1
Introduction
1.1
Discovery
of Sirtuins
The founding
member of sirtuin is the yeast-silencing information regulator 2 (SIR2)
protein, one of four proteins (SIR1–4) required for silencing
the mating-type information loci in yeast.
1
SIR2–4, but not SIR1, are also required for gene silencing
at telomeres.
2
SIR2 also mediates gene
silencing at the rDNA (rDNA) loci, which was shown to be independent
of other SIR proteins.
3,4
Immunofluorescence imaging showed
that SIR2 is mainly in the nucleolus and telomeres in yeast.
5
It was demonstrated that the silenced genetic
loci have low histone acetylation levels compared to loci that are
not silenced. Mutation in Sir2–4 increased histone acetylation
levels and overexpression of SIR2 but not other SIR proteins led to
decreases in histone H4, H2B, and H3 acetylation.
6
Analyzing the sensitivity of yeast chromatin to micrococcal
nuclease and dam methyltransferase indicated that Sir2 mutation affects
the chromatin structure in the rDNA and mating-type loci.
7
These interesting discoveries on SIR2 were
further elevated by two discoveries made by Guarente and co-workers.
The first one was that SIR2 is important for the replicative life
span of yeast cells,
8
a finding that was
later extended to higher eukaryotic species,
9,10
although
the role of sirtuins in life span is highly controversial
11
and may depend on genetic background and diet
conditions.
12,13
The second discovery was that
SIR2 is an NAD-dependent histone deacetylase (Figure 1),
14
which established SIR2 as
a mechanistically novel lysine deacetylase and revealed the connection
between gene silencing and cellular metabolism. Indeed, it was later
found that the life span extension effect of SIR2 is dependent on
NAD level and NAD metabolism.
15−17
Several other groups similarly
reported the enzymatic activity of SIR2.
18
These landmark discoveries established sirtuins as important players
in epigenetics and triggered the explosion of research interest in
sirtuins.
Figure 1
Enzymatic function of sirtuins. (A) NAD-dependent protein lysine
deacylation activity of different sirtuins. (B) Enzymatic reaction
mechanism of sirtuins. (C) Structure of a ternary sirtuin-NAD-acetyl
peptide complex (PDB ID 2H4F). NAD, acetyl lysine, and the key catalytic His residue
are shown in stick representation. Bound zinc is shown as a gray sphere.
Protein structure picture is generated using PyMol.
1.2
Classification and Enzymatic
Activity of Sirtuins
Sirtuins are evolutionarily conserved
in all domains of life. On
the basis of sequence similarity, sirtuins from different species
are classified into at least four classes, classes I–IV.
19
Mammalian SIRT1–3 and all yeast sirtuins
belong to class I. Mammalian SIRT4 is in class II, SIRT5 is in class
III, while SIRT6 and SIRT7 are both in class IV.
The enzymatic
reaction mechanism for sirtuin-catalyzed NAD-dependent protein lysine
deacetylation (Figure 1) has been well understood
through a series of elegant biochemical and structural studies.
20,21
The conserved catalytic core of sirtuins consists of a zinc-binding
domain and a Rossmann fold domain (Figure 1C).
22−24
The active site lies at the interface of the two
domains. It was thought that the acetyl lysine peptide binds first,
followed by the binding of NAD.
25
Once
the tertiary complex is formed, the carbonyl oxygen of the acetyl
group attacks the C1′-position of the nicotinamide ribose,
displacing nicotinamide and forming the alkylamidate intermediate
(intermediate I, Figure 1B).
20
A conserved histidine residue then serves as a general
base to deprotonate the ribose 2′-OH, which then attacks intermediate
I at the carbonyl carbon, generating the 1′,2′-cyclic
intermediate (intermediate II, Figure 1B).
Intermediate II is then hydrolyzed to produce 2′-O-acetyl-ADP-ribose (2′-O-Ac-ADPR),
which can be nonenzymatically
isomerized to 3′-O-Ac-ADPR.
20,21
Using mechanism-based
inhibitors (thioacyl-lysine peptides), an S-alkylamidate
intermediate similar to intermediate I was captured in Thermotoga
maritima SIR2 (TmSIR2) and human SIRT3 crystal structures,
26,27
and an intermediate similar to intermediate II was captured in SIRT5
crystal structure.
28
Among the seven
mammalian sirtuins, only the class I members (SIRT1–3)
have shown robust deacetylase activity in vitro. SIRT4–7, in
contrast, have very weak deacetylase activity in vitro.
29,30
It has been proposed that some of them may function as ADP-ribosyltransferases.
30,31
However, this activity is also very weak in vitro, and its physiological
significance is still under debate.
32,33
Recently,
it was demonstrated that mammalian SIRT5, one of the three mitochondrial
sirtuins in mammals, functions to remove negatively charged acyl groups,
such as succinyl and malonyl, from protein lysine residues (Figure 1A).
34
Protein lysine
succinylation and malonylation were not previously known as common
protein post-translational modifications (PTMs). These initial studies
pointed out that they are widely occurring PTMs in both bacterial
and mammalian cells. Recent proteomic studies have identified close
to a thousand proteins that are succinylated.
35,36
Many substrate proteins of SIRT5 are metabolic enzymes, and interestingly,
SIRT5 can either activate or inhibit the substrate enzymes, depending
on the enzymes being modified.
35
These
studies also suggest that any abundant acyl-CoA molecule may be used
to modify proteins.
37
Indeed, protein lysine
glutarylation has recently been reported, and SIRT5 can also remove
glutaryl group from proteins.
38
Similarly,
protein lysine crotonoylation was also reported.
39
SIRT5 recognizes the negatively charged acyl groups
using an Arg
residue and a Tyr residue, which interact with the carboxylate group
via electrostatics and hydrogen-bonding interactions.
34
The Arg and Tyr residues are conserved in most class III
sirtuins, raising the possibility that all class III sirtuins with
the conserved Arg and Tyr residues may have the ability to remove
negatively charged acyl groups.
34
Indeed, E. coli COBB has been recently shown to efficiently hydrolyze
succinyl lysine.
40
However, different from
SIRT5, COBB also has efficient deacetylase activity in vitro, suggesting
that certain sirtuins can be promiscuous and multifunctional.
SIRT6, which also has weak and sequence-specific deacetylase activity
toward peptide substrates in vitro,
41−43
was shown to have more
efficient activity in removing long chain fatty acyl groups, such
as myristoyl and palmitoyl (Figure 1A).
44
This activity is similar to the previously reported
activity for the Plasmodium falciparum SIR2 protein,
PfSIR2A.
45
One physiological substrate
for the defatty-acylation activity of SIRT6 is tumor necrosis factor
α (TNFα), which was known to be myristoylated on K19 and
K20.
44
SIRT6 promotes the secretion of
TNFα by defatty-acylation of K19 and K20 of TNFα. Although
SIRT6 is a poor deacetylase in vitro, the deacetylation of histone
H3K9 and H3K56 by SIRT6 has been intimately linked to the physiological
function of SIRT6 in genome maintenance,
43
metabolism,
46
and inflammation.
47
Recent studies showed that the deacetylase activity
of SIRT6 could be activated by nucleosomal substrates
48
or free fatty acids.
49
These
findings may partly explain the discrepancy between in vitro and in
vivo studies on SIRT6 activities and also raise the possibility that
SIRT6 acts as a metabolic sensor with multiple switchable functions.
SIRT1–3 can also remove long chain fatty acyl groups in vitro.
49
However, the physiological relevance remains
to be confirmed. Similar to SIRT6, SIRT1–3 may exhibit context-dependent
specificity toward certain acylation on Lys residues.
The different
activities of SIRT1–3 (class I, mainly deacetylation
but can also remove long chain fatty acyl groups), SIRT5 (class III,
desuccinylation, demalonylation, deglutarylation), and SIRT6 (class
IV, more efficient at defatty-acylation but can also remove acetyl)
demonstrate that different classes of sirtuins may have different
acyl lysine substrate specificity. Thus, the classification of sirtuins
based on sequence homology is informative of their biochemical functions,
especially acyl group specificity.
1.3
Biological
Functions of Sirtuins
Although sirtuins were initially studied
in the context of gene silencing
and aging, in mammalian cells, many other biological functions have
been revealed.
50−52
The biological function of sirtuins depends on the
substrate proteins they modify. Because mammalian sirtuins have a
very diverse set of substrates, it is not surprising that they affect
many different biological processes. Sirtuins regulate many aspects
of chromatin biology, such as transcription, recombination, and genome
stability, by modifying histones, transcription factors, and epigenetic
enzymes. Sirtuins also regulate metabolism by modifying a diverse
set of metabolic enzymes, both in the cytosol and in the mitochondria.
The defatty-acylation activity of SIRT6 on TNFα also revealed
that sirtuins can regulate protein secretion and membrane trafficking.
The diverse substrate proteins of sirtuins also dictate that sirtuins
may be involved in various human diseases, such as cancer, neurodegeneration,
diabetes, and other metabolic disorders.
50−52
Below, we will
focus on the role of yeast and mammalian sirtuins in epigenetic regulation.
2
Yeast Sirtuins in Epigenetic Regulation
2.1
Role of SIR2 in Regulating Transcriptional
Silencing
SIR2-mediated silencing is achieved by recruitment
of SIR2 to different chromatin locations. SIR2 recruitment to the
silent mating loci and telomeres requires RAP1 (repressor activator
protein 1), which binds to specific silencer DNA sequences (Figure 2A). RAP1 interacts
with SIR4, which in turn recruits
SIR2 and SIR3.
53
ORC1 (origin recognition
complex 1), a subunit of the origin recognition complex, is responsible
to recruit SIR1 at the silent mating-type loci and helps to stabilize
the silencing complex.
54−56
Once recruited to the silent mating loci and telomeres,
SIR2 is thought to deacetylate various histones (H2B, H3, and H4)
on multiple sites
6,57
in nearby nucleosomes, which
creates sites with higher binding affinity for SIR3,
58
leading to spreading of the silencing complex across the
heterochromatin regions
59−61
(Figure 2A). A histone H4K16 acetyltransferase, SAS2 (something about silencing
2), functions to oppose the role of SIR2 to prevent the spreading
of the SIR complex to euchromatin.
62,63
Paradoxically,
a zinc-dependent histone deacetylase (HDAC), RPD3 (reduced potassium
dependency 3), was also reported to be important for preventing the
spreading of SIR complex.
64,65
One proposal was that
RPD3 removes SIR2 acetyl lysine substrates, thus eliminating the production
of O-Ac-ADPR, which promotes SIR complex spreading.
65,66
However, the role of O-Ac-ADPR in SIR complex spreading is debated.
67
Figure 2
Models for the role of SIR2 in gene silencing in yeast.
(A) SIR2
and SIR complex at the silent mating-type loci and telomeric heterochromatin.
(B) SIR2 at silenced rDNA loci.
At the rDNA loci, SIR2 is recruited by NET1 (nucleolar-silencing
establishing factor and telophase regulator 1).
68
NET1, SIR2, and CDC14 together form a complex termed RENT
(regulator of nucleolar silencing and telophase exit).
68,69
RENT binds to rDNA in mainly two regions: in one of the nontranscribed
spacers (Nts1) and around the Pol I promoter. Binding to Nts1 requires
FOB1 (fork blocking less 1), while binding to Pol I promoter requires
RNA polymerase I
70
(Figure 2B).
How do SIR2-catalyzed histone deacetylation and
the SIR complex
repress transcription? Initial studies pointed to a model that the
SIR complex silences transcription by promoting the formation of a
more protected chromatin structure that is not accessible to the transcription
machinery.
7
However, later studies suggested
that silenced chromatin is actually permissive to activator binding
and preinitiation complex formation.
71,72
It was proposed
that SIR proteins silence gene transcription by blocking the transition
from transcription initiation to elongation.
73
It was further demonstrated that short cryptic RNA Polymerase II
transcripts are produced at the silenced rDNA loci. These cryptic
transcripts are terminated by the NRD1/SEN1 (nuclear pre-mRNA downregulation
1/splicing endonuclease 1) complex and degraded by the exosome. Disruption
of the NRD1/SEN1/exosome pathway leads to decreased gene silencing.
