic, but in vitro experiments suggest that the
poly(ADP-ribosyl)ated form of a specific genic variant of H1, H1e, is
present in decondensed chromatin structure where the housekeeping genes
are located and inhibits the methylation of their CpG islands
(46)
.
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Core histone acetylation |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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, 49)
. Interest in this modification has intensified
with recent discoveries of acetylase and deacetylase functions
associated with the transcriptional machinery (50
, 51)
.
The possible significance of core histone tail acetylation has
generally been attributed to the role of these histone tails in
maintaining higher order structure. Indeed, the recent suggestion from
the crystal structure of the core particle (52)
that the
H4 tail of one core particle contacts an H2B molecule in an adjacent
core particle has sparkled renewed interest in this view, even though
there is no obvious connection between core particle crystal structure
and chromatin fiber structure.
The effect of core histone acetylation could be, at least in part, on
the interaction of core histone tails with LHs. Indeed, earlier
observations that highly acetylated chromatin fractions are
significantly depleted in LH (53)
and that histone
acetylation alters the capacity of H1 to condense transcriptionally
active chromatin (54)
may reflect such an effect. In this
context, the results of a paper by Juan et al. (55)
are of
especial interest. These authors studied the H1-mediated inhibition of
upstream factor binding to reconstituted chromatosomes, and found that
inhibition of factor binding was essentially lost when the core histone
tails were removed and considerably alleviated by their acetylation.
More recent experiments have confirmed that although the tails of the
core histones do not affect the location of the LH in the chromatosome,
they do affect its binding affinity (56)
. Thus, these
experiments suggest that at least one way in which core histone
acetylation promotes transcription is by facilitating the release of
specific LHs adjacent to the modified nucleosome.
Although the results described above are clear, there remains an
observation in apparent contradiction to the idea that core histone
acetylation can facilitate the release of LH. Ura et al.
(57)
found that reconstitution of histone H5 on
mononucleosomes was insensitive to core histone acetylation. At least
two arguments may be addressed toward this observation. First, the
binding studies were not quantitative; there could well be differences
in affinity that would go undetected [note that Juan et al.
(55)
and An et al. (56)
observe quantitative,
not qualitative, effects]. Second, the use of H5 rather than H1 in
these experiments seems especially inappropriate to questions involving
potential mechanisms of transcriptional activation. Perhaps H5 is
simply insensitive to such effects, consistent with its role as a
permanent repressor of transcription (see above). Clearly, further
studies are needed to clarify the way core histone acetylation works in
transcriptional regulation as well as the functional interconnection,
if any, between LH binding and such acetylation.
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DNA methylation |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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, 59)
.
In particular, the ‘CpG islands’ (so named due to their high content
of CpG dinucleotides) in the promoter regions of constitutively
expressed housekeeping are unmethylated, this condition being essential
for the expression of the associated gene (60)
. Note that
CpG islands have been found recently in several tissue-specific and
imprinted genes, within the genes themselves (61
, 62)
;
however, methylation of these newly discovered CpGs does not block
transcription.
A chromatin-mediated mechanism by which DNA methylation may be involved
in gene expression is through methylation-dependent chromatin
condensation, which creates a structure inaccessible to the
transcription machinery. Much research (63
, 64)
identifies
the presence of methyl groups on DNA as the focus where chromatin
condensation is nucleated.
As H1 histone is involved in chromatin compaction, clearly a hypothesis
to verify was whether the methylation-dependent chromatin condensation
could be explained by a stronger preference of H1 histone for
methylated DNA. The literature on this point is highly controversial.
H1 has been described as a protein strongly preferring to bind to
methylated DNA (65
, 66)
, but evidence to the opposite has
also been reported (67
68
69)
.
To explain the ambiguity of the LH binding studies it is important to
remember that H1 histone is not a single molecular entity, but a family
of protein variants encoded by five to seven members of the H1 histone
gene family in mammals (70)
. Some variants (H1a, H1b, H1c,
H1d, and H1e) are present in all somatic cells, whereas others (e.g.,
H5, H1°, H1t, and H1s) are species- and cell type-specific. Even the
genes encoding the somatic variants are expressed at significantly
different levels depending on the species, tissue, cell-cycle phase,
and development stage, so that the relative abundance of each single
variant could be different in the H1 histone mixture used in various
experiments. This is particularly important if we consider that
variants differ in their ability to bind different DNA sequences
(71
72
73)
.
Despite the ambiguity concerning the effect of DNA methylation on LH
binding, the results of Meehan et al. (31)
on the binding
of another protein, MeCP2, to methylated DNA are highly relevant to the
displacement issue considered here. This protein, which binds
specifically to methylated DNA (74)
, is capable of
displacing histone H1 in order to gain access to its preferred binding
sites on the methylated template: the binding of MeCP2 exerts a
repressive effect on transcription. Thus, MeCP2 can be seen as a
closing factor specific for methylated promoters in our
activation/repression scenario (see Fig. 1
).
Recent experiments have identified another role for MeCP2. It has been
reported that MeCP2 acts as a shuttle interlocking DNA methylation and
core histone deacetylation in inducing gene silencing (75
, 76)
. The protein recognizes methylated DNA (77)
and
transfers onto it the histone deacetylase, which in turn deacetylates
the core histones, inducing the methylation-dependent chromatin
condensation. In this case, methylated CpGs seem to serve as the
recognition feature of the genes to be silenced by the action of
histone deacetylases.
But is acetylation (or deacetylation inhibition) enough to activate
repressed genes? This question is still open, although some data seem
to indicate that the induction of chromatin acetylation is not enough
to reactivate repressed genes; in addition, the genes should undergo
DNA demethylation before being reactivated (78)
.
Thus, the overall picture that emerges from the data discussed
above allocates LHs a central position in a complicated network of
molecular interactions modulating specific gene expression (Fig. 2
). The affinity of LH binding to chromatin is inversely related to the
ease with which the protein will be displaced, or its binding modified
by transcription regulating protein factors. The LH binding affinity
is, in turn, a function of postsynthetic modifications of the LH itself
and/or of the core histones. Modification (methylation) of the DNA is
not a side viewer either; rather, it participates, through protein
intermediators in the LH-mediated transcription regulation by either
directly affecting H1 displacement by closing factors or by affecting
histone acetylation levels, and through them, the affinity of LH
binding.
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NEW QUESTIONS |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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Questions 2 and 3 ask whether we may finally be approaching the question that has haunted the chromatin field for decades: why are there so many variants and modifications of the histones? Perhaps we have here a key to a gate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONLINKER HISTONES AS REGULATORS...LINKER HISTONES ARE GATES...PROTEIN FACTORS THAT CAN...Further levels of regulation...Linker histone phosphorylationLinker histone poly(ADP...Core histone acetylationDNA methylationNEW QUESTIONSREFERENCES |
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modulates the interaction of histone H1 with chromatin. Nucleic Acids Res 26,3111-3118 is a chromatin-remodelling protein in mammalian cells. Biochem. J. 333,1-3
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