Reviews

This Review is part of a thematic series on Cardiovascular Complications of Diabetes and Obesity, which includes the
following articles:
The Impact of Macrophage Insulin Resistance on Advanced Atherosclerotic Plaque Progression [Circ Res.
2010;106:58 67]
The RAGE Axis: A Fundamental Mechanism Signaling Danger to the Vulnerable Vasculature [Circ Res.
2010;106:842 853]
The Promise of Cell-Based Therapies for Diabetic Complications: Challenges and Solutions [Circ Res.
2010;106:854 869]
Activation of Protein Kinase C Isoforms and Its Impact on Diabetic Complications [Circ Res. 2010;106:1319 1331]
Aldose Reductase and Cardiovascular Diseases, Creating Human-Like Diabetic Complications In An Experimental Model
[Circ Res. 2010;106:1449 1458]
Endoplasmic Reticulum Stress and Inflammation in Obesity and Diabetes [Circ Res. 2010;107:579 591]
Oxidative Stress and Diabetic Complications [Circ Res. 2010;107:1058 1070]
Epigenetics: Mechanisms and Implications for Diabetic Complications
Ann Marie Schmidt, Guest Editor

Epigenetics
Mechanisms and Implications for Diabetic Complications
Mark E. Cooper, Assam El-Osta

Abstract: Epigenetic modifications regulate critical functions that underlie chromosome metabolism. Understanding the molecular changes to chromatin structure and the functional relationship with altered signaling pathways
is now considered to represent an important conceptual challenge to explain diabetes and the phenomenon of
metabolic or hyperglycemic memory. Although it remains unknown as to the specific molecular mechanisms
whereby hyperglycemic memory leads to the development of diabetic vascular complications, emerging evidence
now indicates that critical gene-activating epigenetic changes may confer future cell memories. Chemical
modification of the H3 histone tail of lysine 4 and 9 has recently been identified with gene expression conferred
by hyperglycemia. The persistence of these key epigenetic determinants in models of glycemic variability and the
development of diabetic complications has been associated with these primary findings. Transient hyperglycemia
promotes gene-activating epigenetic changes and signaling events critical in the development and progression of
vascular complications. As for the role of specific epigenomic changes, it is postulated that further understanding
enzymes involved in writing and erasing chemical changes could transform our understanding of the pathways
implicated in diabetic vascular injury providing new therapeutic strategies. (Circ Res. 2010;107:1403-1413.)
Key Words: epigenetics histones chromatin hyperglycemic memory diabetic complications

iabetic vascular complications remain the major cause

of morbidity and mortality in both type 1 and type 2
diabetes. Although much of the original research and indeed
the earliest end points in clinical studies of type 1 and type 2
diabetes often focused on microvascular end points such as

retinopathy and nephropathy, it is increasingly appreciated

not only in type 2 but also in type 1 diabetes that macrovascular disease presenting clinically as myocardial infarction,
strokes, and peripheral vascular disease confers a major
burden in the diabetic population leading to premature death.1

Original received July 10, 2010; revision received October 6, 2010; accepted October 7, 2010. In September 2010, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 13.1 days.
From Diabetic Complications (M.E.C.) and Epigenetics in Human Health and Disease (A.E.-O.), Baker IDI Heart and Diabetes Institute; Department
of Pathology (A.E.-O.), University of Melbourne; and Faculty of Medicine (A.E.-O.), Monash University, Victoria, Australia.
Correspondence to Dr Assam El-Osta, Baker IDI Heart and Diabetes Institute, Epigenetics in Human Health and Disease, 75 Commercial Rd,
Melbourne 3004, Australia. E-mail assam.el-osta@bakeridi.edu.au
2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org

DOI: 10.1161/CIRCRESAHA.110.223552

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Non-standard Abbreviations and Acronyms

AGE
ChIP
DCCT
EDIC
H3K4
H3K9
LSD1
m1
m2
m3
MCP
NF-B
p65
PKC
ROS
RAGE
Set7

UKPDS
VCAM

advanced glycated end products

chromatin immunopurification
Diabetes Control and Complications Trial
Epidemiology of Diabetes Intervention and Complications
histone tail of H3 lysine 4
histone tail of H3 lysine 9
lysine-specific demethylase 1
monomethylation
dimethylation
trimethylation
monocyte chemotactic protein
nuclear factor light chain enhancer of activated B cells
transcription factor p65 encoded by the RELA gene
protein kinase C
reactive oxygen species
receptor for advanced glycation end products
enzyme that can methylate lysine residues including H3
lysine 4 and a member of the SET domain-containing
family
United Kingdom Prospective Diabetes Study
vascular cell adhesion molecule