73
These studies point to a more complex model
on how SIR2 and SIR complex regulate gene silencing. Interestingly,
the involvement of RNA in gene silencing seems to be a general theme.
In the fission yeast, which has a very different gene-silencing mechanism,
noncoding RNA and the RNAi machinery are required for the formation
of heterochromatin.
74
2.2
Role of SIR2 in Regulating rDNA Recombination
SIR2,
as a member of the RENT complex, was also found to be important
to prevent recombination events at the rDNA loci.
75
Interestingly, available experimental evidence support
a model that the SIR2’s role in suppressing rDNA recombination
is achieved through regulating the transcription of noncoding RNA
under the control of bidirectional promoter called “E-pro”.
It is thought that bidirectional E-pro transcription eliminates cohesin
occupancy at the rDNA loci. SIR2, by suppressing E-pro transcription,
maintains cohesin attachment and thus suppresses rDNA recombination
between sister chromatids.
76,77
SIR2-dependent silencing
is not sufficient to inhibit recombination within the rDNA locus,
and it was demonstrated that perinuclear rDNA positioning also plays
an important role in rDNA stability.
78
The
perinuclear rDNA positioning is achieved via interaction between the
nucleolar Cohibin complex and two inner nuclear membrane proteins
HEH1 (helix extension helix 1) and NUR1 (nuclear rim 1).
78
2.3
Role of SIR2 in Regulating
DNA Repair
The SIR proteins are also required for repair
of double-strand DNA
breaks.
79
Deletion of Sir2–4 impaired the ability of yeast cells to repair double-strand DNA
breaks. Furthermore, the introduction of DNA double-strand breaks
led to the relocalization of the SIR complex from telomeres to DNA
double-strand breaks, which further led to derepression of telomeric
genes.
80,81
Similarly, mammalian SIRT1 was also found
to be important for repairing DNA double-strand breaks and undergo
DNA damage-induced relocalization.
81
It
was proposed that the DNA damage-induced SIRT1 relocalization and
the corresponding gene expression change may contribute to aging.
81
This proposal is in line with the “heterochromatin
island” hypothesis of aging proposed earlier.
82
A similar age-associated depression of silenced genes was
also reported in flies.
83
SIR complex
relocalization was also observed when the yeast telomeres and silent
mating-type loci clustering were disrupted.
84
Yeast telomeres and silent mating-type loci are clustered and form
foci in the nuclear envelope. The SIR complex localizes to these foci.
When the clustering was disrupted by deletion of yeast DNA repair
factor KU70 and ESC1 (establishes silent chromatin 1), the SIR complex
was released from the foci, which consequently changed gene transcription
(derepression of subtelomeric genes and repression of genes in other
locations).
84
These studies suggest
that SIR proteins may exist in limiting concentrations
and preferentially occupy high-affinity binding sites on chromatin.
However, under conditions that disrupt this normal localization or
under conditions that create higher affinity binding sites, the SIR
complex can relocalize to other places on the chromatin and lead to
repression or derepression of transcription.
What is the role
of histone acetylation/deacetylation in DNA double-strand
break repair? It has been well documented that histone acetylation
is required for the repair of DNA double-strand breaks.
85−87
It has been shown that localized acetylation and deacetylation occur
during homologous recombination to repair DNA double-strand break
introduced by the HO endonuclease.
88
Several
acetyltransferases (GCN5 and ESA1) and deacetylases (RPD3, SIR2, HST1)
were recruited to double-strand breaks, and deletion of GCN5 and RPD3
led to inviability after induction of HO endonuclease. Consistent
with this, it was observed that chromatin containing DNA double-strand
breaks underwent local expansion and decondensation.
89
The chromatin decondensation may allow DNA repair factors
to access the damaged DNA. In Drosphila, the acetylation
of phosphorylated H2Av by TIP60 (60 kDa Tat-interactive protein) is
thought to promote the exchange of phosphorylated H2Av by unphosphorylated
H2Av following DNA repair, which serves as a mechanism to indirectly
dephosphorylate H2Av at the damage site to complete DNA repair.
90
In mammals, the histone acetyltransferase HAT1
was also reported to function in DNA double-strand repair by promoting
histone turnover.
91
Whether such a mechanism
also applies in yeast and other eukaryotes is not clear. However,
histone H3K56 acetlylation, catalyzed by RTT109 and histone chaperon
ASF1, has been shown to be important for reassembly of nucleosomes
after DNA double-strand break is repaired.
92
Thus, even though a detailed picture is still unavailable, progress
toward such a picture is emerging.
2.4
Interaction
between SIR2 and Other Epigenetic
Markers/Enzymes
2.4.1
SIR2 Interacts with Other
Proteins That
Control Histone Acetylation
As discussed before, the histone
H4K16 acetyltransferase, SAS2, functions to oppose the role of SIR2
to prevent the spreading of the SIR complex to euchromatin.
62,63
BDF1 (bromodomains factor 1) bromodomain binds to acetylated histone
H3 and H4 and competes with SIR2. This is thought to be important
in maintaining a transcriptional active euchromatin.
93
These observations are easy to understand as histone acetylation
and deacetylation should have opposite effect.
The more puzzling
observation was that a zinc-dependent HDAC, RPD3, was important for
preventing the spreading of SIR complex.
64,65
One proposal was that RPD3 removes SIR2 acetyl lysine substrates,
thus eliminating the production of O-Ac-ADPR, which promotes SIR complex
spreading.
65,66
However, the role of O-Ac-ADPR
in SIR complex spreading is debated.
67
Similarly,
ESA1, a MYST family acetyltransferase that is generally associated
with transcription activation and double-strand DNA repair, is also
reported to be important for silencing at the rDNA loci, similar to
SIR2.
94
Therefore, mechanistically, more
needs to be further elucidated.
2.4.2
SIR2
and Histone Methylation
SIR2-dependent
gene silencing is regulated by the protein Arg methyltransferase HMT1
(HnRNP methyltransferase 1) in yeast. Lack of HMT1 activity leads
to decreased histone H4R3 methylation, increased histone acetylation,
and increased transcription from silent chromatin regions and increased
mitotic rDNA recombination.
95
H3R2
methylation is reported to be required for gene silencing at the same
sites that are silenced by SIR2.
96
H3R2
methylation suppresses H3K4 trimethylation and transcription. However,
H3R2 methylation does not affect SIR2 recruitment to these sites.
Thus, it is not clear how H3R2 methylation promotes gene silencing.
It would be interesting to see whether H3R2 methylation regulates
the histone H3 or H4 acetylation level.
SIR2 occupancy at the
silenced rDNA loci decreased when K79 was
mutated to Ala/Pro/Gln.
97
At the silent
mating loci, SIR3 occupancy and silencing increased when DOT1 (disruptor
of telomeric silencing 1, methylates H3K79) and SET1 (SET domain-containing
1, methylates H3K4) were deleted.
98
When
H3 K4/K79 were mutated to Arg, the SIR complex recruitment increased
and inhibited the nucleoside excision repair at heterochromatin.
99
In vivo, H3 K79 is hypomethylated in silent
loci.
100
In vitro, DOT1-mediated H3K79
methylation decreases SIR complex binding.
101
These evidence suggest that the SIR complex prefers the unmethylated
H3K4 and H3K79 (or the K to R mutation, which mimics the unmethylated
K), while methylation or other mutations will decrease SIR complex
binding. However, the situation was complicated by reports that deletion
of Dot1 decreased telomeric silencing.
97,102
It has been proposed that H3K4 and H3K79 methylation in euchromatin
help to prevent nonspecific binding of the SIR complex, thus promoting
silencing at heterochromatin. When Dot1 or Set1 is deleted, SIR complex may have increased
nonspecific
binding to euchromatin and thus decrease silencing at heterochromatin.
27,103
Recently, it was reported that the complication might be due to
an artifact caused by the use of the Ura3 reporter
gene to read out gene-silencing effects at specific telomeric positions
and DOT1’s effects on gene silencing at heterochromatin is
rather limited and not general.
104,105
2.5
Functions of Other Yeast Sirtuins in Epigenetic
Regulation
Four SIR2 homologues (HST1–4) were found
in yeast, and three of them, HST1, HST3, and HST4, were thought to
have similar gene-silencing activity.
106
HST1 is the closest homologue of SIR2 and was thought to have distinct
but partial overlapping functions.
107
Under
certain genetic conditions, HST1 overexpression can restore repression
that was lost due to Sir2 deletion.
108
HST1 is important for the repression of middle sporulation-specific
gene expression during mitosis.
109
It also
represses genes involved in de novo NAD and thiamine biosyntheses.
107,110
HST1 is recruited to the promoters of the repressed genes by a sequence-specific
transcriptional repressor SUM1, which recognizes DNA sequences called
Mse (middle sporulation element), and a tethering factor called RFM1
(repression factor of middle sporulation element 1).
109,111
The SUM1-RFM1-HST1 complex is also thought to be important for replication
initiation at the origin of replication via histone H4K5 deacetylation.
112
However, the detailed mechanism is not clear.
HST2 is mainly cytosolic
113
but can
shuttle between the nucleus and the cytoplasma.
114
Not much is known about its physiological role. However,
overexpression of HST2 can partially suppress the silencing defects
in Sir2 deletion.
113
It
has also been reported that SIR2-independent life span extension is
through the silencing effect of HST2.
115
However, this is controversial.
116
HST3 and HST4 have been reported to deacetylate histone H3K56 in
a cell cycle-dependent manner.
117,118
Both deletion and
overexpression of HST3/HST4 increased the sensitivity of yeast cells
to genotoxic reagents that interfere with replication fork progression.
118
During S phase, virtually all newly synthesized
histone H3 is acetylated on K56
117
and
are incorporated into newly formed nucleosome. Furthermore, both the
lack of deacetylation and the lack of acetylation of on H3K56 lead
to hypersensitivity to genotoxic reagents,
117,118
but the exact molecular mechanism is not clear.
119
HST3 and HST4 have also been reported to contribute to
gene silencing. HST3 is required for silencing the 2mu replication
origin.
120
Deletion of both Hst3 and Hst4 leads to a defect in telomeric-silencing
defects, which can be rescued by deletion of the H3K56 acetyltransferase
gene Rtt109.
121
3
Mammalian Sirtuins in Epigenetic Regulation
Among the
seven mammalian sirtuins, SIRT1, SIRT2, SIRT6, and SIRT7
have all been demonstrated to have important epigenetic roles. In
contrast, SIRT3–5 are considered to localize mainly in the
mitochondria, where they regulate numerous mitochondrial proteins.
Therefore, the roles of SIRT3–5 are mainly in metabolic regulation.
However, several reports suggested the role of SIRT3 in epigenetic
regulation,
122−124
although it remains controversial.
125
Similarly, SIRT5 also have nonmitochondrial
desuccinylation substrates,
35,36,126
and histones can be succinylated,
127
but
it is not clear whether SIRT5 can catalyze histone desuccinylation.
Therefore, knowledge about the epigenetic roles of SIRT3–5
is very limited at this point, and we will focus on the epigenetic
roles of SIRT1, SIRT2, SIRT6, and SIRT7 here.
3.1
Epigenetic
Functions of SIRT1
SIRT1
is by far the most understood mammalian sirtuin and has the greatest
homology to yeast SIR2. SIRT1 is predominantly present in the nucleus,
29
yet it shuttles between the nucleus and the
cytoplasm in a context-dependent manner. SIRT1 deacetylates lysine
residues in both histones and nonhistone proteins, thereby regulating
transcription, protein stabilities, and activities. SIRT1 controls
various cellular processes (Table 1), such
as chromatin organization, metabolism, cell survival, differentiation,
and development, as well as stress responses. Although the role of
SIRT1 is far more expansive (Table 1), the
majority of its functions are intimately connected to epigenetic regulation.
The role of SIRT1 in epigenetics is achieved via several different
mechanisms, such as regulating chromatin structure by histone deacetylation,
regulating the activity of transcription factors by deacetylation,
and regulating the activity of other epigenetic enzymes by deacetylation
(Figure 3).
