RNA silencing, DNA methylation and post-translational modifications PTMs of the core and linker histones are the mechan-isms that collectively define epigenetics, the latter of which invol
Trang 1Post-translational modifications of the linker histone
variants and their association with cell mechanisms
Christopher Wood1, Ambrosius Snijders2, James Williamson2, Colin Reynolds1, John Baldwin3 and Mark Dickman2
1 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, UK
2 Department of Chemical and Process Engineering, University of Sheffield, UK
3 STFC Daresbury Laboratory, Warrington, UK
Introduction – epigenetic mechanisms
and involvement with disease
Epigenetics is the study of heritable changes in gene
expression that occur without changes in DNA
sequence and, as well as being of fundamental
impor-tance in embryonic development, transcription,
chro-matin structure, X-chromosome inactivation, and
genomic imprinting, it is also now recognized as having
a fundamental role in disease [1] RNA silencing, DNA
methylation and post-translational modifications
(PTMs) of the core and linker histones are the
mechan-isms that collectively define epigenetics, the latter of
which involve the addition of small chemical groups
The PTMs that are created by this mechanism include, but are not limited to, acetylation (lysine), phosphoryla-tion (serine, threonine), methylaphosphoryla-tion (lysine, arginine), sumoylation (lysine), and ubiquitination (lysine) Other epigenetic mechanisms may emerge in the future
Small RNAs MicroRNAs (miRNAs) are RNA molecules that are about 22 nucleotides long and encoded into the Homo sapiens (hereafter ‘human’) genome [2] They
Keywords
abundance; acid extraction; cancer; cell
cycle; disease; linker histone; MS;
post-translational modification; PTM;
PTM function
Correspondence
C M Wood, School of Pharmacy and
Biomolecular Sciences, Liverpool John
Moores University, Liverpool, UK
Fax: +44 0 51 298 2624
Tel: +44 0 51 231 2565
E-mail: c.m.wood@ljmu.ac.uk
(Received 10 December 2008, revised 23
March 2009, accepted 30 April 2009)
doi:10.1111/j.1742-4658.2009.07079.x
In recent years, a considerable amount of research has been focused on estab-lishing the epigenetic mechanisms associated with DNA and the core histones This effort is driven by the fact that epigenetics is intimately involved with genomics in a whole range of molecular processes However, there is now a consensus that the epigenetics of the linker histones are just as important The result of that consensus is that the post-translational modifi-cations (PTMs) for most of the linker histone variants in human and mouse have now been established by a number of experimental techniques, foremost
of which is mass spectrometry (MS) MS was also used by our group to establish the PTMs of the linker histone variants in chicken erythrocytes Although it is now known which types of PTM occur at particular locations
on the linker histone variants, there is still a large gap in the knowledge of how this data relates to function The focus of this review is an analysis of the PTM data for the linker histones from several species, but with an empha-sis on human, mouse, and chicken Our analyempha-sis reveals that certain PTMs can be clearly correlated with specific functions of the linker histones in par-ticular cell types, and that unique PTM patterns exist for different cell types
Abbreviations
CDK, cyclin-dependent kinase; DNMT, DNA methyltransferase; HDAC, histone deacetylase; miRNA, microRNA; MS, mass spectrometry; PTM, post-translational modification.
Trang 2are transcribed by RNA polymerase II into primary
miRNAs and afterwards processed by RNase III
Dro-sha and DGCR8 in the nucleus into precursor
miR-NAs These precursor miRNAs are then exported by
Exportin-5 to the cytoplasm, where they are further
processed by RNase III Dicer into the mature
miR-NAs [2] Each miRNA is thought to have many targets
and can bind its target mRNA completely or partially
If there is complete binding, the mRNA is silenced
and degraded; partial binding leads to downregulation
of a gene It is known that miRNAs are related to
small interfering RNAs and have similar functions As
small interfering RNAs have been shown to be
involved with DNA methylation and histone
modifica-tions, it is likely miRNAs operate in the same manner
[2] The fact that miRNAs are located within the
introns of protein-coding genes has led to the belief
that they are activated with their host genes A
poten-tial way that this may be achieved is via an active
chromatin hub [3]
DNA methylation
In humans, DNA methylation occurs at a cytosine that
precedes a guanine in a CpG dinucleotide sequence,
most often occurring in short stretches of CpG-rich
regions known as CpG islands Such regions are about
0.5–2 kb long and can be found in the 5¢-region of
approximately 60% of genes, near to their promoters
[4,5] The cytosine base is modified at the 5-carbon
position of the pyrimidine ring by the covalent
addi-tion of a methyl group (CH3) [5] This modification is
mediated by DNA methyltransferases (DNMTs) acting
in concert with S-adenosylmethionine, which acts as a
methyl donor in the enzymatic reaction It is believed
that the pattern of DNA methylation is established in
germline cells through the action of de novo DNMT 3a
and DNMT 3b This pattern of DNA methylation is
maintained subsequent to DNA replication through
the action of DNMT1 The linker DNA can be
prefer-entially methylated in the absence of H1, but the
pres-ence of the latter will inhibit methylation [6] CpG
dinucleotides are uniformly dispersed in humans,
prob-ably because 5-methylcytosine can be spontaneously
deaminated to form the DNA base thymidine CpG
dinucleotides outside islands are essentially
continu-ously methylated, leading to the genes where they
