Monday, 6 June 2016

Editing and Interpreting Chromatin Modifications

Molecular tagging of chromatin marks structural and functional genome regions, enhancing transcriptional responses to distinct signaling cues.

A useful conceptual model for epigenetics is that of writers, erasers, and readers of chemical modifications on histones and DNA.

Specific enzymes modify discrete residues on chromatin, generating marks that designate certain regions for transcriptional regulation.

Another class of enzymes, the so-called erasers, can remove these histone and DNA modifications.

Finally, a third group of proteins—readers—recognises and associates with chromatin marks, facilitating or inhibiting assembly of the transcriptional machinery and subsequently regulating gene expression. 

The best characterised chromatin modification is the classically epigenetic methylation of cytosine (methyl-cytosine) nucleotides immediately adjacent to guanine nucleotides (CpG) in the DNA sequence.

DNA methylation is commonly associated with gene suppression when enriched at or near gene regulatory regions by modulating transcription factor binding to directly interfere with gene activation, or by interacting with specific regulatory proteins such as MeCP2 (methyl-CpG binding protein 2).

Recent characterization of the family of ten-eleven-translocation (TET) proteins, which can generate hydroxymethylcytosine, formylcytosine, and carboxylcytosine from existing methylcytosine has inspired strong interest in demethylating pathways. 

Similarly, the myriad post-translational modifications are written to and erased from predominantly arginine and lysine amino acid residues on the protruding N-terminal tails of chromatinised histones by specialised enzymes and modifying complexes.

Methyl groups also feature heavily on histone tails and are associated with both active and silent genes depending on the specific location and degree of modification. The histone methylating functions of enzymes such as Set7 are opposed by the demethylating activities of enzymes such as LSD1.

Contrasting this dual role of histone methylation, histone lysine acetylation, regulated by opposing roles of acetyltransferases (HATs) and deacetylases (HDACs), is exclusively associated with transcriptional competency.

The influence of histone post-translational modifications on higher order chromatin structure and transcriptional regulation is largely attributed to (1) charge disruption of histone tails in the case of acetylation/deacetylation which alters affinity for adjacent histones and DNA, and (2) the establishment of high-affinity binding sites for recruitment of complexes that actively remodel the chromatin.

In conjunction with transcription factor networks and remodelling complexes, these covalent and post-translational modifications collaboratively drive the functional exchange between repressed and active states of chromatin to contextualise gene activity.

Our review, published in Circulation Research (May 2016), discusses recent key findings in a rapidly burgeoning arena of research into chromatin-dependent mechanisms defining cardiometabolic health and dysfunction. Read the full article here.


Sunday, 29 May 2016

From Sites to Cytes in the Diabetic Vasculature

The life threatening cardiovascular complications of diabetes derive from the formation of fibrofatty atherosclerotic plaques in major blood vessels. Chromatin modifications that sensitise the genome to cardiometabolic risk factors of the diabetic milieu are now widely considered as promising therapeutic targets. However the heterocellular nature of atherosclerosis demands a thorough understanding of cell type-specific chromatin signatures underlying their individual pathologies.

The complex development of atherosclerotic lesions is progressed by three predominant cell types.

Vascular endothelial cells intimately engage the circulating factors of the diabetic milieu, such as hyperglycemia and low-density lipoprotein. Chronic exposure damages the endothelium, eliciting a state of endothelial dysfunction characterised by increased vascular permeability and induction of proinflammatory adhesion and chemotactic molecules that promote immune cell infiltration.

Macrophage recruitment and accumulation in the subendothelial space intensifies the inflammatory state by foam cell formation and cytokine secretion.

As the disease develops, activated vascular smooth muscle cells migrate from the arterial media to the intima to secrete various proliferative, fibrotic, osteogenic, and inflammatory factors.

These pathological changes collaborate in the formation of plaques that in some cases are unstable and prone to rupture, often detaching and entering the circulation to occlude smaller downstream blood vessels.

Intensified research now implicates lasting gene expression changes in the vasculopathies instigated by the metabolic perturbations of diabetes.

Despite sharing a common genetic sequence, endothelial, smooth muscle, and circulating immune cells have distinct epigenomes regulating cell type–specific gene expression and pathologies.