Table 1
SIRT1 Deacetylation
Substrates
substrate
full name
site of modifications
function
of SIRT1-catalyzed deacetylation
histones
H1
histone H1
K26
promote the formation of
heterochromatin and transcriptional repression
81,128
H3
histone H3
K9, K14, K56
deacetylation of H3K9 and
H3K14 promotes transcriptional repression and heterochromatin formation;
14,128
deacetylation of H3K56 helps maintain genome stability
150,151
and also inhibit transcription of Bcalf1 in T cells
152
H4
histone H4
K16
promote the formation of
heterochromatin and transcriptional repression
14,81,128
chromatin
modifying enzyme and structural proteins
DNMT1
DNA (cytosine-5)-methyltransferase
1
K160, K188, K259,
K366,
K749, K891, K957, K961, K975, K1054, K1111, K1113, K1115, K1117, K1349,
K1415
deacetylation
of K1349 and
K1415 in the catalytic domain increases DNMT1 activity; deacetylation
of lysine residues in the GK linker decreases DNMT1’s methyltransferase-independent
transcriptional repression function; deacetylation of all identified
acetylated lysine sites in DNMT1 abrogates its binding to SIRT1 and
impairs its capability to regulate cell cycle G2/M transition
174
HDAC1
histone deacetylase 1
K89, K220, K432, K438, K439,
K441
stimulate HDAC1
activity
after DNA double-strand break (DSB) induction, which is critical for
DSB repair by the nonhomologous end-joining pathway
207
hMOF
human ortholog
of MOF
K274
increase the chromatin recruitment
of hMOF to Hoxa9 promoter and the H4K16 acetylation
level in Hela cells
158
or inhibit the acetyltransferase
activity and promote its ubiquitination-dependent degradation
159
P300
K1020, K1024
repress its transactivation
156
and destabilize it by promoting ubiquitination
157
PCAF
histone acetyltransferase
KAT2B
unknown
repress its transactivation
and retard muscle differentiation in response to redox stress
140
SATB1
DNA-binding protein SATB1
K136, K175
facilitate the inter-MAR
(matrix attachment region) association and to promote ε-globin
gene expression
208
SUV39H1
suppressor of variegation 3–9 homologue 1
K266
upregulate the methyltransferase
activity of SUV39H1
163
and stabilize SUV39H1,
163
thereby promoting methylation of H3K9
TIP5
transcription termination
factor I-interacting protein 5
K633
promote the binding of NoRC
with promoter RNA (pRNA) and increase the heterochromatin histone
marks at rDNA loci
175
TIP60
60 kDa Tat-interactive protein
K327
inhibit the acetyltransferase
activity and protein stability of TIP60
159,161
transcription factors
AR
androgen receptor
K630, K632, K633
repress dihydrotestosterone
(DHT)-induced AR signaling by inhibiting coactivator-mediated interaction
between the AR N- and C-termini
209
BMAL1
brain and muscle ARNT-like
1
K537
keep CRY from binding to
BMAL1 and facilitate transactivation of CLOCK/BMAL1
204
c-JUN
proto-oncoprotein
c-JUN
K268, K271,
K273
inhibit the
activity of
transcription factor AP-1, leading to T-cell anergy and diminished
T-cell activation
210
c-MYC
proto-oncoprotein c-MYC
K323
facilitate c-MYC/MAX interaction
and stabilize c-MYC, leading to increased c-MYC transactivation activity,
199,211
or destabilized c-MYC
212
CIITA
class II transactivator
unknown
shield CIITA from proteasomal
degradation and promote its nuclear accumulation and transactivation
on MHC II (major histocompatibility complex II) during antigen-dependent
T-cell stimulation
213
CRTC1
CREB-regulated transcription
coactivator 1
K13,
K20, K33, K178, K197
activate CRTC1 by facilitating
its dephosphorylation and interaction with CREB, thus activating the
transcriptional networks in both the normal and the Huntington’s
disease brain
214
CRTC2
CREB regulated transcription
coactivator 2
K628
destabilize CRTC2, resulting
in decreased CRTC2/CREB-mediated gluconeogenesis during long-term
fasting
181
DCOH2
dimerization cofactor of
HNF-1α
promotes its dimerization
with hepatocyte nuclear factor 1alpha (HNF-1α), leading to increased
DNA binding of HNF-1α and intestinal farnesoid X receptor (FXR)
signaling, and subsequent alteration of systemic bile acid homeostasis
215
E2F1
E2F transcription factor
1
K117, K120, K125
inhibit E2F1 transcriptional
and apoptotic activity in response to DNA damage
216
ERα
estrogen
receptor α
K266,
K268
increase
217
or decrease
218
its
DNA-binding
affinity and transactivation
FOXA2
forkhead box protein A2
K6, K259, K264, K273, K275
target FOXA2 toward proteasomal
degradation and inhibit FOXA2-mediated fatty acid oxidation and ketogenesis
during fasting;
219,220
promote the transcription of
Pdx1 (pancreas duodenum homeobox 1) and β-cell formation
221
FOXO1
forkhead box protein O1
K242, K245, K262
promote FOXO1-mediated transcription
during gluconeogenesis,
181
adipogenesis,
222
lipolysis,
223
starvation-induced
autophagy,
224
response to oxidative stress
225
and nitric oxide,
226
muscle growth
227
and apoptosis;
228
restrain the antiangiogenic activity of FOXO1
and promote vascular growth
229
FOXO3a
forkhead box protein O3a
unknown
inhibit its transcription
activation and ability to induce apoptosis
186,230
but increase its ability to induce cell cycle arrest and resistance
to oxidative stress
186
FOXO4
forkhead box protein O4
unknown
activate FOXO4-dependent
transcription of stress-regulating genes
231,232
FOXP3
forkhead box protein P3
unknown
lead to FOXP3 polyubiquitination
and proteosome-mediated degradation and decreased numbers of regulatory
T cells
193−196
FXR
farnesoid X receptor
K217
decrease its stability but
promote heterodimerization with RXRα, DNA binding, and transactivation
activity,
233
promote FXR-mediated hepatitis
B virus transcription
234
and bile acid
homeostasis during liver regeneration
235
HIF-1α
hypoxia-inducible factor
1α
K674
repress the transactivation
of HIF-1α during hypoxia
197
HIF-2α
hypoxia-inducible factor
2α
K385, K685,
K741
activate HIF-2α,
thereby
promoting HIF-2 signaling during hypoxia
198
HSF-1
heat shock factor 1
K80
prolong HSF-1 binding to
the heat shock promoter;
236
promote HSF-1-mediated
transcription of HSP70 in response to alpha-synuclein aggregation-induced
stress
237
and repression of IGF-IIR expression
and cardiomyocyte apoptosis
238
LXRα
liver X receptor protein
α
K432
activate LXRα, leading
to increased expression of various LXR targets involved in lipid metabolism
239
MeCP2
methyl-CpG binding protein
2
K464
deacetylation keeps it from
binding to the brain-derived neurotrophic factor (BDNF) promoter in
hippocampi
240
MyoD
myogenic determining factor
K99, K102, K104
repress its transactivation
and retard muscle differentiation in response to redox stress
140
NFAT
nuclear factor of activated
T cells
unknown
suppress the transcriptional
activity of NFAT, leading to the inhibition of PMA/lonomycin-induced
expression of COX-2
241
NHLH2
helix–loop–helix
protein 2
K49
SIRT1 deacetylates NHLH2
to activate the monoamine oxidase A (MAO-A) promoter, thereby decreasing
serotonin levels in the brain
242
NOTCH
neurogenic locus notch homologue
protein 1
K1764,
K1770, K1771, K1772,
K1785, K1935, K2050, K2068, K2146, K2147, K2150, K2154, K2161, K2164
destabilize NOTCH, thereby
limiting the DDL4/NOTCH signaling in endothelial cells
243
NPM1
nucleophosmin 1
K212, K215, K229, K230,
K257, K267
reduce
its activity to promote
transcription of genes implicated in oral cancer
244
p53
tumor suppressor
p53
K382
negatively regulate transactivation
activity of p53, thereby attenuating p53-dependent apoptosis induced
by DNA damage and oxidative stress,
188
as
well as PML/p53-induced cellular senescence
189
p73
tumor protein p73
unknown
suppress p73-dependent transactivation
245
PER2
period circadian protein
homologue 2
unknown
promote the degradation
of PER2, thereby derepressing CLOCK/BMAL1-mediated expression of circadian
clock gene expression
203
PGC-1α
peroxisome proliferator-activated
receptor-γ coactivator 1α
K77, K144, K183, K253, K277,
K270, K320, K346, K412, K441, K450, K757, K778
stimulate its transcription
coactivator activity on mediating gluconeogenesis,
181,182,246
fatty acid oxidation,
184
mitochondrial biogenesis,
247,248
and expression of BMAL1 and CLOCK;
249
augment PPARγ-PGC-1α-mediated repression of beta-secretase/BACE1;
250
promote hepatitis B virus transcription
234
PTF1A
pancreatic transcription
factor-1a
unknown
SIRT1 colocalizes with PTF1A
at the acinar gene promoters and promotes acinar-to-ductal metaplasia
251
RARβ
retinoic acid receptor-β
unknown
activate RARβ and
promote the transcription of the α-secretase gene ADAM10, thereby
inhibiting β-amyloid production
252
RelA/p65
RelA/p65 subunit of nuclear
factor-κB
K310
repress NF-κB-dependent
transcription, augmenting TNFα-induced apoptosis
192
RFX5
regulatory factor for X-box
RFX5
unknown
promote its nuclear exclusion
and proteasomal degradation, thereby derepressing collagen type I
(COL1A2) transcription by RFX5 in smooth muscle cells
253
SMAD3
mothers against decapentaplegic
homologue 3
unknown
repress the transactivation
of SMAD3 following TGF-β1 in a chronic kidney disease (CKD)
model
254
SMAD4
mothers against decapentaplegic
homologue 4
unknown
repress the effect of TGF-β
signaling on MMP7 and therefore the EMT transition in cancer metastasis
255
SMAD7
mothers against decapentaplegic
homologue 7
K64,
K70
promote SMAD
ubiquitination
regulatory factor 1 (Smurf1)-mediated proteasome degradation and TGF-β-induced
apoptosis in glomerular mesangial cells
256
SREBP-1c
sterol regulatory element-binding
protein 1c
K289,
K309
inhibits its
transactivation
by destabilizing it and reducing its binding affinity for promoters
of lipogenic target genes
185,257
STAT3
signal transducer and activator
of transcription 3
K679, K685, K707, K709
repress the inhibitory effect
of STAT3 on gluconeogenic gene expression during long-term fasting
183
STAT5
signal transducer and activator
of transcription 5
K681, K694, K701, K705
negatively regulate GH-induced
STAT5 phosphorylation and IGF-I production during fasting in the liver
258
TAFI68
TATA box-binding protein-associated
factor RNA polymerase I subunit B
unknown
decrease its DNA-binding
activity and repress RNA polymerase I transcription
259
TAT
human immunodeficiency
virus
(HIV) TAT protein
K50
SIRT1 deacetylates
TAT and
acts as a transcriptional coactivator during Tat-mediated transactivation
of HIV long terminal repeat
260,261
YAP2
mammalian Ste20-like kinase/Yes-associated
protein 2
K76, K90,
K97, K102
increase
the YAP2/TEAD4
(TEA Domain Family Member 4) association in hepatocellular carcinoma
(HCC) cells, resulting in YAP2/TEAD4 transcriptional activation and
increase in cell growth
262
β-catenin
β-catenin
K49, K345
promote its translocation
from nucleus to cytoplasm and destabilize it, limiting its ability
to activate transcription and drive cell proliferation;
251,263,264
restore nuclear localization
of β-catenin in Mesenchymal stem cells (MSCs) and promote gene
transcription for MSC differentiation
265
DNA damage repair-related
substrates
APE1
apurinic/apyrimidinic
endonuclease-1
K6,
K7
promote base excision
repair
of damaged DN.