reside being unexpressed This is a necessary feature,
as there is a large amount of noncoding DNA in the
human genome However, within CpG islands, the
di-nucleotides can be either unmethylated, if the gene is
expressed, or methylated, if it is not expressed There
are two exceptions to this rule: imprinted genes, and
genes associated with X-chromosome inactivation will always have their CpG islands methylated
Histone modifications The N-terminal tails of the core histones extend beyond the nuclesomes and can have their characteris-tics significantly altered by PTMs H3 has the greatest number of modifications currently identified, followed
by H4, H2B, and H2A The C-terminal tails also con-tain PTMs, but they are few in number, as are those for the non-tail regions Lysine acetylation weakens electrostatic DNA–histone interations, allowing the recruitment of factors containing bromodomains such
as SWI⁄ SNF and TFIID [5] Methylation of H3 Lys10, H3 Lys28 and H4 Lys21 has been associated with gene silencing, whereas H3 Lys5, H3 Lys37 and H3 Lys80 (genomic position numbering) correlate with actively transcribed genes It is not only the core histones that are subject to PTMs; the linker histone H1 can also be modified (see later)
Epigenetic mechanisms in disease
As specific pathologies (syndromes) can be associated with problems in the epigenetic machinery, and epi-genetics is fundamental to chromatin structure, those diseases have become generically known as diseases of chromatin For example, abnormal DNA methylation can cause errors in genomic imprinting, with an increased risk of Angelmann’s syndrome [7] However, epigenetic problems are also implicated in many more frequently-occurring diseases, such as cancer
Many cancer types have been shown to have gains in methylation at CpG islands in the promoters of some key genes Such modifications are associated with tran-scriptional inactivation [8] The gains in DNA methyla-tion, or hypermethylamethyla-tion, are responsible for the underexpression of tumour suppressors such as p16INK4a and BRCA1 [5] Early methylation of DNA may be a sign of tumorigenesis, as happens to the Wnt pathway in colon cancer [9], and DNMTs are often overexpressed in solid and wet cancer types [10] Muta-tions and amplification of the androgen receptor gene, without loss of gene expression, play a key role in the development of advanced, androgen-independent pros-tate cancer [4] Methylation of the androgen receptor promoter is prevalent in androgen-independent prostate cancer, but less so in androgen-dependent prostate can-cer [4] As well as hormonal genes, cell cycle genes are also affected in prostate cancer; an example is the methylation-mediated inactivation of the CDKN2A gene [4] Methylation also goes awry in haematopoietic
Trang 3malignancies, and hypermethylation of p16INK4a has
been observed in non-Hodgkin’s lymphoma, multiple
myeloma, and acute lymphocytic leukaemia [8]
It is widely accepted that DNA methylation should,
in the right circumstances, be a target for clinical
treat-ment Accordingly, nucleoside inhibitors that inhibit
DNA methylation, such as azacitidine, decitabine, and
zebularine, have been developed All three are cytidine
derivatives that irreversibly inhibit DNMTs [11] As
decitabine contains a deoxyribose group, it is
incorpo-rated into DNA [12] However, because azacitidine
contains a ribose group, it is initially incorporated into
RNA [12] Incorporation into DNA occurs when
aza-citidine is converted into 5-aza-2¢-deoxycytidine
diphosphate by ribonucleotide reductase, which is then
phosphorylated, the triphosphate form being
incorpo-rated into DNA in place of the natural base cytosine
[12] The use of such analogues results in the global
depletion of DNMTs and a subsequent reduction in
DNA methylation
Although DNA methylation is the most studied
epi-genetic modification in terms of clinical diagnostics,
the mechanism is also important for histone
modifica-tions DNMTs can interact with histones in two ways
First, DNA methylated by DNMT can attract proteins
such as MeCP2 that are able to recruit histone
deacet-ylases (HDACs); and, second, DNMTs can themselves
directly recruit HDACs to help silence gene expression
[4] Most of the literature on interactions with
methy-lated DNA has centred on the core histones H2A,
H2B, H3, and H4, but a complete picture of epigenetic
modifications cannot be obtained until linker histone
PTMs have been factored in This review analyses the
research effort expended thus far on linker histone
PTMs For consistency, amino acid positions in
a sequence are referred to by their actual genomic
position, as given in Swiss-Prot and similar databases
Structure and function of the linker
histone variants
Location and structure of the linker histones
Historically, the location of the globular domain of the
linker histone has been a matter of contention [13]
Currently, although there is a good degree of
agree-ment about the overall parameters of the fibre formed
by folding the zig-zagging chain of nucleosomes in
inactive chromatin, the location of the linker histone
in relation to the nucleosome core particle and linker
DNA is still not known to high resolution (Fig 1)
However, recent studies [14–16] suggest that the linker
histone is close to the dyad axis of the core particle at
the entry and exit of the DNA Similarly, the geometry
of one nucleosome in the fibre relative to a DNA-con-nected nucleosome is also unknown [17–20]
The structure of the linker histone H1 in humans is characterized by a relatively short N-terminal tail, a longer C-terminal tail, and a conserved globular domain [21] This model extends to most other organ-isms, two exceptions being Tetrahymena thermophila and Saccharomyces cerevisiae [22] The linker histone H1 variants show the greatest diversity when compared