Further understanding the multitude of epigenomic profiles from distinct cell populations in the vasculature will reveal new insights in to the development and progression of atherosclerosis that can be translated to novel therapies in the clinic.

Our review, published in Circulation Research (May 2016), discusses recent key findings in a rapidly burgeoning arena of research into epigenetic mechanisms defining cardiometabolic health and dysfunction. Read the full article here.

Distinguishing epigenomic profiles from distinct cell populations in the vasculature. In this example, histone modifications and genomic DNA methylation sequences are derived from human monocyte and endothelial cells using epigenomic profiling methodology combined with computational analyses and integration. Genes associated with differential histone acetylation at the promoter are indicated. Click to enlarge.


Friday, 26 February 2016

Current Epigenetic Perspective on Diabetes - Who Regulates the Regulators?

Samuel T. Keating and Assam El-Osta

This article was originally published in Cellular & Molecular Medicine: Open Access on December 18 2015 under a Creative Commons Attribution 4.0 International License


Intensified interest in the development of pharmacological compounds that manipulate gene control highlights the importance of understanding the key players and molecular events driving gene function. Epigenetic research too frequently focuses on a single chromatinized modification, often at a limited number of loci, and without context for other determinants of transcription. This perception is problematic because it implies an oversimplification of the vastly complex and multidimensional network of gene control. It overlooks the interactions of chromatin modifying enzymes and transcription factors, and seldom addresses the molecular events and signaling cues that influence the executive enzymatic machinery that regulate the epigenome. Here we discuss the connectivity and complexity of epigenetic regulation in the context of chromatin modifications and transcription factors. Using the example of the Set7 (also Set9 or Set7/9) methyltransferase, we describe recent observations that expand the understanding of chromatin biology.

Dynamic Chromatin

Site-selective DNA-binding proteins interact with genomic regulatory regions as a central mechanism to direct cell-specific transcriptional programs. Though initially considered a benign scaffold to package DNA, the nucleoprotein structures of chromatin are now recognized as integral to the accessibility of transcription factors to gene regulatory elements [1]. A relaxed euchromatin structure where the DNA template is loosely associated with histone proteins allows the transcriptional machinery of the cell to access and read the genetic code. This contrasts with transcriptionally repressive conformations where DNA is tightly associated with histones and buried within the chromatin. By influencing the chromatin-penetrating potential of transcription factors, the structural re-organization of chromatin underpins the selective activation and silencing of gene programs that give rise to an astounding array of cell types from a single source of genetic information. Just as importantly, chromatin dynamics underlie a cell’s ability to manage environmental variations with rapid changes in gene activity [2].

Active and silent regions of the genome are distinguished by small chemical modifications to histones and the DNA itself that markedly shape the chromatin architectures by recruiting or precluding other factors that remodel the histone-DNA complex. Because the chemical tags can regulate changes in gene expression independently of the underlying DNA sequence, they are called epigenetic (literally in addition to genetic). These molecular signatures have an enormous capacity to control the functional state of chromatin and transcriptional activity of genes encoded therein for both dividing and terminally differentiated cells. Chromatin modifications can be influenced by various environmental factors, hence epigenetic grammar contextualizes the language of the DNA code [3].

The importance of this environment-epigenome axis is emerging for many disease states [4-8]. This is exemplified by recent large clinical studies of diabetes. Indeed the pathogenesis of type 2 diabetes has a particularly strong association to environmental factors that are proposed to alter the chromatin landscape. Likewise, diabetes-associated perturbations in metabolism and hemodynamics are known to influence the development of vascular complications by epigenetic modifications. Moreover, some of these chromatinized changes are implicated in the phenomenon of metabolic memory in type 1 and type 2 diabetes, where antecedent periods of hyperglycemia drive persistent vascular complications many years after blood glucose control is achieved [9]. To this end, epigenetic profiling of circulating blood monocytes has revealed a persistent histone acetylation signature at genes implicated in diabetes complications that was closely associated with glycemic history in patients with type 1 diabetes [10].

While numerous enzymes and specific modifications have been described, defining the cell’s ability to sense the variety of diabetic signaling cues at the chromatin level remains an important challenge. Precisely how is information communicated to the orchestra of factors controlling the chromatin landscape? How does the epigenetic machinery interact with transcription factors to control gene expression?