There have been a large number of epidemiological studies

as well as more recent clinical trials exploring the role of
intensive glycemic control in reducing the development and
progression of vascular complications in type 1 and type 2
diabetes.27 Of particular interest were the findings from the
Diabetes Control and Complications Trial (DCCT, 1983 to
1993) and its follow up observational study, the Epidemiology of Diabetes Intervention and Complications (EDIC, 1994
to 2006) trial where the term metabolic memory was
introduced.8 10 This term was used to explain a phenomenon
where the long-term vascular benefits of a previous period of
good glycemic control persist despite a return to usual, often
worse metabolic control. This phenomenon was reported in
the DCCT/EDIC trial not only for nephropathy and retinopathy but more recently also with respect to macrovascular
disease.11 Contrary to the initial report from the DCCT phase
of the trial indicating no differences in cardiovascular events
were observed between the intensive and conventionally
treated groups, in the EDIC phase, clear differences were
demonstrated, with the intensive group having a much lower
risk of cardiovascular as well as renal and retinal disease.11
With a cohort of 1441 individuals and a mean follow-up of
6.5 years,12 the DCCT clinical study demonstrated that
intensive blood glucose control reduced the onset and progression of long-term diabetic complications, including retinopathy, nephropathy, and neuropathy.1318 The frequency of
cardiovascular events was too low to determine whether the
interventions had significantly different effects and therefore
the follow-up EDIC study examined the long-term effects of
the original DCCT group. The completion of the DCCT study
in 1993 transitioned with the launch of the EDIC observational
trial in 1994 with some surprising results. During the EDIC

study, the difference in glycemic control measured by hemoglobin A1c between the intensive treatment group compared with
the conventional treatment group was 2% (7.2% compared
9.1%, respectively) but was now indistinguishable toward the
end of the EDIC follow-up study. The study group identified that
the benefit of intensive therapy for 6.5 years conducted early had
persisted for at least 10 years despite the normalization of
hemoglobin A1c, raising the intriguing concept of long-term
metabolic memory of previous therapies.9,10,19
Over the last few years, this issue linking glucose levels to
diabetic complications has been extensively examined in type
2 diabetes. The phenomenon of metabolic memory has been
given the term as the legacy effect by investigators from the
United Kingdom Prospective Diabetes Study (UKPDS). The
authors of the report indicated that the initial modest effect of
borderline statistical significance of glucose lowering on macrovascular disease described at the termination of the UKPDS
were now much clearer more than 10 years later, despite both the
intensive and conventionally treated groups returning to usual
glucose lowering management after the completion of the active
phase of the trial.20 Thus, based on the findings of the DCCT/
EDIC and UKPDS trials, it appears that there is evidence of
metabolic memory in type 1 and type 2 diabetes, respectively.
More recently, 3 studies, the ADVANCE (Action in Diabetes
and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation), ACCORD (Action to Control
Cardiovascular Risk in Diabetes), and VADT (Veterans Affairs
Diabetes Trial), have reported, in general, unremarkable effects
of glucose lowering on cardiovascular events, cardiovascular
death, and all-cause mortality as a result of glucose-lowering
strategies in type 2 diabetes.2123 However, although the glucoselowering phase of these trials is completed, the mean follow-up
of these trials is much shorter to date than that of the DCCT/
EDIC, which identified persistent benefits years later. In view of
the potential long-term end-organ effects of metabolic memory,
it is predicted that the ongoing follow up of individuals in these
trials, as is indeed already in progress for the ADVANCE trial,
will confirm whether these trials, which involved periods of at
least several years of improved glycemic control, will ultimately
confer reduced cardiovascular disease.
The underlying molecular explanation for metabolic memory remains to be fully elucidated. This concept was elegantly
demonstrated more than 20 years ago in the seminal studies
by Engerman and Kern.24 After 2.5 years of poor glucose
control in diabetic dogs, these animals were switched to good
glycemic control. However, these animals had the same
evidence of retinopathy when compared with dogs subjected
to poor glycemic control for the entire period of the study.
The study was terminated at 5 years, and therefore any
longer-term benefit of intensive treatment might have had on
retinopathy progression or regression was not assessed. Although these experiments in diabetic dogs were focused on
retinopathy, it is likely that this phenomenon, as reported
clinically in the DCCT/EDIC and UKPDS trials, is also seen
with respect to nephropathy and atherosclerosis. Indeed, in
recent studies by our group in diabetic apolipoprotein E
knockout mice, restoration of near normoglycemia after a
period of hyperglycemia was associated with persistent ath-

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Cooper and El-Osta

erosclerosis,25 findings that are reminiscent of the original
retinal findings in the diabetic dogs.24
It has been difficult to identify the mechanisms responsible
for metabolic memory, but, surprisingly, studies performed
20 years ago provide potential clues in this area. In a series of
cell culture experiments, transient exposure of endothelial
cells to high glucose followed by a return to low glucose was
associated with persistent upregulation of various extracellular matrix proteins including fibronectin and type IV collagen.26 In these experiments, the authors postulated a role for
epigenetic pathways. However, at the time, these investigators did not have the methodologies now available to adequately investigate this possibility.