to the core histones There is also a great diversifica-tion in the H1 variants within a single species such as
H sapiens, predominantly in the N-terminal and C-ter-minal tails, with, as stated, a conserved globular domain However, when similar H1 variants are com-pared between species, there is a remarkable similarity The nearer the species, the less is the divergence, such that H1.2 in Pan troglodytes has just one amino acid difference from its human counterpart, and the H1.4 variant in the human and Mus musculus (hereafter
‘mouse’) genomes has 93.6% sequence identity (Fig 2) The reason for this is that the H1-variant genes within a species are paralogues, originating from gene duplication events, whereas the same H1 gene between species is an orthologue, originating from an ancestral gene [23] In humans, the variants consist of the following: the somatic subtypes, H1.1–H1.5; a spermatogenesis subtype, H1t; an oocyte-specific subtype, H1oo; and a replacement subtype, H1o H1.1– H1.5, along with H1t, are known as the replication-independent group, and are mainly expressed in S-phase The remaining two, H1oo and H1o, are known
as the replication-dependent variants H1.1–H1.5 and
Fig 1 The possible locations of the linker histone in relation to the nucleosome core particle The globular domain of the linker histone will be located either symmetrically (left image), or asymmetrically (right image) [14,15] Colour assignments are as follows: magenta, nucleosome core particle; blue, 146 bp of DNA; red, globular domain of linker histone H1; green, 22 bp of linker DNA; orange,
11 bp of linker DNA.
Trang 4H1t reside on the short arm of chromosome 6; H1oo
is located on chromosome 3 and H1oon chromosome
22 H1.2 and H1.4 predominate in most cell types The
affinity of the various types seems to depend on their
C-terminal tails H1.1 and H1.2, with the shortest
C-terminal tails, a low density of positively charged
residues, and the lowest number of cyclin-dependent
kinase (CDK) sites, have the lowest affinity for
chro-matin A CDK site is identified by the consensus
sequence (S⁄ T)PXZ, where X is any amino acid and Z
is a basic amino acid H1.4 and H1.5, with longer
C-terminal tails and more than two (S⁄ T)PXZ sites,
have the highest affinity for chromatin With the
high-est content of positively charged residues, H1.3 has an
intermediate affinity for chromatin [23] The precise
functions of the H1 variants within a cell are only just
starting to be elucidated
Functions of the linker histones
It was evident from work with unicellular organisms
that the linker histones were not critical for growth
and cell division [23–25] Following these experiments,
it was speculated that the H1 variants were not global
repressors of transcription, and this has now been
shown to be the case [25] Depletion of H1 in
mam-mals causes significant changes to chromatin structure
When chromatin is depleted of H1, there is a reduction
in the nucleosome-repeat length globally and a
reduc-tion in local chromatin compacreduc-tion [25] The reducreduc-tion
in repeat length arises from having fewer than one
linker histone per nucleosome [25] Depletion of H1 in
mammals also causes a reduction in H3 Lys28
acetyla-tion, with a smaller reduction in H3 Lys28
trimethyla-tion, and also leads to a reduction of methylation at CpG islands in some of the H1-regulated genes [25] The H1 variants tend to associate with specific tran-scriptional regulators [23] For example, H1.1 specifi-cally associates with BAF, which regulates chromatin structure [23], and H1.2 has been shown to associate with p53 [26] It is thought that the specificity of indi-vidual variants stems partially from their sequence diversity, but mostly from PTMs [23] Thus, the evi-dence emerging is that the H1 variants have specific functions First, individual H1-variant knockout mice gave rise to specific phenotypes, with distinct effects on gene expression and chromatin structure [27] Second,
in the knockout mice referred to, there was no equal upregulation of the remaining variants, with only par-ticular variants being able to compensate Third, there are differences in the localization of the H1 variants within the nucleus, and there are variations in their relative amounts between different cell types [28] Fourth, the H1 variants have different affinities for chromatin, can be recruited to specific transcription factors, and, as we shall see, have particular PTM patterns
Significantly, H1.2 is now associated with an extra-nuclear function This variant will, upon a DNA dou-ble-strand break, translocate to the cytoplasm and then permeabilize the mitochondrial membrane, causing the release of apoptotic compounds [29–31] It has been shown that H1.2 in a cell infected with a virus displays
an increase in mobility [32], and this property may play
an important role in the treatment of cancer [33]
A specific mutation in H1.4 was detected in Raji cells, but was not detected in 103 healthy individuals
or other Burkitt’s lymphoma cell lines [34] This could
ARMCX4_HUMAN H1FX_HUMAN
H1F0_HUMAN HIST1H1T_HUMAN HIST1H1A_HUMAN HIST1H1B_HUMAN
HIST1H1C_HUMAN HIST1H1E_HUMAN
HIST1H1D_HUMAN
H1fx_MOUSE
Hist1h1t_MOUSE Hist1h1a_MOUSE Hist1h1b_MOUSE
Hist1h1c_MOUSE Hist1h1e_MOUSE Hist1h1d_MOUSE XP_899364_MOUSE
Fig 2 Phylogeny tree of human and mouse linker histones Speciation events are indicated by blue dots and gene duplication by red dots HIST1H1C and HIST1H1E are the genes that code for human linker histones H1.2 and H1.4, respectively Note how the human HIST1H1C and HIST1H1E genes have a last common ancestor that is a duplication node, which makes these two genes paralogues However, HIST1H1C in human and mouse originate from a speciation node, and are therefore orthologues The phylogeny tree was generated by
TREEFAM [69].