Who regulates the genome regulators?

Regulating chromatin accessibility and gene expression. Cytosine bases covalently modified by addition of methyl groups are recognized by methyl-CpG binding proteins that associate with chromatin remodeling factors to establish transcriptionally incompetent chromatin architectures. The nucleosome is comprised of approximately 147 bp of DNA encircling an octamer of 2 copies of each of the 4 core histone proteins H2A, H2B, H3, and H4. Post-translational modification (PTM) of nucleosomal histones further regulates transition between active, structurally open euchromatin and silent, condensed heterochromatin. Shown here is a summary of acetyl (purple) and methyl (red) modifications of specific amino acid residues on N-terminal tails of the heavily modified H3 and H4 histones. Click image to enlarge.


Oxidative stress alters the vascular chromatin landscape 

The functional relationship between chromatin architecture and changes in gene expression conferred by chronic and prior hyperglycemia has proven to be an important avenue of investigation for explaining persistent vascular complications of diabetes. We previously described the critical role of H3 histones lysine 4 mono-methylation (H3K4m1) in the high glucose-mediated transcriptional activation of human endothelial NFκB-p65 (encoded by the RELA gene), a key pro-inflammatory transcription factor that regulates the expression of genes implicated in inflammation associated with vascular complications of diabetes [11,12]. Moreover this specific chromatin signature, written by the Set7 lysine methyltransferase, persisted for up to 6 days in normal glucose conditions, suggesting it could confer future cell memories. The clinical relevance of these seminal in vitro findings was recently validated in the peripheral blood mononuclear cells of a cohort of patients with type 2 diabetes [13].

Identification of the methyl writer in the chromatinization of glucose signaling cues raised a new question. How are changes in ambient glucose transmitted to Set7? Indeed Set7 is mobilized to the nucleus with increasing glucose concentration [14]. Mitochondrial overproduction of superoxide has long been known to initiate many hyperglycemia-induced mechanisms related to the pathogenesis of diabetic complications [15]. Accordingly, the up-regulation of RELA induced by transient hyperglycemia was abolished by overexpression of either uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD), both of which prevent hyperglycemia-induced superoxide accumulation [11]. Further, Paneni and colleagues identified epigenetic changes driving up-regulation of the mitochondrial adapter protein and critical mediator of oxidative stress p66Shc in vascular endothelial cells cultured in high glucose conditions [16]. The resulting superoxide production activates PKCβII, which in turn maintains elevated p66Shc levels, ultimately stimulating and sustaining epigenetic changes by enzymes such as Set7 [17]. While this mechanism has the capacity to explain the sustained legacy of hyperglycemia in diabetes, it raises further challenges in identifying how high glucose signals to the epigenetic machinery regulating p66Shc expression.

Chromatin modifiers interact with transcription factors

Large datasets reveal striking overlaps between transcription factor binding sites and chromatin modifications [18]. Coregulatory interactions between transcription factors and chromatin-modifying enzymes may at least partly account for this co-localization [19]. Set7 was shown to be co-recruited with TAF10 to activating gene promoters [20]. In fact, along with its role in methylating histones, Set7 interacts with numerous transcription factors across various cell types, often promoting methylation reactions on regulatory lysine residues at the surface of the transcription factor [21].

Our recent characterization of human vascular endothelial cells depleted of Set7 revealed widespread changes in gene expression across numerous pathways associated with vascular function that were only partly explained by changes in H3K4m1 at promoters and distal enhancer regions [22]. By intersecting the transcriptome profile with publicly available datasets, we identified strong associations between deregulated genes and six transcription factors previously described as Set7 methylation substrates: NFκB, STAT3, IRF1, p53, ERα, and TAF7 [21]. In addition, many deregulated genes were associated with numerous transcription factors not previously connected with Set7 function. By applying a consensus formula derived from Set7 substrates previously used to accurately predict several biochemically validated in vivo non-histone substrates [23], we predicted that Set7 post-translationally regulates transcription factors associated with vascular endothelial expression through the presence of Set7 amino acid methylation motifs. Amino hydrophobicity analysis indicated most predicted sites to be accessible to post-translational modification. Further, in vitro peptide methylation assays suggest that Set7 can indeed modify a predicted site on the STAT1 transcription factor, demonstrating the predictive value of our method to identify novel candidate substrates to analyze in vivo. Further characterization of putative substrates identified in these studies has the capacity to identify not only functional modulation of transcription factors, but also substrate-driven co-recruitment of the enzyme to specific promoters to potentiate H3K4m1 enrichment.