Pathogenesis of Diabetic Complications

Although it has been well known that metabolic factors such
as glucose and hemodynamic stimuli such as vasoactive
hormones or stretch, manifested clinically as hypertension,
are critical in the development and progression of vascular
complications, the key intermediates in preventing end-organ
injury have only recently been increasingly defined.27 Seminal studies reported a decade ago by Brownlee and colleagues
demonstrated a central role for the generation of mitochondrial reactive oxygen species as a key pathway whereby
glucose confers injury on cell populations implicated in
vascular disease.28 The original studies were performed in
endothelial cells and many of the subsequent in vitro studies
exploring hyperglycemic memory have used similar cell
populations. Mitochondrial reactive oxygen species (ROS)
generation, as a result of hyperglycemia, is considered upstream of many of the pathways implicated in the pathogenesis and development of diabetic complications such as
advanced glycation and protein kinase C activation.29
It is appreciated that the advanced glycation pathway participates in diabetes-associated vascular disease, as determined in
vivo in models where the pathway has been inhibited at various
levels including pharmacological approaches to inhibit formation of advanced glycated end products (AGEs) or at the level of
the key receptor,30 RAGE, either deleted genetically or inhibited
by a competitive antagonist, soluble RAGE.31 Furthermore, a
key precursor of AGE formation, the dicarbonyl intermediate,
methylglyoxal appears to be critical in the development of
diabetic complications.29 This molecule is degraded by the
enzyme glyoxylase-1, with manipulation of this enzyme being
an approach that has been used experimentally to directly
modulate methylglyoxal levels, independent of glucose.32 Therefore, in view of the increasing body of evidence implicating
mitochondrial ROS and methylglyoxal in the development of
diabetic vascular complications, these were 2 major targets
considered worth investigating with respect to the phenomenon
of metabolic memory.33 The possible role of classic pathways of
glucose-induced injury being involved in this phenomenon was
also recently identified in retinal endothelial cells, which could
be attenuated by a range of inhibitors of these pathways
including antioxidants.34

Nuclear Factor B, Inflammation, and

Diabetic Complications
In recent years, it has become evident that inflammation is a
prominent feature of diabetic vascular disease. Indeed, au-

Epigenetic Hyperglycemic Memory

1405

topsy studies have suggested that plaques examined from

diabetic individuals show increased inflammatory changes
with enhanced macrophage infiltration when compared with
plaques from nondiabetic subjects.35 These findings have
stimulated research in exploring links between hyperglycemia and key proinflammatory pathways. Such studies have
led to the view that the transcriptional determinant, nuclear
factor (NF)-B, which is readily activated by hyperglycemia,
plays a pivotal role in diabetic vascular complications.29
Furthermore, NF-B activation leads to the upregulation of
molecules such as the chemokine, monocyte chemotactic
protein (MCP)-1, and adhesion molecules such as vascular
cell adhesion molecule (VCAM)-1, which have been extensively investigated in atherosclerosis, albeit predominantly in
a nondiabetic context.36 In view of these relatively recent
findings, subsequent experiments exploring the molecular
mechanisms involved in hyperglycemic memory have focused on NF-B and its dependent proteins including MCP-1
and various adhesion molecules.

Importance of MCP-1 and VCAM-1 in

Diabetic Complications
Recent in vitro studies have clearly defined the ability of
epigenetic mechanisms to modulate glucose-induced gene
expression of the key subunit of NF-B, p65 (transcription
factor p65 encoded by the RELA gene) with subsequent
effects on NF-B activation and in particular on expression of
the key proinflammatory molecules VCAM-1 and MCP-1.33
This builds on a large body of experimental data that have
identified in diabetic blood vessels, as well as in other sites of
end-organ injury including the kidney and retina. For example, in models of diabetes associated atherosclerosis there is
prominent upregulation of the adhesion molecule VCAM-1
and the chemokine MCP-1.37,38 Furthermore, classic pharmacological approaches such as interruption of the reninangiotensin system with angiotensin-converting enzyme inhibitors37 and angiotension II receptor antagonists38 can attenuate
diabetes-associated upregulation of VCAM-1 and MCP-1. In
addition, more novel experimental strategies that have not yet
been introduced to clinical practice, such as interrupting the
AGE/RAGE axis, have also been reported to block NF-B
activation and in particular to reduce vascular expression of
chemokines such as MCP-1 and adhesion molecules including VCAM-1 and intercellular adhesion molecule-1.39 It
remains to be determined whether targeting more upstream
molecular events including the inhibition of epigenetic pathways, specifically blocking key enzymes mediating epigenetic change, will lead to a more effective approach to
attenuate diabetes associated vascular inflammation.

Role of Oxidative Stress in Mediating

Diabetes-Associated Epigenetic Modifications
It is well known that enhanced ROS generation predominantly of mitochondrial origin is considered to play a key role
in the pathogenesis of diabetic complications and in particular
in endothelial abnormalities observed in diabetes.29 To further
define how glucose could modulate epigenetic events, it was
postulated that ROS could be key mediators of this phenomenon. The role of mitochondrial ROS generation was ex-

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December 10, 2010

plored using molecular approaches involving the transfection

of endothelial cells with adenovirus overexpressing the constructs for manganese superoxide dismutase and uncoupling
protein-1.33 These molecular approaches led to the inhibition
of glucose-induced NF-B activation as a result of reduced
p65 gene expression. This attenuation of gene expression
appeared to occur as a result of prevention of glucose-induced
histone methylation of the p65 gene. To complement this
approach, experiments in our laboratory using idebenone,40 a
relatively selective mitochondrial antioxidant, reduced recruitment of the Set7 histone methyltransferase to chromatinized p65
template (Assam El-Osta, unpublished observations, 2008). It
remains to be determined whether other sources of ROS, such as
cytoplasmic ROS generated by enzymes such as NADPH
oxidase, can also influence epigenomic events. This is relevant
because NADPH oxidase has been clearly demonstrated to play
a role in NF-B activation in the diabetic context.41
Another factor that can influence NF-B is the key intracellular signaling molecule protein kinase (PK)C.29 To explore the
role of PKC, pilot studies were performed using the nonselective
PKC inhibitor bisindolylmaleimide, which demonstrated reduced H3K4m1 (monomethylation of the histone tail of H3
lysine 4) of the p65 gene sequence with associated abrogation of
the glucose induced activation of p65 gene expression (Assam
El-Osta, unpublished observations, 2008). With increasing evidence that PKC, specifically the , , and isoforms, may be
relevant to the diabetic context,42 it will be of interest to further
define the role of these enzymes in mediating chromatin remodeling events in the diabetic vasculature.