Trang 5be an example of H1 sequence variation acting as a
marker for a particular phenotype However, the main
aim of Sarg et al [34] was to demonstrate the use of a
particular chromatography technique, rather than to
firmly establish H1 sequence variation with disease
Thus far, then, no in-depth analysis has been
per-formed that attempts to correlate linker histone
sequence or PTM variation with disease This is not
surprising, especially in the case of the latter, as there
is a potential for many permutations Nevertheless,
this must be the next phase in the work on H1 PTMs
H1ois a general differentiation-dependent linker
his-tone, and has a similar sequence to the avian H5
vari-ant H1o will accumulate in a cell, reaching a peak at
terminal differentiation, being initially synthesized in
oocytes and early embryos [35]
Epigenetic control of linker histones was discovered
relatively early, with studies of synchronously dividing
nuclei in the plasmodia of Physarum polycephalum
[36], when it was shown that phosphorylation of linker
histones is strongly implicated in cell cycle control and
that phosphorylation is a precursor of mitosis It is
now widely accepted that patterns of PTMs on the
core histones can influence transcriptional activity For
example, acetylation of H3 Lys10 has an inverse
rela-tionship with the amount of DNA methylation [37,38]
As it is also known that the linker histones can affect
DNA methylation [25,39], it is not unreasonable to
conclude that there must be key PTM patterns that
govern – or at least significantly contribute to – the
function of H1, as they do in the core histones
Although much work has been done to identify specific
PTMs in the linker histones, there is still a gap in our
knowledge of how these affect function
Phosphorylation of CDK and non-CDK
consensus sites
A PTM pattern for mitosis?
Early work on identifying the sites of phosphorylation
in core and linker histones indicated that there was no
correlation between cell cycle status and the number or
location of phosphorylation sites [40] Later work,
how-ever, has shown that this is not the case [41], and that
the H1 linker histone of human lymphoblastic T-cells
has phosphorylation states that correlate with the
phase and mitosis stages of the cell cycle During
inter-phase, it was found that the H1.5 variant was
phosphorylated at Ser18, Ser173 and Ser189, which all
reside in a CDK consensus motif of the form (S⁄ T)PXZ,
as previously defined It was found that, during mitosis,
the same three serine phosphorylations were present,
but were also accompanied by phosphorylations at Thr11, Thr138, and Thr155 The first of these three threonines is not located within a TPXZ consensus sequence, but the latter two are The same pattern of cell cycle dependency of phosphorylations was found in the linker histone variants H1.2, H1.3, and H1.4 So, for all tested linker histone variants, it was established that only serines were phosphorylated during interphase, but
in mitosis, threonine residues were additionally phos-phorylated It was found that, during interphase, the human lymphoblastic T-cells had a proportion of H1.5 molecules monophosphoryated at a particular residue and a smaller proportion that was monophosphorylated
on another residue It was also found that the ratio of these two subgroups of H1.5 occurred in other cell types During mitosis, it was found that H1.5 existed as two species with five phosphorylations either on Thr11, Ser18, Thr138, Ser173, and Ser189, or on Thr11, Ser18, Thr155, Ser173, and Ser189 Therefore, it was concluded that Thr138 and Thr155 of H1.5 can never be phosphor-ylated at the same time There is support for this hypothesis [42], where the only phosphorylations to be found on H1.5 were at Thr138 and Thr155 If these two modifications occurred at the same time, it would be reasonable to expect that they would be found in equal abundance However, whereas the H1.5 peptide with a phosphorylation on Thr155 was readily detected, that with a phosphorylation at Thr138 could only be detected after methanolic HCl was used to convert car-boxylic groups to their corresponding methyl esters Thus, the suggestion is that H1.5 exists as two separate species, with either Thr138 or Thr155 phosphorylated, but not both Wisniewski et al [43] (Table 1) identified phosphorylation at Thr138 on H1.5, but not at Thr155, even though, like Sarg et al [41] and Garcia et al [42], they used HeLa cells Wisniewski et al [43] agree with Sarg et al [41] that Ser18 of H1.5 is phosphorylated, but could find no such modifications at Thr11, Ser173 or Ser189 in human or mouse tissue
Correlation of N-terminal tail PTMs with function Sarg et al [41] could not detect any N-terminal tail phosphorylations for H1.2, H1.3 and H1.4 for cells that were in the interphase part of the cell cycle The reason given is that there are no (S⁄ T)PXZ motifs in the N-ter-minal tails of those variants This is in conflict with the work of Garcia et al [42], who found phosphorylations
on H1.2 at Thr31 and Ser36, on H1.3 at Ser37, and on H1.4 at Ser2, Thr4, Thr18, Ser27, and Ser36 In com-paring the work of Wisniewski et al [43] (Table 1) and Garcia et al [42], it can be seen that: H1.4 is phosphory-lated at Thr18, but not on Ser2 or Thr4; H1.3 is
Trang 6similarly phosphorylated at Ser37; H1.2 is not
phos-phorylated at Thr31 Thus, it is possible to conclude
that phosphorylation of the N-terminal tails of the H1
variants does occur, but why do some researchers detect
them but others do not? Before addressing this question,
there is also the issue as to what variety of PTMs occur
in the N-terminal tails Sarg et al [41] found that only
H1.5 was modified in the N-terminal tail in human cells;
more recent work by Wisniewski et al [43] and Snijders