266
KU70
DNA repair factor KU70
K539, K542
cause it to sequester the
pro-apoptotic factor Bax (BCL2-Associated X Protein) away from mitochondria,
thus inhibiting stress-induced apoptotic cell death;
267
promotes its DNA repair activity
268
MCM10
protein MCM10 homologue
K312, K390, K683, K745,
K761, K768, K681 + K682, K737 + K739, K847 + K849, K868 + K874, K683
+ K685, K674 + K682
modulate its stability and
ability to bind DNA; promote its function in DNA replication fork
initiation
269
NBS1
Nijmegen Breakage Syndrome
1
unknown
deacetylation of NBS1 is
required for ionizing radiation-induced NBS1 phosphorylation at Ser343,
which is essential for the activation of S phase checkpoint and for
efficient DNA damage repair response
270
WRN
Werner syndrome ATP-dependent
helicase
unknown
promote its helicase and
exonuclease activities and facilitate its translocation from nucleoplasm
to nuclei in response to DNA damage
271
XPA
xeroderma pigmentosum group
A
K63, K67
SIRT1-mediated deacetylation
of XPA is essential for optimal nucleotide excision repair (NER) pathway
during UV-induced DNA repair
272
other substrates
AceCS1
acetyl-CoA synthetase 1
K661
activate AceCS1 to convert
acetic acid to acetyl-CoA for use in fatty acid synthesis
273
AKT
RAC-alpha serine/threonine-protein
kinase
unknown
activate AKT and promote
axonogenesis
274
ATG5
autophagy-related protein
5
unknown
promote the induction of
autophagy
275
ATG7
autophagy-related protein
7
unknown
promote the induction of
autophagy
275
ATG8
autophagy-related protein
8
unknown
promote the induction of
autophagy
275
BCL6
B-cell lymphoma 6 protein
K379
activate its oncogenic activit.
276
cortactin
cortactin
unknown
facilitate cancer cell migration
277
CREB
cAMP response
element binding
protein
K136
repress its activity by
preventing its phosphorylation, which leads to decreased expression
of gluconeogenic genes and increased hepatic lipid accumulation and
secretion
278
eNOS
endothelial nitric oxide
synthase
K496, K506
activate eNOS in the cytoplasma,
which increases NO level, leading to vasodilatation, increased blood
flow, and nutrient delivery to tissues
279
EVI1
ectopic viral integration
site I
unknown
lead to destabilization
of the protein
280
HMGB1
high-mobility group box
1
K55, K88, K90,
and K177
repression
of SIRT1 induced
by inflammation disables deacetylation of HMBG1 and promotes the nuclear-to-cytoplasmic
translocation and release into circulation, thereby maintaining inflammation
281
HMGCS1
hydroxymethylglutaryl-CoA
synthase 1
unknown
unknown
282
IRS-1
insulin receptor
substrate
1
unknown
promote systemic insulin
resistance in neurons
283
IRS-2
insulin receptor substrate
2
unknown
promote its phosphorylation
and activate the IGF-1/Ras/ERK1/2 pathway,
sensitizing neurons to oxidative damage
284
LIN28
protein LIN-28
unknown
restore its stability
285
MMP2
matrix metalloproteinase-2
unknown
stabilize MMP2, thereby
increasing tumor cell invasion in prostate cancer
286
PARP1
poly(ADP-ribose)
polymerase
1
K498, K505, K508,
K521,
K524
attenuate PARP1
activity
and protect cells from PARP1-dependent cell death under stress conditions
287
PGAM1
glycolytic enzyme phosphoglycerate
mutase-1
K251, K253,
K254
decrease PGAM1
activity
in glycolysis
288
PIP5Kγ
phosphatidylinositol-4-phosphate
5-kinase gamma
K265,
K268
activate PIP5Kγ
and
promote the secretion of thyroid stimulating hormone (TSH) from pituitary
cells
289
PTEN
phosphatase and tensin homologue
unknown
SIRT1 deacetylates PTEN
to inhibit AKT and trigger apoptosis under antioxidant-free conditions
290
RB
retinoblastoma tumor suppressor
protein
K873, K874
allow phosphorylation of
RB and relieve RB-mediated repression of E2F-regulated cell cycle
genes
291
S6K1
p70 ribosomal S6 kinase
K484, K485, K493
decrease the Thr-389 phosphorylation
and kinase activity of S6K1
292
Tau
microtubule-associated protein
tau
unknown
destabilize tau protein,
resulting in the decrease in tau protein aggregates
293
TDG
thymine DNA
glycosylase
unknown
increase TDG glycosylase
activity and weakly shift its activity toward T/G, 5-formylcytosine/G,
and 5-carboxylcytosine/G compared from 5-fluorouracil/G
294
TIAM1
T-cell lymphoma invasion
and metastasis 1
K1420
promotes activation of DVL/TIAM1/Rac
axis and cell migration in cancer cells
295
zyxin
zyxin
unknown
SIRT1 deacetylates Zyxin
and modulates its activity upon treatment with leptomycin B
296
14-3–-3ζ
14-3-3ζ
K49, K157,
K212
keep 14-3-3ζ from dissociating from caspase-2, thereby
antagonizing
caspase-2-dependent apoptosis
297
Figure 3
Model for SIRT1-mediated
heterochromatin formation and transcriptional
silencing.
3.1.1
SIRT1’s Histone
Deacetylase Activity
Regulates Chromatin Structure and Transcription
SIRT1 is
involved in the formation of both facultative and constitutive heterochromatin.
Like other sirtuins, SIRT1 is not capable to directly bind to DNA
but needs to be recruited by a variety of chromatin-associated factors
to their binding sites. Due to its remarkable ability to bind to many
factors, SIRT1 acts as a coordinator of heterochromatin formation
instead of being merely a histone deacetylase. Recruitment of SIRT1
to chromatin is usually associated with the epigenetic silencing of
target genes and heterochromatin formation.
SIRT1 was shown
to deacetylate lysine residues of the N-terminal tails of H3 and H4,
preferentially H4K16, and to a less extent H3K9. The deacetylation
of histone residues H3K14, H4K8, and H4K12 by SIRT1 was also observed
in a biochemical assay in vitro, but it was shown to occur at a slower
rate.
14,128
RNAi-mediated decrease in SIRT1 expression
in human cells led to hyperacetylation of H3K9 and H4K16, together
with reduction in the repressive chromatin marks, H3K9me3 and H4K20me1,
suggesting that SIRT1 promotes facultative heterochromatin (FH) formation.
128
Further study showed that SIRT1 interacts with
and deacetylates the linker histone H1 at Lys26 (H1K26). Localizing
SIRT1 to the promoter of a Gal4-reproter integrated to euchromatin
led to deacetylation of H3K9 and H4K16, recruitment of H1, loss of
H3K79me2 (a mark associated with active transcription), and subsequent
dramatic decrease in the expression of the reporter gene.
128
Notably, arrival of SIRT1 to chromatin results
in spreading of hypomethylated H3K79, indicating the role of SIRT1
as a coordinator of heterochromatin formation.
Many functions
of SIRT1 depend on its ability to deacetylate H3K9
and H4K16 and to mediate subsequent transcription repression. The
most important mechanism by which SIRT1 promotes cellular response
to stress is the transcription silencing associated with FH formation.
Growing evidence illustrates the role of SIRT1 in metabolism, cell
differentiation and development, cancer, and other diseases through
forming corepressor complexes with numerous transcription repressors.
For example, genome-wide study by Oberdoerffer et al. showed that
SIRT1 represses a large variety of genes across the mouse genome.
81
Upon oxidative stress, SIRT1 redistributes on
chromatin and localizes to DNA strand break repair sites. The relocation
of SIRT1 promotes DNA repair and also results in epigenetic changes
surrounding the break sites. Redistribution of SIRT1 leads to global
changes in the H1K26ac pattern and transcription. Notably, the changes
in transcription of SIRT1-bound genes parallel those in aging mouse
brain, suggesting the role of SIRT1 in maintaining genome stability
and protecting cells from aging.
81
Ghosh
et al. reported that SIRT1 inhibits NF-κB (nuclear factor κB)-mediated
transcription by interacting with TLE1 (transducing-like enhancer
of split-1), which is a non-DNA binding corepressor for NF-κB.
129
Regarding the regulation of metabolism,
SIRT1 stimulates the secretion
of insulin in response to glucose through repressing the expression
of UCP2 (uncoupling protein 2) in pancreatic β-cells. SIRT1
is recruited to Ucp2 promoter and inhibits its transcription.
130,131
Yet how SIRT1 is recruited remains unclear. These findings suggest
that a relationship between loss of SIRT1 activity and age-related
type 2 diabetes.
132
SIRT1-containing corepressor
complexes also target various nuclear receptors. One important example
is the ERRs (estrogen receptor-related receptors), which regulate
genes involved in mitochondria function and energy homeostasis.
133
SIRT1 inhibits the transactivation of ERRγ
by forming a complex with the transcription corepressor of ERRγ
at the ERRE (ERR-responsive element). It was further demonstrated
that the repression of ERRγ target genes is dependent on SIRT1
activity to deacetylate H3K9 at ERRE.
134
In cardiac myocytes, PPARα (peroxisome proliferator-activated
receptor α) binds and recruits SIRT1 to ERRE to inhibit ERR
target genes involved in mitochondrial respiration, thereby mediating
cardiac hypertrophy.
135,136
In white adipose tissue, SIRT1
promotes fat mobilization by suppressing genes controlled by PPARγ.
During fasting, SIRT1 interacts with the corepressors of PPARγ,
NCoR (nuclear receptor corepressor), and SMRT (silencing mediator
of retinoid and thyroid hormone receptors), leading to decreased PPARγ
transactivation and adipogenesis.
137
In
hepatocytes, SIRT1 is recruited to liver receptor homologue-1 (LRH1)
target gene promoters by orphan nuclear receptor SHP (small heterodimer
partner), a transcriptional corepressor of various nuclear receptors.
The recruitment of SIRT1 leads to histone H3/H4 deacetylation and
inhibition of LRH1-dependent Cyp7a1 (cholesterol
7α hydroxylase) gene transcription, thereby regulating
LRH1-dependent bile-acid homeostasis.
138
Moreover, screening for transcription factors that interact with
SIRT1 in response to nutrient restriction identified HNF-1α
(hepatocyte nuclear factor 1-α), a transcription factor that
regulates the expression of several liver-specific genes. Formation
of HNF-1α-SIRT1 complex on the CRP (C-reactive protein) gene promoter results in deacetylation
of H4K16 and subsequent inhibition of HNF-1α-mediated transcription
of Crp gene.
139
Accumulating evidence shows that SIRT1-mediated chromatin silencing
also has critical roles in development. For instance, SIRT1 inhibits
myogenesis in response to redox stress by forming complex with PCAF/MyoD
and deacetylating H3K9/H3K14 at the promoters of myogenin and MHC
(myosin heavy chain).
140
SIRT1 directly
interacts with BCL11 (B-cell leukemia 11) proteins, which are implicated
in hematopoietic cell development and malignancies. Recruitment of
SIRT1 by BCL11 increases deacetylation of H3/H4 and promotes transcription
repression induced by BCL11 proteins.
141,142
SIRT1 also
plays a role in hematopoietic stem and progenitor cell maintenance.
SIRT1 counteracts the upregulation of HOXA9 (Homeobox protein Hox-A9)
in response to hematopoietic stress by binding to the HOXA9 locus
and deacetylating H4K16, thereby blocking the expansion of hematopoietic
stem and progenitor cells.
143
SIRT1 is
also involved in redox-dependent differentiation of neural progenitor
cells by inhibiting the expression of pro-neuronal bHLH (basic helix–loop–helix)
transcription factor MASH1 (mammalian achaete scute homologue 1).
SIRT1 functions together with HES1 (hairy and enhancer of split-1)
as transcriptional repressors. Under oxidative condition, SIRT1 is
upregulated and recruited by HES1 to the Mash1 promoter
region, leading to the deacetylation of H3K9 but not H4K16, as well
as the stabilization of the TLE1-containing repressor complex and
subsequently to Mash1 repression.
144
Additionally, BCL6 (B-cell lymphoma 6) promotes neurogenesis
by excluding the coactivator MAML1 (Mastermind-like 1) and recruiting
SIRT1 to the NOTCH-dependent transcriptional complex at the Hes5 promoter. SIRT1-mediated
epigenetic silencing of Hes5 then leads to neuronal differentiation.
145
Lastly, SIRT1-mediated FH formation and
transcription silencing
has also been implicated in various diseases, including neurodegeneration
diseases and cancer. SIRT1 is involved in the Fmr1 (fragile X mental retardation 1)
gene silencing
in fragile X mental retardation syndrome by deacetylating H3K9 and
H4K16. Inhibition of SIRT1 reactivates Fragile X alleles in neurons.