Like the regulatory function of acetylation, lysine methylation has emerged as an important post-translational modification for modulation of transcription factors [24]. Our novel method of mapping transcriptional changes to transcription factors for the identification of putative substrates with strong associations to functional changes is applicable to substrate prediction for other broad-substrate histone modifiers [22]. Both the histone and non-histone-modifying activities of epigenetic enzymes are important considerations for future strategies of pharmacological targeting in the clinic.

Chromatin modifiers regulate each other

A growing body of evidence points to post-translational modifications as regulators of chromatin modifying enzymes themselves [2]. The prevalence of these modifications suggests a highly ordered and dynamic network of components capable of writing, reading, and erasing modifications at both the chromatin template as well as each other.

Counted among the expanding catalogue of experimentally validated methylation substrates of Set7 are SUV39h1 and DNMT1 - enzymes that methylate histones (at a distinct site to Set7) and DNA respectively. Methylation at lysines 105 and 123 by Set7 impairs the repressive histone modifying capacity of SUV39h1 [25], whereas the stability of DNMT1 is regulated by Set7-mediated lysine methylation [26]. Similarly Set7 also methylates multiple lysines on the p300/CBP-Associated Factor (PCAF) histone acetyltransferase [27]. A single epigenetic enzyme therefore has the ability to control many chromatin modifications. Mapping the inter-enzyme modification network of epigenetic regulators has an enormous capacity to increase our understanding of gene regulation and further raises important considerations for therapeutic strategies aimed at editing the epigenome.


Immense interest surrounds efforts to modulate gene expression, to restore the activity of silenced genes or attenuate unscheduled gene expression. However, the complexity of gene regulation is vast and researchers are only beginning to gain an appreciation for the biochemical determinants and genome-wide interconnectivity of epigenetic enzymes and transcription factors. Using the example of Set7, we have described recent observations that expand the understanding of chromatin biology beyond the immediate histone methyl-writing event. Indeed this is just a scratch on the surface and further studies are required to completely understand this important enzyme. Similar questions of biochemistry and interactivity remain for many other classes of chromatin modifiers considered useful in the clinic, including methylases, demethylases, acetylases and deacetylases, as well as protein components responsible for reading the chromatin mark such as bromodomains. While chromatin modifications can be informative of gene regulation at specific loci, the challenge of understanding their cell-specific function remains unmet. Myeloid-specific genetic deletion of histone deacetylase 3 is associated with stable atherosclerotic plaques [28], whereas deletion of the same enzyme in endothelial cells enhances atherosclerosis in mice [29].

More recently emerged concepts could offer further insight into epigenomic regulation. For example several intermediates of cellular metabolism are critical substrates for chromatin modifying enzymes. Fluctuating levels of these metabolites could therefore signal for continual adjustment and contextualization of gene expression (recently reviewed [2]). In addition long noncoding RNA molecules could play a role in the localization of chromatin signatures. By simultaneously recruiting two different histone modifiers to the chromatin – one a writer and the other an eraser of histone methylation – the HOTAIR long noncoding RNA facilitates the coordinated addition of a repressive modification and removal of an activating one to silence specific genes [30].

Technological and scientific advances have rapidly expanded the field of epigenetics to the point where chromatin modifiers are seriously considered as therapeutic targets for numerous diseases. The challenge for the next decade is to develop a comprehensive understanding of the biochemical and molecular events controlling the genome’s regulators.



Friday, 13 February 2015

Mapping Cell-Specific Histone Acetylation

Changes to intracellular metabolism can alter the expression of specific histone methyltransferases and acetyltransferases conferring widespread variations in epigenetic modification patterns. Although important enzymes and chromatin changes have been described, defining the specific process governing the cell’s ability to sense environmental change in development and adulthood at the level of gene expression is an important challenge facing the field. 