et al.[44] (Table 1) has, excluding the N-terminus acety-lations, identified eight N-terminal tail PTMs in cultured human cells and seven in Gallus gallus (hereafter
‘chicken’) erythrocytes, respectively Although the over-all number of these modifications is low, the density of modifications is much the same as in the rest of the lin-ker histones It is the shortness of the N-terminal tails
Table 1 Alignment of chicken, mouse and human PTMs Each PTM-containing sequence in humans has been aligned with the similar sequence in the other two species, which may or may not contain a PTM Symbols: a, acetylation; d, deamidation; f, formylation; m, methyl-ation; p, phosphorylmethyl-ation; u, ubiquitinmethyl-ation; 2m, dimethylmethyl-ation; 2m ⁄ f, dimethylation and ⁄ or formylation; a ⁄ m, acetylation and ⁄ or monomethy-lation; a ⁄ f, acetylation and ⁄ or formylation; 2m ⁄ f, dimethylation and ⁄ or formylation The ‘a-’ in the second column (first PTM location) refers
to N-terminal acetylation The data for human and mouse were taken from [43], and the data for chicken were taken from [44].
Chicken
Mouse
Human
Chicken
Mouse
Human
Trang 7that accounts for the low numbers Therefore, it is now
possible to say that the N-terminal tails do, in fact,
contain a range of different types of PTM
Returning to the issue of abundancy, Garcia et al
[42] had to use two techniques to increase the number
of peptides with certain PTMs First, protein digests
were treated with propionylation reagent to convert
monomethylated and endogenously unmodified amino
groups on the side chains of lysine residues and
N-ter-mini to propionyl amides Second, it was found that
certain phosphorylated peptides (predominantly
origi-nating from the N-terminal tail) were of such low
abun-dance that, in order to obtain stronger spectra, they
were subjected to enrichment by immobilized metal
affinity chromatography [45] Prior to using this
tech-nique, Garcia et al [42] converted the carboxylic
groups to methyl esters with the use of methanolic
HCl This modification decreases the strength of
bind-ing of nonphosphorylated linker histones to the
immo-bilized metal affinity chromatography column They
can then be washed off before eluting the
phospho-linker histones It should be noted that the practice of
methyl esterification is currently not widely used, owing
to problems with side reactions [46] Deterding et al
[47], who only analysed the H1.4 linker histone variant
in human and mouse tissue, found that, in both species,
only Thr18 in the N-terminal tail region was
phosphor-ylated However, the signal for this in the human tissue
was so noisy in comparison with signals for
phosphory-lated residues in the C-terminal tail that confirmation
could only be obtained by reference to the mouse signal,
which was less noisy With high-mass accuracy mass
spectrometers now readily available, this should be less
of a problem in the future We can perhaps, then,
hypothesize that although N-terminal tail modifications
of linker histones do occur, they may be less abundant
than those of the globular domain and C-terminal tail
The degree to which they are less abundant remains to
be established, as does the biological significance of that
fact
Correlation of C-terminal tail PTMs with function
Sarg et al [41] found that H1.2, H1.3 and H1.4 had
fewer phosphorylations in the C-terminal tail region
than H1.5 In H1.2, Ser173 is phosphorylated, as are
Ser189 in H1.3 and Ser172 and Ser187 in H1.4 Garcia
et al [42] found that H1.2 was phosphorylated at
Ser173, Thr146, and Thr154 It is worth noting that
these two latter modifications occur in TPXZ motifs
and, if Sarg et al [41] is correct, may reflect the fact that
the cells are in mitosis The H1.3 variant in Garcia et al
[42] was found to be phosphorylated at Ser189, Thr147,
Thr155, and Thr180 The latter threonine is in a non-CDK consensus site, and seems to be an anomaly The occurrence of phosphorylated Thr147 and Thr155 in the C-terminal tail of H1.3 probably has the same explanation as their occurrence in H1.2 Deterding et al [47] identified the same H1.4 C-terminal tail phosphory-lations in human and mouse tissue as Sarg et al [41]
As can be seen from Table 2, Wisniewski et al [43] detected no phosphorylation on Ser172 of H1.4 For H1.2 and H1.3, the work of Wisniewski et al [43] agrees with that of Garcia et al [42], noting that in Table I of the former paper the phosphorylation on Thr173 of H1.2 is a typographical error (should be Ser174) Table 2 lists the phosphorylations that have been detected more than once in the research described above It therefore represents those sites that are most likely to be modified at reasonable levels of abundance Most mass spectrometry (MS) analysis of PTMs has been performed on cultured cell lines It has been shown that methylation can be readily detected in tis-sue, but is extremely rare in cultured cells [43] Other potential problems with cultured cells are discussed later
Analysis of other PTMs
It is now accepted that acetylation and methylation of the core histones are key regulators of transcription Although phosphorylation of the linker histones has attracted the most attention, recent results from vari-ous MS analyses have shown that acetylation and methylation are also key modifications of the linker histones
Lysine and N-terminus acetylations Acetylation of the Na-terminus involves the cotransla-tional cleavage of a methionine, followed by acetyla-tion of the second residue of the N-terminal tail Initial, non-MS work showed that only H1.0 in human cells and H5 in avian cells existed in forms that had the Na-terminus both acetylated and unacetylated [48,49] However, it has now been shown that the
Table 2 Phosphorylations of the human linker histones Those modifications that have been identified more than once in the papers referred to are shown The numbers in parentheses refer to the appropriate references.