146
SIRT1 also plays an important role during BRCA1-associated
tumorigenesis. BRCA1 binds to SIRT1 promoter and promotes its expression,
which in turn represses the oncogene Survivin by
deacetylating histone H3. Hence, loss or mutation of Brca1 leads to an increase in
the expression of Survivin and subsequently cell proliferation and cell survival.
147
In prostate cancer cells, SIRT1 induces epithelial-to-mesenchymal
transition (EMT) by suppressing E-cadherin expression. The EMT-inducing
transcription factor ZEB1 (zinc finger E-box binding homeobox 1) recruits
SIRT1 to the E-cadherin promoter, leading to H3K9 deacetylation and
E-cadherin transcriptional suppression.
148
Interestingly, SIRT1 could also serve as a transcriptional
activator.
The ING (inhibitor of growth) subunits of mSIN3A/HDAC1 complex can
recruit SIRT1 to the HDAC-dependent transcriptional repression domain
of RBP1 (retinoblastoma-binding protein 1). In addition, SIRT1 activity
is able to negatively regulate RBP1-mediated transcription repression.
149
SIRT1 has also been linked to DNA replication
and DNA damage repair
through deacetylation of H3K56. Acetylation of H3K56 was shown to
play a critical role in assembling newly synthesized DNA into chromatin
following DNA replication and DNA damage repair.
150,151
SIRT1 deacetylates H3K56 in vitro. Moreover, Sirt1 knockdown or knockout mammalian
cells display hyperacetylation of
H3K56
150,151
and genomic instability.
151
Thus, it was proposed that deacetylation of H3K56 contributes
to the role of SIRT1 in the regulation of genomic stability. In addition,
SIRT1 also exerts its transcriptional repression role by deacetylating
H3K56. SIRT1 deacetylates H3K56 at the promoter region of Bclaf1 (Bcl2-associated
factor 1) and represses
its transcription, thereby negatively regulating T-cell activation.
152
3.1.2
SIRT1 Regulates Other
Epigenetic Enzymes
The role of SIRT1 in transcriptional repression
also involves interaction
with and deacetylation of numerous epigenetic factors. One important
aspect is that SIRT1 interacts with other histone-modifying enzymes
and regulates their activities.
3.1.2.1
SIRT1
Regulates the Activity of Various
Histone Acetyltransferases (HATs)
Various studies suggest
that HATs and HDACs are interdependent on each other. By physically
forming complexes, they are able to execute coordinated histone acetylation
and deacetylation rapidly in the same region of chromatin.
153
An important example is CBP/p300, which
has been shown to acetylate various histone lysine residues, including
H2A (K5), H2B (K12, K15), H3 (K14, K18, K27, K36, K56), and H4 (K5,
K8, K12),
154,155
and to act as a limiting transcriptional
coactivator utilized by many DNA-binding proteins, including p53,
E2F, and NF-κB, to facilitate transcriptional activation. SIRT1
regulates the activity of p300 by two different mechanisms. SIRT1
deacetylates p300 at two lysine residues (K1020 and K1024), leading
to the SUMO modification of these two residues and transcriptional
repression of p300.
156
SIRT1 also destabilizes
p300 by deacetylation and promoting ubiquitination in dystrophic heart.
157
Since SIRT1 and p300 regulate cellular function
reciprocally by deacetylating and acetylating proteins, respectively,
SIRT1-mediated negative regulation of p300 might promote its effect
on chromatin silencing and transcriptional repression.
The MYST
acetyltransferase family members, hMOF
158,159
and TIP60,
159,160
are also SIRT1 deacetylase substrates.
SIRT1 binds and deacetylates the enzymatic domains of hMOF and TIP60,
leading to the repression of their HAT activity and ubiquitination-dependent
degradation of these proteins.
159,161
Notably, hMOF is necessary
for the acetylation of H4K16. Since H4K16 is the primary histone target
of SIRT1 and exerts an important role in higher order chromatin organization,
SIRT1 might control the process not only by deacetylating H4K16 but
also by inhibiting hMOF. Interestingly, under DNA damage conditions,
the binding between SIRT1 and hMOF/TIP60 decreases, activating hMOF/TIP60
for DNA double-strand break repair.
159
3.1.2.2
SIRT1 and Histone Methylation
Deacetylation
of nucleosome histones by SIRT1 is able to alter methylation
of histones. SIRT1 recruits and activates histone methyltransferase
(HMT) at the target sites and therefore regulates both histone acetylation
and methylation. As described above, RNAi-mediated SIRT1 depletion
renders hypomethylation of H3K9me3, which is a hallmark of heterochromatin
in eukaryotes. Later, it was elucidated that SIRT1 promotes H3K9me3
during FH formation by activating the main H3K9 methyltransferase,
SUV39H1 (suppressor of variegation 3–9 homologue 1).
162
SIRT1 directly binds to and recruits SUV39H1
to genomic loci and activates its HMT activity by deacetylating the
Lys266 in its catalytic domain.
163
SIRT1,
SUV39H1, and Nucleomethylin exist in a complex and eNoSC (energy-dependent
nucleolar-silencing complex) in the rDNA loci.
164,165
Energy depletion-induced increase in NAD/NADH ratio activates SIRT1,
resulting in deacetylation of H3K9 and SUV39H1-mediated H3K9me2, establishment
of silent chromatin in the loci, and subsequent repression of rRNA
transcription.
164
SIRT1 is also linked
to SUV39H1-dependent constitutive heterochromatin formation and genome
protection in response to oxidative stress. SIRT1 competes with the
E3 ubiquitin ligase of SUV39H1, MDM2, for binding to SUV39H1, thereby
inhibiting its degradation through ubiquitination.
166
In addition to SUV39H1, EZH2, another polycomb group
HMT that targets H3K27 and H1K26 for methylation, was also linked
to the effect of SIRT1 on histone methylation. SIRT1 and EZH2 are
found to coexist in the PRC4 complex (polycomb repressive complex
4), which is detectable only in undifferentiated embryonic stem (ES)
cells and some EZH2-overexpressing cancer cells. Although EZH2-containing
PRC complexes usually preferentially methylate H3K27, with the presence
of SIRT1, PRC4 specifically targets H1K26 for methylation. This finding
suggests that the deacetylation of H1K26ac by SIRT1 may coordinate
the methylation of H1K26 by EZH2.
167
SIRT1 also acts in concert with histone demethylases to control
gene expression. It forms corepressor complex with the histone H3K4
demethylase LSD1/KDM1A (lysine-specific demethylase 1A) and plays
conserved and concerted roles in deacetylation of H4K16 and demethylation
of H3K4 to repress NOTCH target genes during development.
168
Lastly, SIRT1 arrival also promotes H4K20me1
and loss of H3K79me2, suggesting the coordination between SIRT1 and
other methyltransferase and demethylase during heterochromatin formation.
Yet the mechanism remains to be elucidated.
128
3.1.2.3
SIRT1 and DNA Methylation
Multiple
reports suggested that SIRT1 is associated with DNA methylation and
colocalizes with the DNA methylation machinery in hypermethylated
chromatin regions. Pruitt et al. showed that SIRT1 localizes to promoters
of various silenced tumor suppressor genes with 5′ CpG island
hypermethylation but not to the same promoters that are not hypermethylated.
Inhibition of SIRT1 in breast and colon cancer cells resulted in reactivation
of the silenced tumor suppressor genes.
169
Studies by Espada et al. indicated that DNMT1 (DNA-methyltransferase
1) interacts with SIRT1 and serves as an epigenetic caretaker to maintain
the nucleolar structure through recruitment of SIRT1.
170
Using an exogenous reporter construct
containing E-cadherin promoter, O’Hagan et
al. illustrated that SIRT1, EZH2, DNMT1, and DNMT3b were recruited
to DNA double-strand breaks and that SIRT1 is required for transient
recruitment of DNMT3b and subsequent aberrant DNA methylation in
the exogenous promoter CpG island.
171
In
line with this, a follow-up genome-wide study revealed that a large
silencing complex containing DNMT1, DNMT3b, SIRT1, and other PRC4
members are relocalized to CpG islands of gene promoters upon oxidative
damage. Such translocation causes aberrant DNA methylation and transcriptional
silencing, indicating the involvement of SIRT1-dependent DNA methylation
in DNA damage-induced epigenetic silencing in tumors.
172
Stable interaction between DNMT3b and SIRT1
was also detected in the condensed chromatin, suggesting that SIRT1
promotes recruitment of DNMT3b and the onset of DNA methylation.
173
Furthermore, SIRT1 deacetylates DNMT1
and alters its activities.
Interestingly, SIRT1-induced deacetylation of DNMT1 shows domain-specific
consequences. Deacetylation of the C terminus catalytic domain increases
its methyltransferase activity, whereas deacetylation of the GK linker
impairs the transcription repression activity independent of its methyltransferase
activity.
174
These seemingly contradictory
effects might be due to the fact that different domains regulate the
functions of DNMT1 independently and that the deacetylation of distinct
domains of DNMT1 may occur in a context-dependent manner. Although
details of the mechanism remain to be further studied, these observations
provide novel insight into the functional diversity of SIRT1 in DNA
methylation and epigenetic gene silencing.
Additional evidence
supporting the coordinating role of SIRT1 in
heterochromatin formation came from a study by Zhou et al. They showed
that SIRT1 deacetylates TIP5 (transcription termination factor I-interacting
protein 5), the largest subunit of the SNF2h-containing chromatin
remodeling complex NoRC (nucleolar remodeling complex). NoRC silences
rRNA gene expression by establishing histone H4K16 deacetylation,
H3K9 dimethylation, and DNA methylation at the rRNA promoter. Deacetylation
of TIP5 by SIRT1 increases the binding of NoRC to promoter-associated
RNA (pRNA), which is complementary to the rRNA promoter and essential
for NoRC function. Therefore, SIRT1 promotes the NoRC-mediated establishment
heterochromatin histone marks, DNA methylation, and rRNA silencing,
further highlighting its function in heterochromatin formation.
175
3.1.3
SIRT1
and Constitutive Heterochromatin
Growing evidence suggests
that SIRT1 is involved in the formation
of not only FH but also constitutive heterochromatin (CH), including
the pericentromeric and telomeric heterochromatin. SIRT1 was found
to localize to and contribute to the silencing of pericentromeric
major satellite repeats in mouse ES cells.
81
Sirt1
–/– MEFs showed a
complete loss of H3K9me3 and localization of heterochromatin protein
HP1 at the pericentromeric CH loci in over 50% of the cells tested.
163
A similar phenomenon was observed in Sirt1
–/– brain compared with the Sirt1
+/+ brain, suggesting the involvement of
SIRT1 in SUV39H1-dependent CH formation.
176
Supporting the direct effect of SIRT1 on CH, expression of exogenous
wild type but not catalytic mutant of SIRT1 in Sirt1
–/– MEFs rescued the loss of H3K9me3 in
CH regions.
163
Later, it was elucidated
that SIRT1 regulates CH by stabilizing SUV39H1 in CH regions as discussed
above.
166
The role of SIRT1 in telomere
maintenance is still under debate. On one hand, it was observed that
mouse cells deficient in SIRT1 exhibit spontaneous telomeric abnormalities
associated with impaired cell growth.
177
SIRT1 interacts with telomeric repeats in vivo and positively regulates
telomere length by decreasing the rate of telomere erosion.
178
In line with these findings, SIRT1 silencing
causes telomere dysfunction and repressed proliferation of HCC (hepatocellular
carcinoma cells) associated with decreased expression of TERT (telomerase
reverse transcriptase) and PTOP (POT1- and TIN2-organizing protein).
179
However, on the other hand, there is evidence
showing that SIRT1 inhibits the expression of TERT and suppresses
the growth of certain cell lineages, suggesting the role of SIRT1
as a barrier to retard the growth of certain nutrient-sensitive cells.