The Distinguished Genome, Epigenetic

Modifications to the Chromatinized Template
The significance of genome structure and function cannot be
fully explained nor understood unless we begin to untwine
the DNA strands and examine the core nucleosome components. Indeed, not all genomic sequences are born equally.
The genome is in fact characterized by epigenetic sequences
that distinguish the polyvalent chromatin fiber, which we and
other groups have recently exemplified in specified models of
glycemic variability.25,33,43,44 The posttranslational modification of histone proteins represent chemical variations to the
chromatin template, which control sequence structure and
gene function. The diversity of modifications as well as the
pattern and distribution can reflect different structural and
functional roles mediated by epigenetic change.
The multiplicity of modifications represent a complexity of
epigenetic regulation, with the best studied being the lysine
residue of the histone tail. These amino acids can be distinctly
modified by different classes of covalent changes that include
acetylation and methylation, phosphorylation, ubiquitination,
or sumoylation (Table 1). When, in 1977, the structure of the
nucleosome core particle was described, the significance of
the original work on nucleosomal crystals heralded a new
beginning in chromatin biology and an advancement in our
understanding of chromosome structure and function.45 More
than 20 years later, the concept that nucleosomal modifications
regulating chromatinized events, now referred to as the histonecode hypothesis, was published and has since gained considerable momentum with the realization that nucleosomal histones

Table 1.

Posttranslational Modifications of Histones

Covalent
Changes

Histone Residues

Chromosome Function

Acetylation

Lysine

Transcription, repair, replication,

condensation

Methylation

Lysine, m1, m2, or m3

Transcription, repair

Methylation

Arginine, m1 or m2

Transcription

Phosphorylation

Serine, threonine

Transcription, repair,
condensation

Ubiquitination

Lysine

Transcription, repair

Sumoylation

Lysine

Transcription

are distinct in pattern and distinguishable by modification.46 The

acetylation mark was the first posttranslational modification
identified on histones,47 and the impact and significance of
research in this area has exploded with the identification of
enzymes that can either write48 or erase49 these acetylation
moieties. The majority of acetyltransferases belong to the GNAT
(Gcn5-related N-acetyltransferase)50 or MYST (MOZ, Ybf2/
Sas3, Sas2, and Tip60)51 families and catalyze the addition of the
acetyl moiety from acetyl-coenzyme A to the -amine of the
lysine residue (Table 2). These covalent modifications are often
classically associated with changes in chromatin structure, rendering chromatinized templates more accessible and open,
which closely parallel transcriptional competence and gene
expression. Indeed the ability to dynamically regulate chromatin
is facilitated by the recruitment of critical transcriptional coactivator complexes such as p300/CBP (E1A binding protein
p300/CREB binding protein),52,53 PCAF (p300/CBP-associated
factor),54 and TAFII250 (transcription initiation factor TFIID
250-kDa subunit),55 which possess intrinsic histone acetyltransferase activity. The acetylation of histones is also a reversible
process and to initiate the enzymatic reaction, histone
deacetylase proteins catalyze the removal of acetyl groups on
lysine and arginine residues. This dynamic reaction increases
the positive charge on histone tails and the binding affinity of
regulatory proteins to the DNA sequence; this encourages a
more closed chromatin structure and as a consequence represses gene transcription.
Posttranslational modifications of histone tails are integral
elements of chromatin structure, transcription, and regulatory
behavior, and the methyl-group has received considerable
attention in recent years (Figure 1). Histone methylation of
lysine and arginine residues provide added complexity to the
Table 2.

Mammalian Histone Acetyltransferase Enzymes

Histone Target

Enzymes Mediating Acetylation

H3K9

Src1

H3K14

Src1, TAF1, CBP/p300, MOZ/MORF, PACF, GCN5

H3K18

CBP/p300

H3K23

CBP

H4K5

Hat1, p300, Tip60, HB01

H4K8

P300, Tip60, HB01

H4K12

Tip60, CBP/p300, HB01

H4K16

Tip60, Mof

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Epigenetic Hyperglycemic Memory

1407

a
H3K4m1

H3K4m2

m
m
m

H3K4m3

m
m
m

H3

m
m
m

H3

A R T K Q T ..... K ..........

A R T K Q T ..... K ..........

H3

A R T K Q T ..... K ..........

b
H3K4m3

active transcriptional start sites

m
m
m
A R T K Q T ..... K ..... K .....

H3

H3K9m3

constitutive heterochromatin

m
m
m
A R T K Q T ..... K ..... K .....