H1.2 (P16403) H1.3 (P16402) H1.4 (P10412) H1.5 (P16401) pS36 [42,43],
pT146 [42,43]
pS37 [42,43], pT147 [42,43]
pT18 [42,43,47], pS172 [41,42,47]
pS18 [41,43], pT138 [41–43] pS173 [41–43] pS189 [41–43] pS187 [41–43,47] pT155 [41,42]
Trang 8former modification not only occurs on all the linker
histone variants in human and chicken [42–44], but
that is also the most abundant type of acetylation
Linker histones do exist with their Na-termini
unacetylated, but are significantly fewer in number
Although the function of Na-terminus acetylation is
unclear, it was noted in earlier work [48,49] that
whereas the ratio of acetylated to unacetylated
Na-ter-mini remained constant in avian H5 erythrocytes from
newly hatched and adult chickens, it increased for
H1.0 in ageing rat tissues As H1.0 is associated with
differentiation and is most abundant in terminally
dif-ferentiated cells, there may well be a correlation
between Na-terminus acetylation and differentiation
Not all methionines are cotranslationally cleaved and
can therefore become acetylated This process is not
widespread, but it has been shown to be present in
recent work [43,44] (Table 1)
Garcia et al [42] found that H1.2, H1.3 and H1.4 in
human cells all had just one site of lysine acetylation,
and on the same residue, Lys64 (or Lys65, depending
on the variant) Considerably more acetylations – up
to nine – were found in H1.4 [43] (Table 1) The
glob-ular domain was found to contain the largest number
of acetylations: Lys52, Lys64, Lys85 and Lys97; all of
these are thought to be involved with DNA binding
[43] The abundance of lysine methylation can be
attributed to the fact that certain types of human cell
were rapidly proliferating In mouse tissue, the spleen
was found to contain the most acetylations, because
lymphopoiesis is associated with rapid cell division In
mouse tissue containing mostly differentiated cells, e.g
liver, the number of acetylations was much lower
Lysine methylation
Methylation of lysine in linker histone proteins has
been reported in human HeLa cells [42,43], although
there is a difference in the number of identified sites
Garcia et al [42] found that, in H1.4, Lys26 and Ser27
were simultaneously methylated and phosphorylated,
respectively The point is made that the
aforemen-tioned residues occur in the sequence KARKSAGA
(residues 23–30), which is similar to one found in the
core histone H3 (VARKSAPA, residues 25–31)
Within H3 there are well-known adjacent methylation
and phosphorylation sites at Lys9-Ser10 and
Lys27-Ser28 that are involved with transcription Thus, the
same argument is made for H1.4, by virtue of it, too,
having adjacent methylation and phosphorylation sites
There is support for these assertions [49]; however,
Wisniewski et al [43] (Table 1) found no methylations
in this region of H1.4, or of the other variants
Puta-tive sites of methylation were identified at Lys169 in H1.4 and H1.5, or Lys170 in H1.3 In mouse, the H1.4 variant has no modifications at Lys26, and at posi-tion 27 there is an alanine, rather than a serine [43]
Ubiquitination and formylation For the first time, ubiquitinations of the histone lin-ker protein were identified by MS [43] (Table 1) It was found that Lys46 was ubiquitinated in H1.2, H1.3 and H1.4 in human HeLa cells, but not in MCF7 cells In mouse tissue, Lys116 of H1.1 and Lys46 of H1.2 and H1.3 in the spleen were the only sites of ubiquitination The fact that both cell lines were cultured in the same growth medium, and have the same doubling time, increases the probability that these ubiquitinations are unique for HeLa cells Another putative novel modification found is that of formylation [43]; H1.1, H1.2, H1.3, H1.4 and H1.5 in human MCF7 cells were all found to be formylated Whereas H1.5 was uniquely formylated on Lys88, the others were similarly modified on Lys90 (H1.2 num-ber) In mouse tissue, the most frequently occurring formylation site was Lys63 Formylation of lysines has been shown to arise as a result of oxidative dam-age to DNA [50] Snijders et al [44] (Table 1) identi-fied a single site of lysine dimethylation at Lys71, but were unable to distinguish between dimethylation and formylation
Perturbation of phosphorylations
by external mechanisms
It has been clearly shown in several studies that phos-phorylation can be imposed by external influences [41,47,51] This is an important phenomenon, and means that those processes will be able to influence the cell cycle
Garcia et al [42] found that growing T thermophila cells had site-specific higher levels of phosphorylation than when they were being starved Phosphorylated Thr47 was enriched in growing cells by a factor of seven as compared with starved cells Similarly, phos-phorylated Thr35 was also found to be enriched by a factor of four in growing cells It is perhaps impor-tant that these two residues occur in (S⁄ T)PXZ motifs (as defined) It was found that in Drosophila melanog-aster embryos, phosphorylated Ser11 was associated with mitosis and that the proportion of this post-translational modification decreased as those embryos aged [52] These experiments clearly show that the amount of phosphorylated H1 is a function of cell activity Villar-Garea and Imhof [52] concluded that,
