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.

References

Keating ST, Plutzky J, El-Osta A. (2016) Epigenetic Changes in Diabetes and Cardiovascular Risk. Circ Res 118:1706-22

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.

References 

Keating ST, Plutzky J, El-Osta A. (2016) Epigenetic Changes in Diabetes and Cardiovascular Risk. Circ Res 118:1706-22

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. 

References

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

The chromatin code

A dynamic landscape

The chromatin polymer is a dynamic assembly of DNA and various nuclear proteins that spatially regulates eukaryotic gene transcription by structural adaptation and genome compartmentalisation. 

Gene expression is primarily regulated by chromatin accessibility. The repeating unit of chromatin is the nucleosome, comprised of approximately 147 base pairs of DNA encircling an octamer of two copies of each of the four core histone proteins: H2A, H2B, H3, and H4. A relaxed chromatin structure where DNA is loosely associated with nucleosomes (euchromatin) is permissive to the transcriptional machinery, allowing the synthesis of RNA. By contrast, genes are silenced when DNA is tightly wrapped around histones and buried within the chromatin (heterochromatin). It is by selective activation and silencing of different genes that more than 200 distinct cell types of the human body are derived from a single source of genetic information. Furthermore, chromatin dynamics provides the adaptive agency for cells to manage environmental variations with rapid changes in gene expression.

Small chemical modifications that decorate the chromatin landscape distinguish active and silent genomic regions. Modifications frequently occur at specific amino acid residues on histones as well as the DNA itself to collectively comprise the epigenome. 

By several distinct mechanisms, the chemical signatures of chromatin architectures are fundamental to gene expression. Individual modifications can recruit functional complexes responsible for the structural reorganisation of chromatin. Other modifications directly affect the stability of the histone-DNA complex to promote an open conformation. These gene-regulatory effects in combination with substantial functional interplay between histone modifications has lead to the proposition of a chromatin code that greatly extends the information potential of the genetic code by shaping transcriptional competency. 

The epigenome is diverse

Methylation of DNA predominantly at cytosine nucleotides adjacent to guanine residues (CpG) at gene promoters is predominantly associated with gene suppression and corresponds most closely to the etymological interpretation of epigenetics. Regulatory proteins mechanistically interpret this prevalent epigenetic modification by recruiting chromatin-remodeling complexes to establish transcriptionally repressive chromatin. Recent discovery of the Ten Eleven Translocation family of dioxyegnases that oxidise methyl-cytosine to generate hydroxymethyl-cytosine have inspired strong interest in transitions between methylated and unmethylated DNA in mammalian cells.

Equally important are the wealth of chemical groups dynamically written to and erased from the protruding N-terminal tails of histones by specialised enzymes and complexes. Here the methyl modification also occupies a prominent role in gene regulation when assigned to lysine (K) and arginine (R) residues. Moreover the effect on transcription is dependent on the site of modification. For example, methylation at lysines 9 and 36 of H3 histones associates with gene repression and activation respectively. Furthermore, diverse degrees of methylation (lysine residues can be mono-, di-, or tri-methylated, and arginine residues mono-methylated and asymmetrically or symmetrically di-methylated) are differentially distributed across chromatin and ascribed distinct functional roles in gene regulation. A prominent example is the range of methyl modifications occurring on lysine 4 of H3 histones. The tri-methylated form of this modification punctuates the start of actively transcribed genes. On the other hand, di-methylation is dispersed throughout gene bodies, and mono-methylation is frequently observed at both proximal and distal regulatory regions of active genes. Contradicting the activities of histone methylases are enzymes responsible for the removal of methyl groups, such as JmjC-domain-containing proteins and LSD1.   

In contrast, site-specific histone lysine acetylation is ubiquitously associated with open chromatin and transcriptional activation. Regulated by the opposing activities of histone acetylases (HATs) and deacetylases (HDACs), acetyl groups are thought to disrupt the charge states of histone tails, altering their contact with the nucleosome as well as reducing their affinity for DNA. Additionally, acetylation of lysines 9 and 27 of H3 histones directly competes with the transcriptionally repressive methylation of these residues.

Other important histone modifications include phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, and the recently emerged O-linked addition of N-acetylglucosamine (O-GlcNAcylation).

Gene function is primarily regulated by chromatin accessibility (click to enlarge). Shown here is a summary of acetyl (purple) and methyl (red) modifications of specific arginine (R) and lysine (K) residues on N-terminal tails of the heavily modified H3 and H4 histones, as well as DNA methylation at active and silent regions of chromatin. Click image to enlarge

Who regulates the regulators?