Reminiscent of early single-loci genetic studies, many epigenomic investigations have examined specific modifications in isolation. Increased availability of public epigenome-wide datasets has greatly enhanced our understanding of the chromatin landscape. For instance, profiling of chromatin modifications in conjunction with gene expression allows the generation of detailed cell-type and stimuli-specific epigenetic maps. 

Our recent review published in Circulation Research (Feb 2015) discusses the most recent key findings that link cellular metabolism with chromatin-dependent gene changes, including histone acetylation. Read the full article here.

This plot of human chromosome 12 shows the variation of histone H3 lysine 9 acetylation (H3K9ac) among cell types. The outer ring represents ≈134 million base pairs showing some of the estimated 1200 protein coding genes represented in blue and red, including long intergenic nonprotein coding RNA (LINC) and short noncoding or micro RNA (MIR). Differential H3K9ac is represented in each concentric ring shown in blue bar charts proportional to the H3K9ac signal or score for each region. The innermost track represents aortic endothelial (primary human aortic endothelial) showing histone acetylation (green) and deacetylation (orange) conferred by pharmacological histone deacetylase (HDAC) inhibition. Histone acetylation data are generated by chromatin immunoprecipitation combined with sequencing (ChIP-seq) derived from ENCODE and accessed from UCSC. Click image to enlarge. 


Keating ST & El-Osta A. Epigenetics and Metabolism. Circulation Research doi: 10.1161/CIRCRESAHA.116.303936

Saturday, 10 January 2015

Mapping Metabolic Memory

Some experiences are harder to forget than others. 
The genome's ability to adapt to environmental changes also has a dark side. Emerging research is beginning to unravel an extraordinary phenomenon of cellular memory centred on the field of chromatin biology. One where periods of challenging metabolic conditions can initiate programs of gene expression that dramatically increase the risk for disease later in life. 

Your genome remembers the past

Living cells respond to environmental variations with rapid and stable adjustments to gene expression that are fundamental to many aspects of survival, regeneration, and metabolism. The ability to sense and adapt to local changes in nutrient availability, toxicity, stress, and other environmental factors centres on the gene-regulating dynamic assembly of DNA and histone proteins called chromatin.

Many chemical modifications occurring both on DNA and histones, such as methylation and acetylation, can influence if a gene is 'switched on' or 'off'. Specifically, they can determine whether or not the cellular machinery can physically access the biological information encoded in a gene by controlling transitions between relaxed and condensed chromatin configurations. By several recently discovered mechanisms, these modifications to the chromatin template sensitise the genome to changing environmental conditions.

Considerable interest surrounds the direct connections between metabolism and chromatin structure dynamics. Numerous chromatin-modifying enzymes and complexes respond to various metabolic signals. In particular, several components of the epigenetic machinery utilise intermediates of cellular metabolism for their enzymatic function.

For example, addition of epigenetically important methyl groups (CH3) to DNA and histones is influenced by dietary intake of methyl-donors such as folate. Similarly, many chromatin-modifying enzymes can respond to fluctuations in metabolites derived from the processing of fatty acids and glucose for energy. Furthermore, changes to intracellular metabolism can alter the expression of specific histone-modifying enzymes leading to widespread variations in chromatin patterns.

Indeed most modifications are transiently and dynamically written to and erased from chromatin. However it is increasingly appreciated that some modifications can persist beyond the initial metabolic stimulus. Depending on the type of modification and genomic location, such changes can cause a gene to remain activated or silenced, conferring a molecular memory of previous metabolic experiences.

Under certain conditions, maladaptive expression of particular genes contributes greatly to the development and progression of metabolic disease. Understanding the chromatin component of metabolic memory now represents an important conceptual challenge to explain many aspects of metabolic dysfunction.

The importance of first impressions

Our earliest metabolic experiences can have profound effects on health in adulthood.

Groundbreaking clinical observations associating low birth weight with risk for coronary heart disease later in life led Professor David Barker to propose the developmental origins of disease hypothesis in 1989 (often called the Barker hypothesis). Adaptive responses to the intrauterine environment, including over- and under-nutrition, in anticipation of post-natal conditions are hypothesised to confer increased risk of chronic disease in adulthood.