Trang 9in mammalian cells, phosphorylation in mitosis only
occurs in the N-terminal tail However, it has been
shown that, during mitosis, phosphorylations also
occur in the C-terminal tail, and in (S⁄ T)PXZ motifs
[41]
Deterding et al [47] analysed human UL3 cells
(derived from the osteosarcoma cell line U2OS) treated
with dexamethasone, CVT313, or CGP74514
(dexa-methasone is a synthetic glucocorticoid used in the
treat-ment of autoimmune diseases; CGP74514 and CVT313
are CDK1 and CDK2 inhibitors, respectively), and
looked at the phosphorylation state of the linker
histones using MS It was found that treatment with all
of these compounds reduced the global level of
phos-phorylation of the H1.2 and H1.4 isoforms Although
the work did not go so far as to establish site-specific
patterns of phosphorylation related to compound and
dosage, it did establish, by the use of antibodies, that the
level of phosphorylated Thr18 in the N-terminal tail of
H1.4 was reduced by treatment with any one of these
three compounds
In the examples discussed, extensive use was made of
cultured cells There is, of course, nothing wrong with
this in the substantial majority of cases However,
culturing cells may have an impact on overall PTM
pat-terns In particular, differences may arise in the
struc-tural and biochemical properties of a cultured cell (and
hence PTM patterns), particularly when the cells are
grown on a monolayer 2D medium Normal cells grown
in such a medium can display a nuclear structure that is
different to their in vivo structure [53,54] Use of a 3D
culture medium better mimics the extracellular matrix
[53], and the cells should therefore have a nuclear
struc-ture that is more representative of the in vivo strucstruc-ture
If the nuclear structure of cultured cells can be altered,
then there will be a concomitant change in the
biochem-istry of those cells [54]
Existence of global PTM patterns
in different cell types
The strong evidence emerging is that specific PTM
pat-terns occurring on DNA and particular sets of proteins
can be correlated with cell type The inference from
this is that there will be a change in a cell’s PTM
pat-tern when it progresses from a normal to diseased
state, and that, accordingly, such changes can be
detected and made the target of clinical intervention
[55,56] However, although changes in the PTM
pat-terns of particular proteins between normal and
dis-eased cells have been detected [55,56], can the concept
be taken to the lower level of chromatin? This has
already been shown to be the case in three sets of
mouse cells [57] A proportion of murine embryonic stem cells, embryonic fibroblasts and embryonic carci-noma cells were grown in standard cell growth med-ium, with the remainder having trichostatin A, an HDAC inhibitor, added, the aim being to mimic dis-ease-induced hyperacetylation of histones Two changes were detected: (a) PTM patterns alter for ‘dis-eased’ cell lines; and (b) those cell lines have unique and specific PTM patterns The PTMs that were being monitored resided on the H3 and H4 core histones However, there is nothing to suggest that the linker histones should not display the same global property, and such changes involving just phosphorylation were discussed in an earlier section The process of disease-induced alteration of global PTM patterns has also been observed in human colon adenocarcinoma cell lines [58] MS is of fundamental importance when it comes to detecting combinations of PTMs on a single protein This has been demonstrated on human embry-onic stem cells [59]
Table 1 shows the PTMs detected in human, mouse and chick cells [43,44] From the data for chick cells, it can clearly be observed that six of the linker histones have identical PTMs at amino acids
71, 84, 147, and 189 Unlike human linker histones, the chick variants have very similar sequences, and
it would be easy to dismiss this observation with the argument that near-identical sequences will inevitably have the same PTM pattern However, this line of argument would ignore two important facts First, the chick linker histones can – like their human counterparts – be associated with specific and differ-ent functions; and, second, the cells, being erythro-cytes, are terminally differentiated It is therefore possible to say that the PTM patterns in Table 1 for chick cells can be identified as being unique for terminally differentiated chick erythrocyte cells How-ever, as previously mentioned, although MS can detect many modifications, it does have restrictions, such as difficulty in distinguishing PTMs that have near-identical masses [44]
It was mentioned earlier that mouse tissue with the higher replication rate has higher levels of linker his-tone acetylation This can be taken as evidence that unique linker histone PTM patterns also exist in live tissue, and not just in cultured cells [43] It is possible
to come up with a list of PTMs that are either absent