180
3.1.4
SIRT1 Deacetylates and
Regulates Transcription
Factors
SIRT1 can also interact with and deacetylate numerous
transcription factors, thereby modulating their transactivation activity
to activate or suppress genes. By modulating these transcription factor-dependent
expression programs, SIRT1 is involved in the regulation of stress
response, metabolism, cell differentiation, cell survival, and circadian
clock. These transcription factors include p53, PGC-1α (peroxisome
proliferator-activated receptor gamma coactivator 1-α), FOXOs
(forkhead box O transcription factors), HIF-1α and HIF-2α
(hypoxia-inducible factor 1α and 2α), NF-κB, and
MYC. Since numerous transcription factors have been reported as SIRT1
substrates, we list them in Table 1 and only
discuss some of them in the following section.
3.1.4.1
SIRT1
Regulates Transcription Factors
Controlling Metabolism
SIRT1 is known to be involved in various
metabolic processes in response to nutrient availability. During fasting,
SIRT1 regulates gluconeogenesis and fatty acid oxidation by deacetylating
several transcription factors, including CRTC2 (CREB-regulated transcription
coactivator 2), PGC-1α, FOXO1, and SREBP1 (sterol regulatory
element-binding protein 1). Thisis one of the best established models
about how SIRT1 modulates stress response through deacetylating specific
transcription factors. During the early stage of fasting, the fasting
hormone glucagon activates CRTC2 by facilitating its nuclear localization
and acetylation by p300, which then stimulates the gluconeogenic gene
expression. During late fasting, SIRT1 is activated to deacetylate
CRTC2, which leads to the ubiquitination and degradation of CRTC2.
181
At the same time, SIRT1 deacetylates and activates
FOXO1
181
and its coactivator PGC-1α,
182
resulting in the activation of genes involved
in the late stage of gluconeogenesis. However, STAT3 (signal transducer
and activator of transcription 3) antagonizes the activity of FOXO1/PGC-1α
and inhibits the gluconeogenic gene expression. SIRT1 suppresses the
inhibitory effect of STAT3 by deacetylation, thereby maximizing the
activation of gluconeogenesis.
183
The deacetylation
of PGC-1α also coactivates PPARα to enhance the expression
of mitochondrial fatty acid oxidation genes.
184
Moreover, SREBP1, an important transcription factor that controls
lipid and sterol homeostasis, could also be deacetylated by SIRT1
during fasting.
185
Deacetylation of SREBP1
represses its protein stability and transactivation of target genes,
thereby blocking synthesis of lipid and cholesterol.
3.1.4.2
SIRT1 Regulates Transcription Factors
Involved in Stress Response
A large number of studies have
demonstrated that SIRT1 mediates different types of stress response,
such as oxidative stress, DNA damage, inflammation, hypoxic stress,
and so on. For example, SIRT1 forms a complex with and deacetylates
FOXO3a
186
in response to oxidative stress,
which has a dual effect on the activity of FOXO3a. On one hand, SIRT1
activates FOXO3a to induce cell cycle arrest and resistance to oxidative
stress. On the other hand, SIRT1 inhibits the ability of FOXO3a to
induce cell death. Thus, SIRT1 protects cells from stress-induced
cell death by tipping the balance toward stress resistance and survival.
187
The p53 protein is the first reported
nonhistone substrate of SIRT1. SIRT1 deacetylates p53 at its C-terminal
Lys382 residue and alleviates its transactivation activity. SIRT1
therefore is able to protect cells from p53-induced apoptosis
188
and senescence.
189
Notably, p53 also regulates SIRT1 in a positive feedback loop. The
binding of p53 to the Sirt1 promoter inhibits SIRT1
expression under normal nutrient conditions.
190
Also, p53 stimulates the expression of miR-34a, which represses
SIRT1 and therefore SIRT1-mediated deacetylation and inhibition of
p53.
191
Hence, the SIRT1-p53 axis is implicated
in the development of various cancers.
SIRT1 could also attenuate
inflammation by regulating NF-κB.
SIRT1 deacetylates the RelA/p65 subunit of NF-κB at Lys310 and
represses its transcription activity, thereby sensitizing cells to
TNF-α-induced apoptosis.
192
It has
also been reported that SIRT1 deacetylates and destabilizes FOXP3
(forkhead box protein P3), which is essential for the functionality
of regulatory T cells (Treg).
193−196
SIRT1 binds to and deacetylates HIF-1α
at Lys674 and inhibits
the transactivation of HIF-1α to control the glycolysis in response
to hypoxic stress. By doing so, SIRT1 negatively regulates the growth
and angiogenesis of fibrosarcoma tumors in vivo.
197
SIRT1 also deacetylates HIF-2α, which is closely
related to HIF-1α in structure but differs from HIF-1α
in the transcriptional targets. During hypoxia, SIRT1 deacetylates
and activates HIF-2α signaling and the corresponding hypoxic
stress response.
198
Notably, HIF-1α
and HIF-2α are regulated by SIRT1 oppositely. Given that during
hypoxia, the expression and activity of SIRT1 gradually decrease as
NAD level decreases, it has been proposed that the deactivation of
SIRT1 may trigger a switch from HIF-2α to HIF-1α, thereby
coordinating hypoxic stress response and hypoxic metabolism.
197
3.1.4.3
SIRT1 Regulates Tumor-Promoting
Transcription
Factors
SIRT1 could also promote cell proliferation by forming
a positive feedback loop with the oncoprotein MYC. C-MYC increases
SIRT1 expression, which in turn deacetylates and enhances its transcriptional
activity.
199−202
Constitutive activation of this SIRT1-C-MYC positive feedback loop
promotes C-MYC-induced cell proliferation by suppressing apoptosis
and senescence.
199,202
SIRT1 also promotes N-MYC oncogenesis
in neuroblastoma through a positive feedback loop involving MKP3 (mitogen-activated
protein kinase phosphatase 3) and ERK (extracellular-signal-regulated
kinases). However, unlike C-MYC, N-MYC is not a deacetylase substrate
of SIRT1. Instead, SIRT1 and N-MYC form a transcriptional repressor
complex at gene promoter of MKP3, leading to repression of MKP3 expression,
ERK protein phosphorylation, N-MYC phosphorylation, and stabilization.
200
3.1.4.4
SIRT1 Regulates Transcription
Factors
in Circadian Clock
The core players of the circadian clock
machinery are the transcription factors CLOCK (circadian locomotor
output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1), which
heterodimerize, bind, and activate the transcription of clock controlled
genes (CCGs), such as Per (Period) and Cry (Cryptochrome). When
PER and CRY proteins accumulate to a certain level, they form a complex
with CLOCK-BMAL1 heterodimer and inhibit their own transcription.
In peripheral tissues such as liver, SIRT1 binds to CLOCK-BMAL1 rhythmically
and promotes the deacetylation-dependent degradation of PER2, thereby
derepressing the transactivation of CLOCK-BMAL1.
203
CLOCK has HAT activity and is able to acetylate histone
H3 and its partner BMAL1. Acetylation of BMAL1 facilitates CRY binding
and thus CRY-mediated transcriptional repression. SIRT1 is recruited
by CLOCK/BMAL1 and deacetylates BMAL1 and histone H3K9/H3K14 at the
promoters of CCGs in a timely manner. Deacetylation of BMAL1 releases
it from CRY-mediated transcriptional repression, whereas deacetyaltion
of histone H3 by SIRT1 negatively regulates the CCGs transcription.
Interestingly, it has been shown that the oscillations in the acetylation
pattern of BMAL1 and H3 differ in the timing, which is in line with
the dual role of CLOCK-induced acetylation of BMAL1 (repression) and
H3 (activation).
204
Recently, SIRT1
is linked to central circadian control in the SCN (suprachiasmatic
nucleus). In the brain, SIRT1 activates the transcription of CCGs,
including Bmal1 and Per2, by cooperatively
binding with and deacetylating PGC1-α at the promoters of CCGs.
Notably, SIRT1 modulates the central circadian clock by a mechanism
that becomes less efficient in aged animals. It has been reported
that NAMPT (nicotinamide phosphoribosyltransferase), a key enzyme
that controls the NAD salvage pathway, is also a CCG that is controlled
by the circadian machinery containing SIRT1.
205
Thus, when the NAD levels decrease during aging,
206
SIRT1 becomes less active and the circadian control decays.
Therefore, it is also proposed that SIRT1 acts as a rheostat to transduce
signals generated from cellular metabolism to the circadian clock
control.
204
3.2
SIRT2 in Epigenetic Regulation
SIRT2
is connected with multiple cellular processes, including mitosis,
cell cycle, cell death, metabolism, and aging. SIRT2 was initially
reported to be mainly in the cytoplasm, colocalizes with microtubules,
and deacetylates α-tubulin at Lys40.
298
It was recently reported to regulate many other cytosolic proteins,
including LDH-A (lactate dehydrogenase A), PEPCK1 (phosphoenolpyruvate
carboxykinase 1), ACLY (ATP-citrate lyase), and G6PD (glucose-6-phosphate
dehydrogenase) (see Table 2). It was later
found that SIRT2 can translocate to the nucleus and has important
epigenetic roles. A nuclear specific isoform of SIRT2 was also reported,
but it has no deacetylase activity.
299
Table 2
SIRT2 Deacetylation Substrates
substrate
full name
modified
lysine residues
function
of SIRT2-catalyzed deacetylation
histones
H4
histone H4
K16
regulate chromatin condensation
during metaphase, regulate H4K20 methylation, promote cell cycle progression
and genome stability; suppress transcription of certain genes
300−302
H3
histone H3
K18
L. monocytogenes InlB triggers
SIRT2 nuclear localization to suppress gene transcription;
303
involved in DNA replication and DNA damage
repair
150,304
K56
unclear, may be involved
in DNA damage repair
transcription
factors
P300
many
promote binding of p300
to preinitiation complex
305
FOXO3
forkhead box protein O3
unknown
increase FOXO3 DNA binding
and target gene transcription;
148,306
increase FOXO3 ubiquitinylation
and degradation
234
FOXO1
forkhead box protein O1
unknown
promote FOXO1 interaction
with PPARγ and repress PPARγ target genes;
307,308
inhibit FOXO1 interaction with ATG7 and autophagic cell death
309
HIF-1α
hypoxia-inducible factor
1α
K709
promote hydroxylation and
degradation of HIF-1α
310
NF-κB
nuclear factor κ B
K310
suppress NF-κB-dependent
gene expression
311
PGC-1α
peroxisome proliferator-activated
receptor-γ coactivator 1α
unknown
decrease expression of β-oxidation
and mitochondrial genes
312
cell cycle related
BubR1
mitotic checkpoint serine/threonine-protein
kinase BUB1 β
K688
stabilize
BubR1, improve
cardiac function, and extend lifespan in vivo
313
CDK9
cyclin-dependent
kinase
9
K48
increase CDK9 kinase activity
and decrease sensitivity to hydroxylurea-induced replication stress
response
314
CDH1/CDC20
CDH1/cell-division cycle
protein 20
K69 and
K159 (CDH1), K66
(CDC20)
activate
the E3 ubiquitin
ligase activity, leading todecreased Aurora A level
315
metabolic enzymes
LDH-A
lactate dehydrogenase A
K5
activates LDH-A
316
PEPCK
phosphoenolpyruvate carboxykinase
K70, K71
inhibit the ubiquitinylation
and degradation of PEPCK
317
ACLY
ATP-citrate lysase
K540, K546, K554
promote ATP-citrate lyase
degradation
318
G6PD
glucose-6-phosphate dehydrogenase
K403
promote the formation of
active G6PD dimer and increase NADPH production
319
PGAM
phosphoglycerate
mutase
K100
activate PGAM activity
320
cell
signaling related
PRLR
prolactin receptor
many
facilitate
prolactin receptor
dimerization and activation of STAT5
321
K-Ras
Kirsten rat sarcoma viral
oncogene homologue
K104
promote K-Ras
activity
322
PAR-3
partitioning defective 3
homologue
K831, K848,
K881, K1327
decrease
the activity of
aPKC and regulate myelin formation
323
TIAM1
T-cell lymphoma invasion
and metastasis 1
K1420
promote activation of DVL/TIAM1/Rac
axis and cell migration in cancer cells
295
structural proteins
keratin 8
keratin 8
K207
affect its phosphorylation
and filament organization
324
α-tubulin
α-tubulin
K40
destabilize microtubule
325
3.2.1
SIRT2
Deacetylates Histones and Regulates
Cell Cycle, DNA Repair, and Transcription
SIRT2 can deacetylate
H4K16 and regulate chromatin structure during cell cycle and DNA damage.
