Samuel T. Keating

Epigenetics and Metabolism

The molecular signatures of epigenetic regulation and chromatin architectures are fundamental to genetically determined biological processes. Covalent and post-translational chemical modification of the chromatin template can sensitize the genome to changing environmental conditions to establish diverse functional states. Recent interest and research focus surrounds the direct connections between metabolism and chromatin dynamics, which now represents an important conceptual challenge to explain many aspects of metabolic dysfunction. Several components of the epigenetic machinery require intermediates of cellular metabolism for enzymatic function. Furthermore, changes to intracellular metabolism can alter the expression of specific histone methyltransferases and acetyltransferases conferring widespread variations in epigenetic modification patterns. Specific epigenetic influences of dietary glucose and lipid consumption, as well as undernutrition, are observed across numerous organs and pathways associated with metabolism. Studies have started to define the chromatin-dependent mechanisms underlying persistent and pathophysiological changes induced by altered metabolism. Importantly, numerous recent studies demonstrate that gene regulation underlying phenotypic determinants of adult metabolic health is influenced by maternal and early postnatal diet. These emerging concepts open new perspectives to combat the rising global epidemic of metabolic disorders.

Epigenetic changes in diabetes

Diabetes is a multifactorial disease with numerous pathways influencing its progression and recent observations suggest that the complexity of the disease cannot be entirely accounted for by genetic predisposition. A compelling argument for an epigenetic component is rapidly emerging. Epigenetic processes at the chromatin template significantly sensitize transcriptional and phenotypic outcomes to environmental signaling information including metabolic state, nutritional requirements and history. Epigenetic mechanisms impact gene expression that could predispose individuals to the diabetic phenotype during intrauterine and early postnatal development, as well as throughout adult life. Furthermore, epigenetic changes could account for the accelerated rates of chronic and persistent microvascular and macrovascular complications associated with diabetes. Epidemiological and experimental animal studies identified poor glycemic control as a major contributor to the development of diabetic complications and highlight the requirement for early intervention. Early exposure to hyperglycemia can drive the development of complications that manifest late in the progression of the disease and persist despite improved glycemic control, indicating a memory of the metabolic insult. Understanding the molecular events that underlie these transcriptional changes will significantly contribute to novel therapeutic interventions to prevent, reverse or retard the deleterious effects of the diabetic milieu.

Vascular histone deacetylation by pharmacological HDAC inhibition

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

Transcriptional regulation by the Set7 lysine methyltransferase

Posttranslational histone modifications define chromatin structure and function. In recent years, a number of studies have characterized many of the enzymatic activities and diverse regulatory components required for monomethylation of histone H3 lysine 4 (H3K4me1) and the expression of specific genes. The challenge now is to understand how this specific chemical modification is written and the Set7 methyltransferase has emerged as a key regulatory enzyme mediating methylation of lysine residues of histone and non-histone proteins. In this review, we comprehensively explore the regulatory proteins modified by Set7 and highlight mechanisms of specific co-recruitment of the enzyme to activating promoters. With a focus on signaling and transcriptional control in disease we discuss recent experimental data emphasizing specific components of diverse regulatory complexes that mediate chromatin modification and reinterpretation of Set7-mediated gene expression.

Epigenetic Changes in Diabetes and Cardiovascular Risk

Cardiovascular complications remain the leading causes of morbidity and premature mortality in patients with diabetes mellitus. Studies in humans and preclinical models demonstrate lasting gene expression changes in the vasculopathies initiated by previous exposure to high glucose concentrations and the associated overproduction of reactive oxygen species. The molecular signatures of chromatin architectures that sensitize the genome to these and other cardiometabolic risk factors of the diabetic milieu are increasingly implicated in the biological memory underlying cardiovascular complications and now widely considered as promising therapeutic targets. Atherosclerosis is a complex heterocellular disease where the contributing cell types possess distinct epigenomes shaping diverse gene expression. Although the extent that pathological chromatin changes can be manipulated in human cardiovascular disease remains to be established, the clinical applicability of epigenetic interventions will be greatly advanced by a deeper understanding of the cell type-specific roles played by writers, erasers, and readers of chromatin modifications in the diabetic vasculature. This review details a current perspective of epigenetic mechanisms of macrovascular disease in diabetes mellitus and highlights recent key descriptions of chromatinized changes associated with persistent gene expression in endothelial, smooth muscle, and circulating immune cells relevant to atherosclerosis. Furthermore, we discuss the challenges associated with pharmacological targeting of epigenetic networks to correct abnormal or deregulated gene expression as a strategy to alleviate the clinical burden of diabetic cardiovascular disease.

Chromatin Modifications Associated with Diabetes

Accelerated rates of vascular complications are associated with diabetes mellitus. Environmental factors including hyperglycaemia contribute to the progression of diabetic complications. Epidemiological and experimental animal studies identified poor glycaemic control as a major contributor to the development of complications. These studies suggest that early exposure to hyperglycaemia can instigate the development of complications that present later in the progression of the disease, despite improved glycaemic control. Recent experiments reveal a striking commonality associated with gene-activating hyperglycaemic events and chromatin modification. The best characterised to date are associated with the chemical changes of amino-terminal tails of histone H3. Enzymes that write specified histone tail modifications are not well understood in models of hyperglycaemia and metabolic memory as well as human diabetes. The best-characterised enzyme is the lysine specific Set7 methyltransferase. The contribution of Set7 to the aetiology of diabetic complications may extend to other transcriptional events through methylation of non-histone substrates.