in MCF7 cells and present in HeLa cells, or present in MCF7 cells but missing in HeLa cells (Table 3) As mentioned earlier, the two cell lines were grown in the same media, so it is clearly possible to distinguish the two human cell lines by comparison of the PTMs on their linker histones
Trang 10The evidence accumulating from MS and other
bio-physical experiments considerably strengthens the
hypothesis that not only can the linker histone
vari-ants be associated with specific functions, but PTMs
thereon can also uniquely identify particular cell
types Indeed, this is now becoming the accepted
par-adigm [60] Those functional capabilities even, as in
the case of H1.2, have an extranuclear reach PTMs
modulate the range of functions covered by the linker
histone variants and, by analogy with the core
hi-stones, each of those functions will have a distinct
PTM signature
The extraction of the H1 variants from cells or
tis-sue has the potential to alter PTM states It is
there-fore necessary that gentle procedures should be used
Acid extraction of H1, although efficient, can instigate
the reversal of labile PTMs, such as histidine
phos-phorylation [61–63] A range of different acids have
been used to extract linker histones, including
per-chloric acid [43,52], sulfuric acid [42,64], and
hydro-chloric acid [65] Extraction by salt is gentler and just
as efficient at isolating H1 histones [44,63] Although it
is the extraction of the linker histones from tissue and
cell cultures that has a high potential to alter PTMs,
purification of the extracts can be considered to be a
benign step in the process of isolation Purification of
histones in general can involve a myriad of processes,
and these have been discussed in detail elsewhere [66]
It was found in one case that phosphorylation of
threonines in H1.5, namely Thr138 and Thr155, was
associated with cells in mitosis [41] Some support was provided for this principle [42], where both of these residues were found to be phosphorylated, although Thr138 only with some difficulty In another case, it was found that only Thr138 was phosphorylated [43] There is a clear conflict here, so is it possible to distin-guish the respective cases? In the first case, the cell lines were specifically treated to put them into mitosis; this was not so in the second and third cases How-ever, in two cases [42,43], it seems unlikely that there would have been a significant number of cells in mitosis In addition, in MS experiments, absence of a condition is not proof of its nonexistence
Whereas, initially, it was found that PTMs in the N-terminal tail of most of the H1 variants did not occur – an exception being H1.5 [41] – it is now clear that there are, in fact, numerous modifications, although they seem to be less abundant [43,44] Acety-lation of the N-terminus of H1 is the most abundant modification, although it has been shown that the un-acetylated form does exist [43,44] There seems to be no consensus on the significance of N-terminus acetylation However, as the amount of H1.0 with an acetylated N-terminus has been observed to increase in ageing rat tissue [48,67], and given that H1.0 is most abundant in terminally differentiated cells, there may be a link between N-terminus acetylation and differentiation From work on human cells and mouse tissue [43], it can be clearly seen that the amount of acetylated H1 is
a function of the replication rate, with most acetyla-tions occurring in rapidly replicating tissue, and the least in the most slowly replicating tissue Confirma-tion of this comes from work on chicken erythrocytes [44], where it was found that there are relatively few acetylations in the chicken H1 variants This is because the erythrocyte sample material comprises cells that are largely terminally differentiated
Phosphorylation is correlated with growth rates [64] and can be significantly increased The addition of compounds that influence the cell cycle will cause changes in the levels of phosphorylation of the linker histone isoforms [47]
Particular cell lines can be identified with particular patterns of PTMs on the core and linker histones [43], and variations in those patterns – having been associ-ated with oncogenic progression [68] – are primary candidates for pharmacological intervention
Taken as a whole, the data from the experiments discussed herein clearly show that it is possible to asso-ciate specific PTM patterns in the linker histones with particular functions, and that unique patterns of PTMs exist for diseased cells when compared with normal cells, and between cells of different types
Table 3 PTMs that uniquely identify human MCF7 from HeLa
cells ‘+’ indicates a modification that is present on MCF7 linker
histones but is missing from HeLa linker histones ‘ )’ indicates a
modification that is missing from MCF7 linker histones but is
pres-ent on HeLa linker histones The symbols in italics are as defined
for Table 1.
MCF7 PTMs compared with HeLa PTMs
H1.5: +aK17
H1.1: )aK22
H1.2, H1.3, H1.4: +aK34
H1.2, H1.3, H1.4: )uK46
H1.2, H1.3, H1.4: )aK52
H1.1, H1.2, H1.3, H1.4: )aK64
H1.1, H1.2, H1.3, H1.4: )aK85
H1.5: +a ⁄ fK88
H1.1, H1.2, H1.3, H1.4: )fK90
H1.1, H1.2, H1.3, H1.4: +aK97
H1.2: )pT146
H1.2: +pT165
H1.2: +aK169
H1.4: +aK169