SIRT2 level is regulated by cell cycle. During mitosis, SIRT2 expression
level increases and translocates to the nucleus.
326
Deletion of SIRT2 leads to increased H4K16 acetylation
in mitosis and affects the cell cycle.
327
Interestingly, the hyperacetylation in Sirt2
–/–
mice also leads to loss
of H4K20me1, a histone mark that is established in G2/M phase by the
methyltransferase PR-SET7 and is important for cell cycle progression
and DNA repair.
301
H4K16 acetylation directly
inhibits the methylation of H4K20 by PR-SET7.
301
In addition, SIRT2 can also bind to PR-SET7 and deacetylate
it at Lys90. SIRT2 binding increases PR-SET7 activity, and deacetylation
promotes its recruitment to the chromatin.
301
The decrease in H4K20me1 is thought to be responsible for the defect
in pericentromeric heterochromatin and in DNA repair. Correspondingly, Sirt2
–/–
mice
suffer from greater DNA damage and genome instability.
301
Increased tumorigenesis in Sirt2
–/–
mice was observed in
a DNA-damage-induced skin tumor model. However, in contrast to the
previous report,
315
no increased spontaneous
tumorigenesis was observed in the knockout mice up to 1 year of age.
301
Unlike SIRT1, the gene-silencing function
of SIRT2 is less well known. However, a few examples do exist. SIRT2
is known to suppress the transcription of keratin 15/19,
328
ARRDC3 (arrestin domain-containing 3),
329
and NEDD4 (neural precursor cell expressed
developmentally downregulated protein 4).
302
The H4K16 deacetylation activity likely underlies the repression
function of SIRT2 on these genes. However, it is not clear how SIRT2
is specifically recruited to these genes.
Interestingly, SIRT2
is reported to function as an H3K18 deacetylase
in the context of Listeria monocytogenes infection.
303
In a series of elegant studies, Eskandarian
and co-workers demonstrated that the InlB protein of L. monocytogenes activates the
PI3K/AKT signaling pathway, leading to the nuclear
translocation of SIRT2. The nuclear translocation may be mediated
by a posttranslational modification of SIRT2. However, the detail
is not clear. Most strikingly, SIRT2 in this case does not deacetylate
H4K16, but instead it deacetylates H3K18. The deacetylation of H3K18
is thought to lead to the suppression of a set of genes during L. monocytogenesis
infection. Treatment with a SIRT2-specific
inhibitor, AGK2, inhibited the deacetylation of H3K18 and transcriptional
suppression. It is proposed that L. monocytogenesis uses this mechanism to manipulate
the host cell to maximize their
survival and proliferation. Indeed, inhibition or knockdown of SIRT2
significantly impaired L. monocytogenesis infection.
303
It is tempting to speculate that SIRT2-catalyzed
H3K18 deacetylation and transcriptional regulation also occur in mammalian
cells in the absence of bacterial infection. Eskandarian and co-workers
thought this is unlikely as SIRT2 inhibition by AGK2 did not change
the gene expression profile in the absence of L. monocytogenes infection. Future studies
will be required to further validate this.
SIRT2 has also been reported to deacetylate H3K56.
150,304
H3K56 acetylation level increases during S phase and in DNA damage
foci, colocalizes with double-strand break markers, such as γ-H2AX,
pATM, CHK2, and p53.
150,304
In yeast, histone H3K56 acetylation
has been shown to signal the completion of DNA repair and facilitate
the reassembly of nucleosomes at the repaired sites.
92
If the same applies in mammalian cells, SIRT2 may function
to remove the H3K56 acetylation after DNA repair.
3.2.2
SIRT2 Deacetylates Transcription Factors
and Regulates Transcription
SIRT2 can also deacetylate several
transcription factors or coactivators, thereby exerting effects on
transcription of specific genes. In this case, the effects on transcription
can be either positive or negative. SIRT2 was shown to deacetylate
p300 and promote binding of p300 to the preinitiation complex.
305
FOXO1 and FOXO3 are both known to be deacetylation
targets of SIRT2. FOXO1 deacetylation by SIRT2 promotes FOXO1 interaction
with PPARγ and represses PPARγ target genes.
307,308
Deacetylation by SIRT2 also inhibits FOXO1 interaction with ATG7
and autophagic cell death.
309
For FOXO3,
SIRT2-catalyzed deacetylation increases DNA binding and target gene
transcription in one report
306
and increases
FOXO3 ubiquitination and degradation in another.
148
HIF-1α can be deacetylated by SIRT2, which promotes
its hydroxylation and degradation.
310
NF-κB
and PGC-1α are also deacetylation targets of SIRT2.
311,312
For both proteins, deacetylation suppresses their target gene expression.
The regulation on transcription factors by SIRT2 can also be indirect.
For example, SIRT2 has recently been shown to stabilize the C-MYC
and N-MYC oncogenic transcription factors in neuroblastoma and pancreatic
cancer cells.
302
This is achieved by suppressing
the transcription of the ubiquitin ligase NEDD4 via histone H4K16
deacetylation.
302
3.3
SIRT6 in Epigenetic Regulation
Among
the seven mammalian sirtuins, SIRT6 is the second sirtuin, next to
SIRT1, whose deletion in mice causes very severe phenotypes, including
severe metabolic defects, genome instability, and premature aging.
330
Further studies revealed that SIRT6 regulates
many important pathways via epigenetic mechanisms, mainly histone
deacetylation. SIRT6 itself is regulated by several different mechanism,
including p53, AP-1 (activator protein 1), and SIRT1-mediated transcription
control,
331−333
as well as phosphorylation by AKT1, MDM2-mediated
ubiquitination, and UCP10 (UBX domain-containing protein 10)-mediated
deubiquitintion at posttranslational level.
334,335
3.3.1
SIRT6 in Gene Silencing
SIRT6 has
a very weak deacetylase activity toward peptide substrates in vitro.
By screening a series of histone peptides, Chua and others identified
that it can specifically deacetylate H3K9
41
and H3K56,
42,43
although the activity is still
fairly weak compared to SIRT1. More recently, Cohen and co-workers
found that SIRT6 deacetylates histones when they are packaged as nucleosomes
but not as free histones,
48
suggesting
that the deacetylase activity of SIRT6 is nucleosome dependent. Additionally,
Denu and co-workers’ study revealed that SIRT6’s ability
to remove acetyl group from histone peptides could also be activated
by the existence of free fatty acids, such as myristic, oleic, and
linoleic acids.
49
At present, most
of the gene-silencing effects of SIRT6 have been explained by its
H3K9 deacetylase activity. SIRT6 is recruited to HIF-1α target
gene promoters and suppresses the transcription by deacetylating H3K9.
332,336
Similarly, SIRT6 is shown to be recruited to MYC,
46
NF-κB,
47
C-JUN,
337,338
and FOXO3
339,340
target gene promoters and suppress
the transcription of these genes. The recruitment of SIRT6 by specific
transcription factors to help suppress gene transcription via H3 deacetylation
have been used to explain many phenotypes associated with SIRT6 deficiency,
including tumor development,
46
cardiac
hypertrophy,
338
growth retardation,
341
and lipid metabolism/liver inflammation.
339,340
This mechanism has similarly been used to explain the phenotype
of SIRT6 overexpressing transgenic mice, including the decreased low-density
lipoprotein cholesterol levels
340,342
and increased lifespan
in male mice.
343
The mechanism underlying
the lifespan extension effect caused by SIRT6 overexpression, however,
is not completely understood. The major gene expression change induced
by SIRT6 overexpression that contributes to the lifespan extension
is the increased IGFBP1 (insulin-like growth factor binding protein
1) level.
343
How SIRT6 increases IGFBP1
expression cannot be directly explained using the simple model described
above. It is possible that SIRT6 suppresses the expression of another
factor that can suppress IGFBP1 expression.
A genome-wide CHIP-Chip
analysis showed that SIRT6 binds to the
promoters of about 2000 genes in mouse fibroblasts.
344
SIRT6 chromatin localization has significant overlap with
that of NF-κB. Furthermore, the localization is very dynamic.
TNFα stimulation causes the release of SIRT6 from a large portion
of the 1900 genes and the relocalization to an even larger number
(∼4300) of different genes.
344
Despite
the fact that SIRT6 deletion changes the expression of many NF-κB
target genes, there is a report that overexpression of SIRT6 in mice
does not change the expression of NF-κB target genes.
345
This was also observed in the studies by Cohen
and co-workers on the lifespan-extending role of SIRT6 in male mice.
343
A model that could explain these observations
is that at resting state SIRT6 binds to promoters of NF-κB target
genes to suppress the basal expression. There is perhaps enough SIRT6
to suppress the basal expression of target genes, and thus, overexpression
of SIRT6 does not further increase the suppression. When there is
an external stimulation (e.g., TNFα), NF-κB will be turned
on more strongly, which leads to dissociation of SIRT6, releasing
the suppression.
SIRT6 is demonstrated to be required for the
telomere position
effects in mammalian cells,
346
similar
to the role of yeast SIR2 discussed above. Knockdown of Sirt6 increased the expression
of both an integrated lucifierase reporter
gene and an endogenous telomere-proximal gene. The silencing effect
requires the deacetylase activity of SIRT6 and is associated with
decreased H3K9 acetylation and increased H3K9 methylation.
346
SIRT6 has also been reported to deacetylate
transcription factors
or coactivators. GCN5 was shown to be a deacetylation target of SIRT6.
347
Deacetylation of GCN5 increases its acetyltransferase
activity, leading to increased PGC-1α acetylation and decreased
gluconeogenesis gene expression.
347
This
is in contrast to the role of SIRT1 in PGC-1α regulation, which
directly deacetylates PGC-1α and activates gluconeogenesis gene
expression. SIRT6 has recently been reported to deacetylate transcription
factor FOXO1 and regulates the expression of gluconeogenesis genes.
331
The tumor suppressor p53 upregulates SIRT6
expression, which promotes the deacetylation and nuclear exclusion
of FOXO1, leading to downregulation of gluconeogenesis genes.
331
The deacetylation of other transcription factors
by SIRT6, such as HIF-1α, was suspected but not detected.
102
However, the deacetylation of both GCN5 and
FOXO1 by SIRT6 was only demonstrated in vivo but not in vitro. Thus,
it remains possible that the deacetylation of FOXO1 and PGC-1α
by SIRT6 is indirect, and further validation will be helpful.
SIRT6 has also been indicated to control the circadian clock in
mice liver.
348
The circadian expression
of many liver genes is affected by SIRT6, which is distinct and shows
only partial overlap with circadian gene expression controlled by
SIRT1. This is an interesting observation, but mechanistically this
seems to be different from the silencing role of SIRT6 described above
for other transcription factors, such and NF-κB and HIF-1α.
Existing data suggest that SIRT6 can bind to CLOCK and BMAL1. SIRT6
also decreases the binding of BMAL1 and SREBP1 at target gene promoters.
The role of SIRT6 in BMAL1 and SREBP1 regulation seems to resemble
that in FOXO1 regulation but without deacetylating CLOCK/BMAL1 or
SREBP1. This example also illustrates that the role of SIRT6 in transcriptional
regulation is still not well understood.
3.3.2
SIRT6
in Genome Stability and DNA Repair
SIRT6 plays several distinct
roles in DNA repair and genome stability. Sirt6 mice
display genome instability.
330
SIRT6-deficient
cells have increased sensitivity to ionizing
radiation, monofunctional alkylating reagents, and hydrogen peroxide
but normal sensitivity to UV-induced DNA damage or endonuclease induced
double-strand break, which suggest that SIRT6 is important for base
excision repair (BER).
330
PARP1 is another
protein that is involved in BER; however, PAR foci formation in Sirt6 knockout cells
were not affected, suggesting that
SIRT6 may be acting downstream of PARP1.
330
Sirt6 deletion leads to end-to-end chromosomal
fusion and abnormal telomere structures that resemble defects observed
in Werner syndrome.
41
Later, SIRT6 was
also found to be important for DNA double-strand break repair.