H3

Figure 1. Example of the diverse posttranslational methylation marks on the

histone H3 tail. Amino acid residues are
shown in different colors and (K) lysine
highlighted in yellow. a, The chemical
variation illustrated for simplicity as
mono- (m1), di- (m2), and tri- (m3) methylation of lysine 4 of histone H3. b,
Methylation marks are correlated with
distinct gene expression patterns; for
example, H3K4m3 (H3K4 trimethylation)
is identified on active transcriptional start
sites of gene sequences, whereas
H3K9m3 is tightly associated with the
suppression of gene expression and
constitutive heterochromatin. Facultative
heterochromatin (silent euchromatin) has
distinguishing H3K27 trimethylation
(H3K27m3) identified on specific gene
sequences.

H3K27m3

facultative heterochromatin

m
m
m
A R T K Q T ..... K ..... K .....

H3

epigenome and confer distinct nuclear functions.56 The diversity of chemical variations is also complicated by the number
of modifications; for example, in addition to unmodified
histones and hyperacetylation, lysine residues can be either
mono- (m1), di- (m2), or tri- (m3) methylated (Figure 2).
These modifications can exist in different states with recent
genome-wide analyses57,58 examining histone methylation
using chromatin immunopurification (ChIP) strategies59 coupled with array hybridization or next-generation sequencing
having distinguished regions of chromatinized sequences and
histone methylation. These recent efforts have revealed that
specified histone modifications can be subcategorized; for
example, H3K4 trimethylation (H3K4m3) is frequently associated with gene expression and often distributed at transcription
start sites.60 By contrast, gene suppression is accompanied with
trimethylation of lysine K9 of histone H3 (H3K9m3),61,62
whereas H3K27 trimethylation (H3K27m3) is a prominent
feature of euchromatic gene suppression.
Histone methylation patterns are dynamically regulated
and are written and erased by methylase and demethylase
enzymes with remarkable target and amino acid specificity
(Table 3). The striking feature of many of the histone H3 and
H4 methyltransferase enzymes identified to date is the conserved SET domain sequence which was first characterized in
Drosophila melanogaster and named after Su(var)3-9 (Suppressor of variegation 3 to 9),63 E(z) (Enhancer of zeste),64
and trithorax (Trx).65 The mammalian enzymes responsible
for the methylation of H3K4 include Set7/Set9, MLL, and

Smyd3 and are associated with transcriptional expression and

have been extensively reviewed elsewhere.66

Histone Methyltransferases SET the Scene

At the Second Alan Wolffe EMBO Conference on Chromatin and Epigenetics, held May 19 22, 2005 at the European
Molecular Biology Laboratory in Heidelberg, Germany, we
presented some primary findings that transient hyperglycemia
caused persistent gene-activating epigenetic changes. Almost
5 years on, and despite some of the advances made in the
field, we still know very little of the regulatory mechanism
conferring hyperglycemia and the persistent epigenomic
changes and its role in the expression of key molecules in the
diabetic vasculature. What we do recognize are some insights
from human primary cell culture models and small animal
experiments that associate ambient hyperglycemia with geneactivating events.33 These discoveries highlight that hyperglycemia mediate key biochemical pathways which specify
changes in epigenetic information and gene expression patterns. We had recently published these findings showing the
importance of specified histone methylation and acetylation
changes engaged with gene activity.25
Further research has expanded our view of the importance
of the NF-B dependent pathway mediated by Set7 methyltransferase in monocytes,67 as well as distinguishable regulatory functions in pancreatic islets68 and cytokine-dependent
regulation of NF-B-p65 activity by lysine methylation.69
These studies provide critical insights into the regulatory role
of the Set7 methyltransferase can strikingly catalyze the

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S
1

12

K
15

13

119

120

14

H2A

H2B

20

120

M
A

10

11

14

17

18

23

26

27

28

K
9

R
3

M
A

12

K
16

M
A

36

56

79

H3

H4

20

Figure 2. Posttranslational marks identified on nucleosomal histones. Shown here is a summary of the major modifications located at
the N terminus of the histone tail, which include acetylation (A), methylation (M), phosphorylation (P), and ubiquitination (U). The amino acid
and positions are shown beneath and the globular domain represented in bold. Both N and C termini of the histone tails are shown.

methyl-writing events critical for transcription of histone

H3K470 72 and nonhistone proteins73 such as p53,74 ER75
and TAF10.76 The function of Set7 in transcriptional regulation is emerging, and these studies now provide some
mechanistic insights demonstrating that methyltransferase
activity has broad specificity and more importantly show a
multiplicity of protein substrates for the lysine residue. It is
clear that Set7 involvement in lysine methylation might
follow a similar mechanism connecting methylation of determinants involving signal transduction with gene regulatory
events.66,77 Thus, Set7 has a primary function in the methylation of lysine residues and this is critical in correctly
specifying regulatory roles associated with transcription, as
Table 3.