Because chromatin modifications established in early development can respond to local changes in nutrient availability, and in some cases are remarkably stable, gestational cues have enormous capacity to define and direct future gene expression. Importantly, the chromatin landscape of genes involved in metabolism can be altered by conditions in utero. And it is these changes that are speculated to underlie the elevated risk of developing disease.

A recent Austrian population study described a massive increase in the risk for developing diabetes in people born during and immediately after three major famines during the last century. Similar risk for metabolic disease was also observed in a cohort of individuals conceived in the winter famine of 1944-45 in Nazi-occupied Holland.

Among the best characterised epigenetic marks is the addition of methyl groups to the DNA, which stably modifies gene expression. DNA methylation is a key regulator of genomic imprinting. However its role in the development and progression of metabolic disease has only recently begun to emerge. Importantly, researchers examining the Dutch cohort were able to link maternal starvation around the time of conception with altered DNA methylation patterns in blood cells 60 years later. Specifically, the level of DNA methylation at the maternally-imprinted insulin-like growth factor 2 gene (IGF2) was reduced in people born during this period compared to their unexposed, same-sex siblings. Similar epigenetic changes at this growth-promoting gene were observed in rodent livers from models of intrauterine growth restriction and low protein diet as well as other metabolically important genes in the hippocampus, the pancreas, and skeletal muscle.

Indeed most findings linking transient environmental conditions in gestation to chromatin changes in adult disease are derived from experimental animal models of maternal dietary modulation. With important implications at the level of public health, studies have begun to link maternal over-nutrition with chromatin changes associated with adult disease. 

As an organ that is crucial to fat metabolism, the liver has drawn considerable attention in studies of maternal high fat diets. For example, DNA methylation-dependent silencing of the fatty acid desaturase 2 gene (Fads2) was observed in livers of adult offspring born to rats fed a diet of high fat content during pregnancy. Similarly, transcriptional silencing of a gene associated with mitochondria biogenesis, Pcg-1α, in mice born to dams fed a high fat diet was sustained in adulthood by a DNA-methyl-dependent mechanism.

Other dietary factors are also known to influence gestational epigenetic programming. As the universal substrate for methylation reactions, cellular concentrations of S-adenosyl methionine are fundamental to some of the most prevalent chromatin modifications. The importance of dietary methyl donors such as folate has been examined in rodent models as well as in humans, and the influences of maternal dietary intake are a key focus of recent studies. In fact the effects of maternal folate diet are quite complicated, with both low and high intake associated with increased disease risk in adult offspring. Strikingly, standard recommended maternal daily folate intake of 0.4mg/day was associated with decreased birth weight and altered DNA methylation at the IGF2 gene.

Finally, persistent changes to the expression  of chromatin modifying enzymes are implicated in chromatin modifications induced by maternal dietary fat over-consumption. The livers of non-human primates and rodents exposed to high fat during development exhibited changes in the expression of enzymes involved in the removal of histone acetylation in parallel with a distinctly altered chromatin landscape at genes important for fat metabolism.

The glucose legacy

This remarkable ability of chromatin to incorporate environmental information in to its program of gene regulation also holds tremendous importance beyond early developmental periods. The deleterious effects of periods of poor metabolic conditions in adulthood can persist well beyond the initial exposure. This is particularly relevant to diseases that can remain undiagnosed or unsatisfactorily treated in early stages. The most prominent example in humans is diabetes.

An average of 263 diabetic Australians register each day with the National Diabetes Services Scheme (September 2014). This devastating disease is characterised by loss of insulin producing pancreatic β-cells (type 1 diabetes) or progressive decline in insulin sensitivity and β-cell dysfunction (type 2 diabetes). Because insulin is necessary for the cellular uptake of glucose from the blood and normal metabolic functioning, uncontrolled diabetes leads to elevation of blood glucose concentrations to toxic levels (hyperglycaemia) as well as secondary factors such as high blood pressure and oxidative stress.  

The effects of hyperglycaemia on long-term health are pronounced. They are particularly associated with injury and inflammation of the blood vessels (known clinically as the vasculature). For this reason diabetes remains the leading cause of cardiovascular disease, amputation, kidney impairment, and vision loss in adults.