349,350
The molecular role of SIRT6 in DNA repair is not entirely clear
and likely multifacet. The telomere fusion phenotype has been explained
by a model that SIRT6-catalyzed H3 K9 deacetylation forms a special
chromatin state at telomeres, which is required to recruit WRN, the
factor that is mutated in Werner syndrome. During DNA double-strand
repair, it has been shown that SIRT6 deacetylates H3K9 at DNA damage
site and helps to recruit DNA-PK (DNA-dependent protein kinase).
349
SIRT6 also promotes DNA end resection by deacetylating
CtIP (CtBP-interacting protein), a protein involved in DNA end resection.
350
Mao et al. also reported that SIRT6 can ADP-ribosylate
PARP1 to increase PARP1 activity and promote DNA repair.
376
Recently, SIRT6 is reported to be one of the
earliest enzymes recruited to DNA double-strand breaks and promotes
the recruitment of a chromatin remodeling protein, SNF2H, and deacetylates
H3K56. The involvement of chromatin remodeler and histone deacetylation
suggests that similar to DNA repair in yeast discussed earlier, dynamic
chromatin structure changes are required for DNA repair.
377
3.4
Role of SIRT7 in Epigenetic
Regulation
Similar to SIRT6, SIRT7 is also a class IV sirtuin
that is mainly
localized in the nucleus.
351
It is specifically
enriched in the nucleolus.
351
The nucleolus
localization is dependent on active RNA Pol I transcription.
352
In addition, significant cytoplasmic localization
of SIRT7 has also been reported.
353
Many
interacting proteins have been identified, many of which are associated
with ribosome biogenesis.
354−356
SIRT7 has low deacetylase
activity,
351
and because of this, its first
deacetylation substrate was only identified in 2012. By screening
a set of acetyl lysine peptides, Chua and co-workers discovered that
SIRT7 can specifically deacetylate H3K18.
357
This elegant study demonstrated that SIRT7 is recruited to the promoters
of a set of genes, many of which are controlled by a transcription
factor called ELK4. SIRT7 is recruited by ELK4 to the promoters of
target genes, leading to H3K18 deacetylation and suppression of transcription.
357
Recently, MYC was identified as another transcription
factor that also recruits SIRT7 to target gene promoters (mainly genes
encoding ribosome proteins) to suppress transcription via H3K18 deacetylation.
358
Interestingly, it was shown that SIRT7 attenuate
ER (endoplasmic reticulum) stress and prevents fatty liver formation
in mice by suppressing MYC target genes. The model proposed was that
under ER stress, SIRT7 suppresses translation by suppressing ribosomal
protein synthesis, thus relieving the protein folding pressure in
the ER. However, this is controversial as another report showed that
SIRT7 promotes fatty liver formation under a high-fat diet.
359
This latter report by Yoshizawa et al. showed
that SIRT7 can bind to an E3 ubiquitin ligase complex DCAF1/DDB1/CUL4B
and inhibits its activity in promoting TR4 (testicular receptor 4)
degradation. It has been reported that TR4 deficiency protects mice
from high-fat diet-induced hepatic steatosis. Thus, without SIRT7,
TR4 level decreases, which leads to decreased expression of genes
that promote lipid deposition, such as CD36 and CIDEA, and protects
mice from high-fat diet-induced fatty liver. The SIRT7 effect on TR4
can at least partly explain the phenotype of Sirt7 knockout.
359
SIRT7 is unique in
the sense that it can regulate both RNA Pol
II transcription by working with different transcription factors as
described above and RNA Pol I transcription. One of the earliest effects
of SIRT7 reported was its ability to positively regulate RNA Pol I
transcription.
352
The mechanism underlying
the effect was found to be SIRT7-mediated deacetylation of the PAF53
subunit of RNA Pol I.
360
Deacetylation
of PAF53 leads to increased association of RNA Pol I with rDNA and
increased rRNA transcription.
360
4
Summary and Outlook
The studies on sirtuins discussed
above provided a number of important
lessons in chromatin biology and epigenetics. These studies also revealed
many unresolved fundamental questions. Here we discuss a few of the
lessons and questions that may be useful for guiding future studies.
4.1
Value of Model Organisms
Sirtuins
have attracted many researchers, and the number of publications on
sirtuins have increased dramatically in the past decade. Looking back,
it is stunning that this prosperous research area was initiated from
some basic genetic studies in a simple model organism, the budding
yeast. Obviously, there are important differences between yeast and
mammalian sirtuins. The biological function of mammalian sirtuins
is more complex and quite different from that of yeast. However, the
basic enzymatic function is conserved, and many general trends are
similar. Thus, it is important to appreciate the value of model organisms.
4.2
Connection between Sirtuins, Metabolism, and
Epigenetics
The connection between sirtuins, metabolism,
and epigenetics is manifested in several aspects. First, most of the
protein acyl lysine modifications use acyl-CoA molecules as the acyl
donors, which are common metabolites in cells. Thus, metabolism can
affect these PTMs, including the epigenetic modifications on histones,
by changing the concentrations of the acyl-CoA molecules. The absolute
requirement of NAD as a cosubstrate for sirtuin-catalyzed deacylation
suggests that sirtuins can act as NAD sensors that transduce metabolism
signals to epigenetic regulations of gene expression. It has been
reported that NAD levels increase in muscle and white adipose tissue
upon caloric restriction, thereby activating sirtuins.
361
Indeed, it was found in many studies that SIRT1 activity is low in
conditions of glucose excess and high in conditions of nutrient limitation.
362
As discussed above, it has been well established
that SIRT1 regulates metabolic responses to changes in nutritional
availability in multiple tissues. However, it still remains elusive
how other mammalian sirtuins respond to changes in metabolism and
how the responses affect epigenetic marks in cells. The observation
that SIRT6 can be activated by free fatty acids suggests that sirtuins
may be regulated by metabolites other than NAD. It remains to be elucidated
whether other metabolites regulate sirtuins and how the regulation
contributes to the epigenetic roles of sirtuins.
4.3
Recruitment of Mammalian Sirtuins to Different
Regions of Chromatin or DNA Damage Sites
For yeast SIR2,
the recruitment to the silencing loci is relatively well understood.
Such understanding for mammalian sirtuins is only partial. For example,
how SIRT1 is recruited to certain gene promoters or DNA damage sites
is not completely known. The recruitment of SIRT6 by different transcription
factors is relatively clearer. However, given that SIRT6 interacts
with many different transcription factors, what determines the binding
specificity is not clear. The recruitment of SIRT2 to chromatin is
even more mysterious as no transcription factor is known to bind to
SIRT2. Furthermore, because SIRT2 deacetylates H4K16 in certain cases
while it deacetylates H3K18 in other cases, there may be different
mechanisms to recruit SIRT2, which may affect the selectivity of SIRT2
for H3K18 versus H4K16.
4.4
Possibility of New Epigenetic
Marks
Sirtuins were initially thought to be deacetylases,
but recent evidence
suggests that some of them can remove other acyl lysine modifications
more efficiently.
34,44,45
Succinyl lysine was reported to occur on histones.
127
However, since SIRT5 is mainly in the mitochondria, whether
histone succinylation is an epigenetic mark remains to be elucidated.
A more interesting case is SIRT6. In vitro with peptide substrates,
it is much more efficient at removing long chain fatty acyl groups.
In vivo, the deacetylation of histone H3K9 and H3K56 by SIRT6 has
been firmly demonstrated. The discrepancy between in vivo and in vitro
may be partly explained by the report that SIRT6’s deacetylase
activity is increased on nucleosome substrates
363
or in the presence of free fatty acids (Table 3).
49
However, another
possibility also needs to be further investigated, which is that histones
lysine fatty acylation could be a new epigenetic mark and SIRT6 can
regulate this mark. Given that histones have been shown to contain
Cys palmitoylation,
364
Lys fatty acylation
on histones is also possible.
Table 3
Comparison of the
Deacetylation and
Defatty-Acylation Activities of SIRT6
acyl peptide
k
cat (S1–)
K
m (μM)
k
cat/K
m (S–1 M–1)
H3K9 acetyl
0.0039 ± 0.0006
365
810 ± 160
365
or ∼450
49
4.8
365
or 6.4 ± 2
49
H3K9 acetyl with myristic
acid
∼0.002
49
9 ± 1
49
230 ± 30
H3K9 myristoyl
0.0049 ± 0.0004
365
3.4 ± 0.9
365
1.4 × 103
365
H3K9 palmitoyl
0.0027 ± 0.0002
365
0.9 ± 0.4
365
3.0 × 103
365
4.5
Function
of Different Histone Acetylation
Sites
The available experimental evidence suggest that different
mammalian sirtuins have preference for different histone acetylation
sites. The transcriptional-silencing effect of SIRT2 is achieved through
deacetylation of either H3K18 or H4K16, while the silencing effect
of SIRT6 is achieved mainly through H3K9 deacetylation. Mechanistically,
this is very interesting. Is deacetylation of a single site (e.g.,
H3K18) sufficient to suppress transcription or this is because only
one site is acetylated in vivo at the relevant loci? Do different
acetylation sites have different function (e.g., associated with target
genes of different transcription factors)? The selectivity of sirtuins
toward unique histone acetylation sites may provide a unique opportunity
to address these questions. Addressing these questions will provide
important insights into the fundamental epigenetic mechanisms.
4.6
Drug Discovery Targeting Sirtuins and the
Importance of Biochemical Assays
Since increasing lines of
evidence provide significant support for the importance of sirtuins
in many biological processes, there has been a broad interest in developing
small molecules that regulate sirtuins. Sirtuin activators have attracted
more interest given the importance of SIRT1 in mediating the beneficial
effects of calorie restriction.
366
The
natural polyphenol, resveratrol, was the first molecule shown to activate
SIRT1
367
and extend the life span of S. cerevisiae,
367
Drosophila,
368
and C. elegans.
369
Later, large-scale screening identified more
potent and specific SIRT1 activating compounds (STACs), including
SRT1720, SRT2183, and SRT1460. More studies also showed that STACs
are able to protect mice from high-fat-diet-induced metabolic disease.
370
However, studies on STACs still remain controversial.
It was shown that the SIRT1-activating effect of STACs was only obvious
when using an artificial p53-derived Fluo-de-Lys peptide, and the
STACs did not lead to apparent SIRT1 activation with native p53 peptide
lacking a fluorophore.
371,372
Later re-evaluation
of the STACs revealed an assisted allosteric activation mechanism
by which the hydrophobic fluorophore mimics the properties of endogenous
substrates required for STACs-mediated SIRT1 activation. Analysis
of several peptides with consensus motif carrying the corresponding
properties revealed that their deacetylation by SIRT1 was enhanced
by STACs, suggesting the STACs may work on specific SIRT1 substrates
in vivo.
373
It should be pointed out that
even though controversies exist, the concept of STACs is valid. More
effort is needed to develop STACs that are chemically diverse but
do not act by the assisted allosteric activation mechanism. These
studies on the development of STACs also highlight the potential pitfalls
of biochemical assays (especially high-throughput assays) and suggest
that it is important to use alternative biochemical assays to validate
results.
Sirtuin inhibitors have also been developed, and many
of them have been shown to impact the progression of neurodegenerative
disorders or to exhibit anticancer activity.
374,375
Also, specific sirtuin inhibitors can be excellent tools for in
vivo studies of the physiological roles of sirtuins. However, the
development of sirtuin inhibitors has been mainly limited to academia
so far. The effects of sirtuins are extremely complex given the large
number of biological substrates regulated by sirtuins and the multiple
enzymatic activities possessed by a certain sirtuin. It remains challenging
to determine the precise and disease-related targets of sirtuins and
to fully understand the therapeutic potential of targeting sirtuins.
As the disease relevance of sirtuins gets clearer, sirtuin inhibitor
development may be increasingly taken up by pharmaceutical companies.
The importance of biochemical assays is also reflected in the sirtuin
inhibitor development. This is particularly relevant for sirtuins
with weak deacetylase activities in vitro. For these sirtuins, the
development of inhibitors lags behind because there was no reliable
assay to analyze the inhibition effects of compounds. As more efficient
activities of these sirtuins are discovered, the inhibitor development
for these sirtuins will speed up.
Given the fundamental questions
and the therapeutic potentials
of sirtuins mentioned above, we believe that research interest and
activity in sirtuins will continue to rise and new exciting development
will continue to occur in the next decade.