The enzymes responsible for the transfer and removal of histone modifications demonstrate strong specificity toward particular amino acid positions within histone tails. Research has characterised a substantial catalogue of enzymes that catalyse more than 100 histone tail marks. Indeed, several biological factors are known to influence the expression of epigenetic modifiers. However a number of questions remain unanswered regarding the precise mechanism underlying their enzymatic function. Specifically, how are chromatin-modifying reactions regulated?

A current topic in the field is the effects of metabolism on the epigenome. Several components of the epigenetic machinery require intermediates of cellular metabolism for enzymatic function. Dietary factors such as folate and B vitamins influence cellular concentrations of S-adenosyl methionine, the universal methyl-donor to methylation reactions. Similarly, acetyl-CoA generated from glucose and fatty acid metabolism is the essential acetyl group donor to lysine acetylation reactions. Furthermore, some DNA and histone demethylase enzymes utilise specific intermediates of the citric acid cycle.

Secondly, post-translational modifications are not restricted to the chromatin. In fact, the same enzymes that write and erase epigenetic marks modify a diverse array of non-histone proteins. These include not only transcription factors, which can independently influence gene expression, but also the activities of other histone modifiers. For example, acetylation enhances the enzymatic functions of the P/CAF, p300, and MYST acetylases, as well as lowering the methylase activity of SUV39H1.  On the other hand, SUV39H1 is activated, and the DNA methylase DNMT1 destabilized by lysine methylation. Characterisation of this network of modifications controlling enzyme function and consequent epigenetic chromatin modulation holds immense potential to further our understanding of gene regulation.

Finally, an important role for non-coding RNA molecules in the establishment of cell-type and gene-specific chromatin modification patterns has recently emerged. Advances in nucleic acid sequencing technologies have revealed that while approximately 90% of the human genome is transcribed, only 1-2% of RNA transcripts encode proteins. Non-coding RNA vary in length and function, with short transcripts such as mircoRNAs playing numerous regulatory roles in gene expression primarily at the mRNA level. Importantly, long non-coding RNAs (lncRNAs) interact with epigenetic enzymes to direct their chromatin binding. For example, the HOTAIR lncRNA simultaneously recruits the Polycomb Repressive Complex 2 and the LSD1 demethylase for coordinated methylation of lysine 27 and demethylation of lysine 4 on H3 histones. Future research is anticipated to uncover many scaffolding and tethering roles for lncRNAs in the specific localisation of chromatin changes.

Finally, an important role for non-coding RNA molecules in the establishment of cell-type and gene-specific chromatin modification patterns has recently emerged. Advances in nucleic acid sequencing technologies have revealed that while approximately 90% of the human genome is transcribed, only 1-2% of RNA transcripts encode proteins. Non-coding RNA vary in length and function, with short transcripts such as mircoRNAs playing numerous regulatory roles in gene expression primarily at the mRNA level. Importantly, long non-coding RNAs (lncRNAs) interact with epigenetic enzymes to direct their chromatin binding. For example, the HOTAIR lncRNA simultaneously recruits the Polycomb Repressive Complex 2 and the LSD1 demethylase for coordinated methylation of lysine 27 and demethylation of lysine 4 on H3 histones. Future research is anticipated to uncover many scaffolding and tethering roles for lncRNAs in the specific localisation of chromatin changes.

Mapping the chromatin landscape

Whether these observations constitute a true code is often questioned.  The existence of a strict chromatin code implies that definite patterns of post-translational histone modifications instruct a rigid functional outcome.  Unlike the causal nature of the genetic code, it is more likely that combinatory patterns of histone modifications create a biased chromatin landscape that generally favours a particular transcriptional outcome. Nonetheless, genetic knock-out animals and similar approaches in cells demonstrate the necessity of epigenetic regulation.

Chromatin modifications are increasingly studied in development and disease and considerable interest surrounds the pharmacological targeting of epigenetic enzymes for therapy. Particularly HDAC inhibitors are rigorously investigated for their clinical use in the treatment of human disease such as cancer and heart disease. Clearly, the thorough characterisation of the epigenome holds immense promise for the clinic. 

Increased availability and use of massive parallel sequencing has allowed the epigenetic analysis of various cell types and biological contexts. Accelerated generation of epigenomic datasets has driven the accumulation of large repositories of data.This shift to systems-level perspective signifies a fundamental change in the way cell biology is investigated, rapidly propelling our modern knowledge of medicine and biology. The generation of cell-specific epigenomic maps and transcriptome profiles is fundamental to a truly comprehensive understanding of gene regulation.

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