Mammalian Histone Methyltransferase Enzymes

Histone Target

Enzymes Mediating Methylation

H3K4

SET7/SET9, MLL, Smyd3

H3K9

SUV39h1, SUV39h2, G9a, Eu-HMTase1, ESET, SETDB1

H3K27

EZH2

H3K36

SETD2, NSD1

H3K79

DOT1L

H4K20

Pr-SET7/SET8, SUV4-20

H3R2

CARM1

H3R26

CARM1

H4R3

PRMT1

well as establishing histone methylation patterns, namely,

H3K4m1 and gene expression events in models of inflammation and diabetes. Furthermore, even though the precise
molecular machineries that cooperate with the Set7 enzyme
are not well understood and the anticipated modes of action
discovered to date remain unclear, recent experimental discoveries indicate a common theme with associated with
genomic methylation. The Set7 methyltransferase enzyme
also regulates the stability of the major maintenance DNA
cytosine-5 methyltransferase 1 or DNMT1 (DNA cytosine-5
methyltransferase 1) enzyme, demonstrating functional consequence associated with DNA demethylation.78,79 It is perhaps not difficult to imagine how the Set7 methyl-lysine
writer can function in the signaling and transcriptional pathways given the broad specificity of protein substrates, yet,
this clearly does not explain how the protein determinants are
catalyzed by the Set7 methylase. Important to our research
goals and of particular interest to us is to understand the
regulatory determinants that segregate and distinguish histone
substrate preferences derived from biochemical and mechanistic experiments in primary models of the endothelium and
diabetes-induced inflammation (J. Okabe and A. El-Osta,
manuscript in preparation). The challenge now is to identify
in different cell types how the determinants and the molecular
mechanisms that mediate Set7 and associated machineries
localize specified proteins and segregate histones in establishing genome-wide methylation and regulatory events.70

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Transient Hyperglycemia Causes Sustained

Recruitment of Set7 and H3K4 Methylation

We explored the endogenous promoter region of the NF-B-p65

subunit and identified mono-methylation of histone 3 lysine 4
(H3K4m1). Interestingly, neither di- nor trimethylation at this
amino acid residue of histone H3 was altered on the NF-B-p65
gene sequence in response to glucose. This specific methylation
change suggested a potential role for certain methyl-writing
enzymes including the Set7 protein.70 Therefore, we examined
recruitment of Set7 to the chromatinized template of NF-B-p65
demonstrating increased binding of this histone methyltransferase to the NF-B-p65 sequence. Importantly, the association
of other H3K4 methyltransferases such as MLL180 on the
promoter of NF-B-p65 were not specifically changed. Because
gene expression occurs as a result of a balance of activating and
suppressive gene regulating events, H3K9 methylation is also
altered in models of hyperglycemic variability indicating that
decreased methylation is inversely associated with gene expression (Figure 3). Indeed, macrophages exposed to high glucose
concentrations show reduced H3K9 methylation marks on genes
implicated in vascular inflammation.43,44 Interestingly, in our
own studies using endothelial cells, we observed a dynamic
cooperation between H3K4 and H3K9 marks associated with
gene activating epigenetic events.25 Although this does not
explain how histone code changes other than H3K4 are conferred, the primary endothelial model of hyperglycemic variability allowed direct investigation of extracellular signals such as
glucose in determining other epigenetic changes. We next
determined what the role of hyperglycemia on asymmetrical
methylation exemplified on H3K4 and H3K9 lysine residues
and asked the question; what is the significance of a transient
hyperglycemic gradient on histone tail demethylation?

Transient Hyperglycemia Causes Sustained

Recruitment of Lysine-Specific Demethylase 1
and H3K9 Demethylation
In a follow-up study, we extended the precedent beyond the
H3K4 tail and identified specific changes to H3K9 during
transient hyperglycemia and subsequent normal glucose homeostasis.17 Experimental evidence indicates that antecedent
hyperglycemia is associated with persistent upregulation of
NF-B-p65 gene expression and is associated with specific
reduction in H3K9 methylation on the promoter region of this
gene (Figure 4). Interestingly, transient hyperglycemia causes
a sustained reduction in both H3K9 dimethylation (H3K9m2)
and H3K9m3 on the NF-B-p65 promoter, and these events
coexist with the recruitment of the lysine-specific demethylase
(LSD)1 H3 demethylase81,82 on gene promoters. These results
suggest that persistent epigenetic-transactions specify asymmetrical lysine methylation of K4 and K9 of histone H3 and
strikingly reminiscent of probable multivalent events conferred
by hyperglycemia, which will provide critical insights into the
relationship of specified histone signatures.83,84

Short- and Long-Term Epigenetic Persistence

Hyperglycemic exposure per se does not carry a set of
instructions; instead, we postulate that glucose exposure and
more specifically, the continuation of hyperglycemia can

Epigenetic Hyperglycemic Memory

1409

signify epigenetic-context dependent signaling that regulates

either short- or long-term persistence (Assam El-Osta, unpublished observations, 2008). Since the initial discoveries that
glycemic cues can modulate gene-activating epigenetic
changes our understanding of histone code changes has
challenged many of the original concepts suited for hyperglycemic memory. Indeed, although hyperglycemia can activate gene expression patterns, the reciprocal is also evidenced
with suppressed mRNA abundance85 as well as diverse
epigenetic modifications including increased H3K433 and
attenuated H3K925,44,86 methylation events as adaptive regulatory processes. The general concept that gene environment
interactions not only represent current glycemia, but also
include precedent hyperglycemia, which could transmit conserved epigenetic fates87 that are spatially and temporally
consistent with persistent gene activities. This is highlighted
by striking plasticity of extracellular signals such as glucose
exposure which is dynamically regulating gene activity,
indicating that sustained short-term glucose exposure is
integrated over time to adapt transcriptional events in pancreatic -cells.88 Although the role of hyperglycemia regulating epigenetic determinants or more specifically, methyltransferase function in regulating and specifying histone code
fate remains to be determined, there appears to be integrative
signaling networks connecting adaptive transcriptional memory mediated by epigenetic cues. Indeed, core pathway
components have been identified in converting transient
signals into longer lasting-persistent responses that are robustly expressed following stimulus reexposure. For example
the incorporation of histone variant H2A.Z into nucleosomes
represent memory of epigenetic states associated with transcriptional activity.89 The coordination of adaptive transcriptional memory mediated by the persistence of epigenetic
changes would also explain how the re-exposure of environmental cues such as sugar-specifying signaling cascades
determine future cell memories that are integrated by chromatin structure regulating transcriptional events.90,91