The deleterious effects of hyperglycaemia are associated with injury and inflammation across multiple organs and tissues. Diabetes is the leading cause of vascular complications in adults leading to cardiovascular disease, limb amputation, kidney impairment, and vision loss. Incorporates image by Mikael Häggström/Wikimedia commonsClick to enlarge

Pioneering studies of animal models first highlighted the difficulties of reversing the damage induced by hyperglycaemia. In 1987 Engerman and Kern reported persistent vascular damage to the retinas of diabetic dogs despite more than two years of insulin therapy and strict blood glucose control. Similarly, pancreatic islet transplantation and subsequent correction of blood glucose levels failed to prevent pathological changes in the kidneys of diabetic rats.

Large-scale epidemiological studies underscore the importance of early and strict metabolic control in preventing vascular complications of diabetes in humans. Moreover, these studies exemplify that early periods of poorly controlled diabetes can instigate the development of complications that present later in the progression of the disease.

The Diabetes Control and Complications Trial (DCCT) compared conventional and intensive regimens of blood glucose control in type 1 diabetes. At the conclusion of the DCCT, the same cohort of patients were all assigned intensive therapy as part of the Epidemiology of Diabetes Intervention and Complications (EDIC) 10-year follow-up study. Throughout this period, vascular complications were significantly reduced in patients that received intensive therapy during the DCCT. On the other hand, patients initially assigned to the conventional treatment group were found to be at significantly higher risk of developing vascular complications despite the tightening of blood glucose during the EDIC phase. Large-scale trials of type 2 diabetes therapies such as the United Kingdom Prospective Diabetes Study similarly point toward a deleterious memory of previous periods of inadequate metabolic control.

A memory of metabolic control. Significantly lower cumulative incidents of retinopathy were observed in patients that received intensive insulin therapy during the DCCT phase, despite normalisation of blood glucose in both groups during the EDIC phase. Click to enlarge

So what is the main driving force behind glucose memory? Clearly it is not a simple matter of poor metabolic or blood glucose control, as even with intensive intervention complications still occur. Further still, it is not simply having the wrong genes, as genetic studies have demonstrated that the DNA code explains only a fraction of the risk for diabetic complications. The most likely explanation is a complex interaction between the cellular environment and genes

Endothelial cells line the inner most surface of blood vessels. Because they are in constant contact with blood and are critical to vascular health, endothelial cells are a useful starting point to examine metabolic memory.  

Seminal findings published in 2008 and 2009 were the first to implicate persistent chromatin modifications in the sustained activation of inflammatory genes in endothelial models of glycemic variability. Professor Assam El-Osta and colleagues at Baker IDI Heart and Diabetes Research Institute in Melbourne demonstrated that the high glucose-mediated induction of a gene critically associated with vascular inflammation persisted in cultured human endothelial cells despite restoration of normal glucose conditions. Moreover, a specific histone methyl modification written by the Set7 lysine methylase was found to be essential to this metabolic memory.

Glucose memory in vascular cells. Cultured human vascular endothelial cells transiently exposed to high glucose concentrations exhibited strong activation of inflammatory genes that persisted despite restoration of normal glucose conditions. Click to enlarge

The same research group reported that restoring normal glycemic conditions per se does not reduce atherosclerosis in animal models of diabetes, and also that transient induction of hyperglycaemia in vivo was associated with persistent up-regulation of genes implicated in vascular disease, resulting from chromatin changes.

Currently it is unclear if transient spikes in blood glucose are sufficient to confer a vascular legacy in human diabetes, and the level of hyperglycaemic exposure required to establish future cell memories is yet to be clearly defined.

Very recently however, the same Set7-driven chromatin signatures were observed in peripheral blood monocytes isolated from patients with type 2 diabetes, providing strong clinical evidence for the importance of this enzyme in diabetic vascular disease. Similarly, epigenome-wide analysis recently revealed substantial increases in monocyte histone acetylation in the conventional treatment group compared to the intensive treatment group subjects of the DCCT/EDIC, indicating a further possible mechanism of metabolic memory.

Future strategies to counter memories of the past

Modification of the chromatin template can sensitise the genome to changing environmental conditions to establish diverse functional states of gene expression. Despite recent advances, scientists are only beginning to scratch the surface of this evidently complex system. The rapidly increasing prevalence of obesity and metabolic dysfunction underscores the imperative challenge of mapping the metabolically responsive chromatin landscape.