Chromatin modifications remodel cardiac gene expression.

Signalling and transcriptional control involve precise programmes of gene activation and suppression necessary for cardiovascular physiology. Deep sequencing of DNA-bound transcription factors reveals a remarkable complexity of co-activators or co-repressors that serve to alter chromatin modification and regulate gene expression. The regulated complexes characterized by genome-wide mapping implicate the recruitment and exchange of proteins with specific enzymatic activities that include roles for histone acetylation and methylation in key developmental programmes of the heart. As for transcriptional changes in response to pathological stress, co-regulatory complexes are also differentially utilized to regulate genes in cardiac disease. Members of the histone deacetylase (HDAC) family catalyse the removal of acetyl groups from proteins whose pharmacological inhibition has profound effects preventing heart failure. HDACs interact with a complex co-regulatory network of transcription factors, chromatin-remodelling complexes, and specific histone modifiers to regulate gene expression in the heart. For example, the histone methyltransferase (HMT), enhancer of zeste homolog 2 (Ezh2), is regulated by HDAC inhibition and associated with pathological cardiac hypertrophy. The challenge now is to target the activity of enzymes involved in protein modification to prevent or reverse the expression of genes implicated with cardiac hypertrophy. In this review, we discuss the role of HDACs and HMTs with a focus on chromatin modification and gene function as well as the clinical treatment of heart failure.

Glycemic Memories and the Epigenetic Component of Diabetic Nephropathy

A strong case for the deregulation of epigenetic chromatin modifications in the development and progression of various chronic complications of diabetes has emerged from recent experimental observations. Clinical trials of type 1 and type 2 diabetes patients highlight the importance of early and intensive treatment and the prolonged damage of hyperglycemia on organs such as the kidney. The functional relationship between the regulation of chromatin architecture and persistent gene expression changes conferred by prior hyperglycemia represents an important avenue of investigation for explaining diabetic nephropathy. While several studies implicate epigenetic changes at the chromatin template in the deregulated gene expression associated with diabetic nephropathy, the molecular determinants of metabolic memory in renal cells remain poorly understood. There is now strong evidence from experimental animals and cell culture of persistent glucose-driven changes in vascular endothelial gene expression that may also have relevance for the microvasculature of the kidney. Exploration of epigenetic mechanisms underlying the hyperglycemic cue mediating persistent transcriptional changes in renal cells holds novel therapeutic potential for diabetic nephropathy.

Interplay of chromatin modifications and non-coding RNAs in the heart

Precisely regulated patterns of gene expression are dependent on the binding of transcription factors and chromatin-associated determinants referred to as co-activators and co-repressors. These regulatory components function with the core transcriptional machinery to serve in critical activities to alter chromatin modification and regulate gene expression. While we are beginning to understand that cell-type specific patterns of gene expression are necessary to achieve selective cardiovascular developmental programs, we still do not know the molecular machineries that localize these determinants in the heart. With clear implications for the epigenetic control of gene expression signatures, the ENCODE (Encyclopedia of DNA Elements) Project Consortium determined that about 90% of the human genome is transcribed while only 1-2% of transcripts encode proteins. Emerging evidence suggests that non-coding RNA (ncRNA) serves as a signal for decoding chromatin modifications and provides a potential molecular basis for cell type-specific and promoter-specific patterns of gene expression. The discovery of the histone methyltransferase enzyme EZH2 in the regulation of gene expression patterns implicated in cardiac hypertrophy suggests a novel role for chromatin-associated ncRNAs and is the focus of this article.

Deep sequencing reveals novel Set7 networks

AbstractBackground Methyl-dependent regulation of transcription has expanded from a traditional focus on histones to encompass transcription factor modulation. While the Set7 lysine methyltransferase is associated with pro-inflammatory gene expression in vascular endothelial cells, genome-wide regulatory roles remain to be investigated. From initial characterization of Set7 as specific for methyl-lysine 4 of H3 histones (H3K4m1), biochemical activity toward non-histone substrates has revealed additional mechanisms of gene regulation.ResultsmRNA-Seq revealed transcriptional deregulation of over 8,000 genes in an endothelial model of Set7 knockdown. Gene ontology identified up-regulated pathways involved in developmental processes and extracellular matrix remodeling, whereas pathways regulating the inflammatory response as well as nitric oxide signaling were down-regulated. Chromatin maps derived from ChIP-Seq profiling of H3K4m1 identified several hundred loci with loss of H3K4m1 at gene regulatory elements associated with an unexpectedly subtle effect on gene expression. Transcription factor network analysis implicated six previously described Set7 substrates in mRNA-Seq changes, and we predict that Set7 post-translationally regulates other transcription factors associated with vascular endothelial gene expression through the presence of Set7 amino acid methylation motifs.ConclusionWe describe a role for Set7 in regulating developmental pathways and response to stimuli (inflammation/immune response) in human endothelial cells of vascular origin. Set7-dependent gene expression changes that occurred independent of H3K4m1 may involve transcription factor lysine methylation events. The method of mapping measured transcriptional changes to transcription factors to identify putative substrates with strong associations to functional changes is applicable to substrate prediction for other broad-substrate histone modifiers.