Perspectives on Genome-Wide Determination

of Epigenetic Signatures
The human genome is almost 2 m in length and is packaged
in the nucleus as chromatin that regulates and confers critical
metabolic functions to each chromosome. Robustness in
chromatin crosstalk and transcriptional coordination allows
for the interconnection of epigenetic information to regulate
gene sets in a broader program, similar to process networks
rather than disconnected-linear regulatory events. For example the coordination of methyl-writing and methyl-erasing
enzymes provides a dynamic and robust mechanism of
transcriptional control conferred by transient hyperglycemia.
Not surprisingly, the genes identified to date have been
empirically determined that include members of proinflammatory cytokines. A key challenge in genome-wide analyses
is the identification of distinguishable epigenomic patterns
that feature transcriptional output and consequence, this
means that specifying histone code changes that are associated with gene-regulatory events will require the precise
epigenomic mapping if we are to clearly understand the
importance of the continued development of diabetic compli-

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1410

Circulation Research

December 10, 2010

Hyperglycemia confers gene activating events

that are associated with changes in chromatin
structure and function

H2B

H2A

H3

H4

NFB-p65 promoter H3K9m2 & H3K9m3 enriched

closed structure
gene suppression

repressive co-factors
hyperglycemia

NFB-p65

glucose
variability

histone
modifications

Set7

Pol II

closed structure
gene suppression

H3
m

euglycemia

chromatin
architecture

LSD1

coordinated recruitment of Pol II

and methyl-writing Set7
and methyl-erasing LSD1 enzymes

open structure
gene activity

A R T K Q T ..... K
4
9

normoglycemia

Pol II

H3
hyperglycemia

m m
m
A R T K Q T ..... K

Set7

Pol II

H3

H3K14ac

m m
m
A R T K Q T ..... K

return to
euglycemia

H3K4m1
H3K9ac

LSD1

longlived

m m
m
A R T K Q T ..... K
4
9

open structure
gene activation

open structure
gene activity
NFB-p65 promoter H3K4m1 with reduced H3K9m2 & H3K9m3

Figure 3. Hyperglycemia causes persistent gene activating

changes mediated by chromatin remodeling and the posttranslational modification of histones. In this schematic, the
NF-B-p65 gene is simplified to show the nucleosome core in
yellow consisting of the histones H2A, H2B, H3, and H4
wrapped by the DNA duplex highlighted in blue. The promoter
sequence of the NF-B-p65 gene is contrasted in red to illustrate the changes in chromatin structure are primary architectural features corresponding with gene regulatory events. Glucose variability, and more specifically transient hyperglycemia, is
associated with methyl-writing and methyl-erasing events on
histones H3K4 and H3K9, respectively. These molecular events
were monitored by ChIP strategies to immunopurify soluble
chromatinized NF-B-p65 sequences using specific antibodies
that recognize methylated histones. In parallel with these histone modifications are key architectural changes that correlate
with gene expression. The closed chromatin template is inaccessible to regulatory cofactors and functionally transcriptionally
suppressed, whereas the open conformation allows entry and
recruitment of key enzymes such as RNA pol II for transcriptional activity. These gene regulatory features were recently validated for NF-B-p65 gene using nuclease accessibility, partial
chromosome walk, and ChIP assays.33

cations mediated by transient hyperglycemic exposures.92

Recent technological advances in genome-wide analyses
using ChIP-Seq (ChIP strategies coupled with massive parallel sequencing) now realizes this formidable challenge with
the potential to map and atlas the epigenome of diabetes.
There are several advantages to unbiased determinations of
epigenetic signatures. Genome-wide screens provide unprecedented epigenomic-information that are functionally interconnected by intersecting signaling networks with interactive
extracellular cues with distinct epigenomic and transcriptional fates. For example, distinguishing the breadth of

Figure 4. Persistent epigenetic changes to the chromatinized NF-B-p65 gene include the recruitment of the Set7
methyl-writing and LSD1 methyl-erasing enzymes that mediate correspondent histone modification and gene expression. Hyperglycemia associated with endothelial dysfunction
and alterations in blood vessel growth is the primary cause of
vascular complications in diabetes. Furthermore, these vascular
complications often persist and may progress despite improved
glucose control, possibly as a result of prior episodes of hyperglycemia. In this schematic, we simplify the signaling pathways
mediated by hyperglycemia to show some of the key transcriptional events associated with gene activation and the concept of
epigenetic persistence. Clinical studies suggest that the injurious effects of exposure to high glucose levels persist for years
after better treatment, a phenomenon typically referred to as
hyperglycemic memory. Indeed, experimental evidence indicates
that short-term memory and the persistent gene expression
events are associated with the recruitment of the methyl-writing
enzyme Set7 and confer H3K4 methylation on the NF-B-p65
promoter. In parallel, hyperglycemia is postulated to mediate
demethylation of H3K9 by the LSD1 enzyme, demonstrating
reciprocal exchange of methyl-writing and methyl-erasing
enzymes, recruitment of pol II, and hyperacetylation events correlating with gene expression.