There is now strong evidence that chromatin-dependent gene regulation underlying phenotypic determinants of adult metabolic health is influenced by maternal and early postnatal diet. Numerous studies emphasise the importance of dietary balance during development and researchers are increasingly focussing their attention on this important period of foetal development and nutrition. In particular the past few years have seen a surge in the number of published studies examining the epigenetic effects of maternal diet. What we learn from historical human famine cohorts and experimental animal models is pivotal to our ability to cope with the after-effects of recent, current, and future famines around the globe. Of equal importance to public health are the findings that link maternal fat over-consumption with future metabolic dysfunction.

This adaptive agency is also known to influence gene expression in adulthood, where chromatin-dependent mechanisms drive persistent pathophysiological changes. Vascular complications remain the major cause of mortality and morbidity in diabetes with increasing evidence that prior glycemic exposure is a major determinant of susceptibility and progression of these disorders. Despite the promise of the genetic revolution, progress using genome-wide association approaches has thus far been limited. It is becoming increasingly clear that genetic factors simply do not explain diabetes susceptibility and its clinical sequelae. 

Generation of epigenetic maps using newly developed deep-sequencing technologies will greatly advance our understanding of gestationally-programmed chromatin changes as risk factors for adult disease. Equally, accurately defining the molecular events underlying diabetic complications will offer new strategies and specific targets for the development of therapies to prevent, retard, or reverse the long-term deleterious end-organ effects of chronic, intermittent, and prior hyperglycaemia.

To this end, molecular therapeutic approaches aimed at directly inhibiting enzymes that modify DNA and histones have shown promise in various pre-clinical models of disease. However several key challenges delay a comprehensive understanding and impede the efficacy of such treatments.

Distinguishing persistent and transient responses to environmental change remains a key challenge to defining metabolic memory. Precisely how are metabolic changes signalled to the chromatin modifying machinery? Specific intermediates of cellular metabolism such as acetyl-CoA, NAD+, and α-ketoglutarate are mechanistically associated with gene regulation by their use in chromatin-modifying reactions. Fluctuations in the cellular concentrations of these metabolites could therefore connect the chromatin with environmental signalling.

Furthermore the chromatin modifying machinery comprises a multifaceted network of enzymes capable of modifying both the chromatin as well as each other. Understanding the metabolically contextualised effects of inter-enzymatic modifications on the chromatin holds immense promise for greater understanding of environment-gene interactions and the development of effective intervention strategies to treat persistent metabolic dysfunction.

These emerging concepts open new perspectives to combat the rising global epidemic of metabolic disorders. The extent that pathological chromatin changes can be reversed remains controversial. However novel research approaches are aspiring to ensure that our metabolic past is not our destiny.

References and further reading

Originally published in The Australia Times Science magazineVol.3 No.1 (January 2015)
  1. Keating ST & El-Osta A, (2013) Glycemic memories and the epigenetic component of diabetic nephropathy. Current Diabetes Reports 8(4):361-72
  2. Keating ST & El-Osta A, (2013) Epigenetic changes in diabetes. Clinical Genetics 84(1): 1-10
  3. Barker DJ et al., (1989) The intrauterine and early postnatal origins of cardiovascular disease and chronic bronchitis. Journal of Epidemiology and Community Health 43(3): 237-40
  4. Thurner S et al., (2013) Quantification of excess risk for diabetes for those born in times of hunger, in an entire population of a nation, across a century. PNAS 110(12): 4703-7
  5. Heijmans BT et al., (2008) Persistent epigenetic differences associated with prenatal exposure to famine in humans. PNAS 105(44): 17046-9
  6. Gong L et al., (2010) Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation. Epigenetics 5(7): 619-26
  7. Hoile SP et al., (2013) Maternal fat intake in rats alters 20:4n-6 and 22:6n-3 status and the epigenetic regulation of Fads2 in offspring liver. Journal of Nutritional Biochemistry 24(7): 1213-20
  8. Laker RC et al., (2014) Exercise prevents maternal high-fat diet-induced hypermethylation of the Pcg-1α gene and age-dependent metabolic dysfunction in the offspring. Diabetes 63(5): 1605-11
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