covalent epigenetic modifications to the histone tail is a

formidable challenge, not only because of technology, but
also because the list of modifications continues to grow.56
Empirically identifying histone modification patterns in response to subtle extracellular stimuli such as glucose would
be impracticable if considering the epigenomic landscape is
more than a linear on/off closed circuit switch,93 clearly there
is significant signaling crosstalk and epigenetic pathways do
intersect.94

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Cooper and El-Osta

Chr 8q11.23

hyperglycemia

euglycemia
TIS

H3 acetylation

H3 acetylation

Chr 3p25.1

hyperglycemia

euglycemia
TIS

position map

position map

hyperglycemia

euglycemia
TIS

H3 acetylation

Chr 18q12.1

Chr 14q12

H3 acetylation

Epigenetic Hyperglycemic Memory

hyperglycemia

euglycemia
TIS

position map

position map

We are interested in understanding how gene-activating

epigenetic changes are regulated by extracellular signals and
one project receiving considerable attention is the precise
mapping of glucose induced epigenetic signatures (El-Osta et
al, manuscript in preparation). The project is aimed to map
and intersect epigenomic signatures of diabetes and key
experiments using primary aortic cells show distinguishable
histone modifications that parallel transcriptional competence. Chromatin immunopurification coupled with next
generation-sequencing methodologies distinguish precisely
hyperacetylation profiles regulated by hyperglycemia, illustrated and exemplified on human chromosomes (Chr) 3p25.1,
8q11.23, 14q12 and 18q12.1 (Figure 5). Surprisingly, shortterm glucose exposure confers dynamic changes in covalent
histone modifications that closely correspond to transcriptional output and reveal signatures reminiscent of endothelial
cells with proatherogenic features.

Conferring Future Memories

In summary, it has become increasingly apparent that in both
type 1 and type 2 diabetes, there is evidence of ongoing
vascular injury as a result of prior transient episodes of poor
metabolic control, as demonstrated in both the DCCT/EDIC
and UKPDS trials. This has been termed as metabolic
memory or a legacy effect. With an increasing understanding of epigenetic mechanisms and, in particular, how environmental factors such as glucose can affect the genome, it
may now be possible to interrupt epigenetic pathways to
promote vascular protection. It is now critical to build on
these key findings from in vitro studies, and we can expect
the inhibition of specified methyl-writing and methyl-erasing
enzymes as targets in attenuating glucose-induced activation
of proteins that play critical roles in vascular inflammation. It
is anticipated that over the next few years, this hypothesis,
which postulates a central role for epigenetic pathways in
modulating the development and progression of vascular
complications in diabetes, will be greatly strengthened by
preclinical studies and clinical investigations that will include
molecular and pharmacological approaches to attenuate these
glucose-induced changes in chromatin structure and gene

1411

Figure 5. Mapping glucose-induced

histone acetylation signatures.
Genome-wide profiles of histone acetylation patterns determined by highthroughput ChIP-Seq (ChIP strategies
coupled with massive parallel sequencing) approach to studying gene regulating epigenetic modifications. Exemplified
here are the hyperacetylation profiles of
human chromosomes (Chr) 3p25.1,
8q11.23, 14q12, and 18q12.1. Soluble
chromatin was prepared from primary
aortic cells using euglycemic (low glucose) or hyperglycemic (high glucose)
conditions and acetylated histone H3
immunopurified and genomic libraries prepared for next generation sequencing.
Hyperacetylation signatures are shown for
euglycemia (blue) and hyperglycemia (red)
relative to the transcription initiation start
site (TIS).

function. We are beginning to unravel some of the distinct

changes associated with the histone code and the epigenome,
which could partly explain why transient elevations of glucose
often lead to progressive diabetic vascular complications.

Conclusions
Although there are increasingly new therapeutic approaches
to improve metabolic control, it remains difficult to achieve
near-normal glucose levels in clinical practice. This issue of
refractory hyperglycemia with the concomitant impact of
hyperglycemic memory implies that over the near to medium
term, health systems will continue to be burdened by the
social and medical impact of diabetic vascular complications.
One innovative strategy to attenuate the burden of complications as a result of prior hyperglycemia is to target the
molecular pathways that promote hyperglycemic memory. As
our understanding increases in this area, and as these pathways are further elucidated, it is predicted that targeting
enzymes implicated in chromatin structure and gene function,
such as histone methyltransferases, could be a promising
approach. With increased exploration of these targets in
various medical contexts, it may be possible in the near future
to apply such strategies to preventing and treating diabetic
vascular complications.

Sources of Funding
Supported by the Juvenile Diabetes Research Foundation International (JDRF), the Diabetes Australia Research Trust (DART), the
National Health and Medical Research Council (NHMRC), and the
National Heart Foundation of Australia (NHF).

Disclosures
None.

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Epigenetics: Mechanisms and Implications for Diabetic Complications

Mark E. Cooper and Assam El-Osta
Circ Res. 2010;107:1403-1413
doi: 10.1161/CIRCRESAHA.110.223552
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