What exactly are epigenetic modifications?

Epigenetics describes any changes in gene expression that occur without alteration in a DNA sequence. Epigenetics is why identical twins tend to become less identical with age- their genomic DNA sequence does not change but the epigenetic landscape that regulates gene expression naturally diverges over time. These changes may be cell or tissue-specific and may be heritable through multiple generations. Even though epigenetic mechanisms primarily function through the nucleus, they can be induced by a range of environmental signals including physiological and nutritional stress, cellular damage, environmental signals, and cell signaling molecules such as hormones. Epigenetic changes can be mediated through modifications of the DNA directly, or of the protein components of the chromatin structure.

As our understanding of epigenetics is advancing, its influence has expanded across many fields of research including cancer and stem cells, aging, and neuroscience, opening the possibility of exciting and new therapeutic applications at the epigenetic level.  

Epigenetic Changes in Chromatin Structure

All of the functional processes of DNA occur in the form of chromatin rather than on naked DNA (Fig. 1). Chromatin describes a complex of genomic DNA and several associated proteins that are located in the nucleus, that collectively form the nucleosome. The proteins involved in the formation of chromatin include histones (H1, H2A, H2B, H3, and H4) as well as a diverse group of proteins such as transcription factors, polymerases, hormone receptors, and nuclear enzymes.  Site-specific modification of these proteins alters the chromatin structure to regulate the transcription of genes, as well as DNA repair, recombination, and replication by changing the local chromatin architecture. 

Epigenetic modifications can take several different forms from methylation, acetylation, phosphorylation, to ubiquitination, citrullination, and ADP-ribosylation, each of these are regulated independently to modify the chromatin structure and subsequent transcription activities within that region. Alterations in the genes regulating epigenetic processes are frequently found to be cancer drivers and appear to be caused by changes in DNA and RNA modifications, histone modifications, and nucleosome structure. Various studies have identified common somatic mutations in epigenetic regulators that appear to drive the initiation and maintenance of cancers. Therefore, epigenetic therapy represents an emerging field of medicine, and currently, three inhibitors targeting DNMTs, HDACs, and JAK2 have been approved by the FDA.  

Epigenetic Changes Through Histone Modification

Acetylation 

Histone regulation through acetylation is mediated through two enzyme systems: histone acetylases (HATs) that acetylate histone lysine residues, adding a negative charge to these residues that opens up the chromatin structure to promote transcription, and deacetylase (HDACs) that remove histone acetyl groups and decrease transcription.  Histone acetylation is a dynamic process that is regulated by these competing enzymatic systems.  HDACs have been shown to be overexpressed in numerous cancers, in which global reduction of histone acetylation is observed, and tumor-suppression gene expression is silenced. Histone acetylation on lysine residues is read by protein modules called bromodomains (BRD2, BRD3, BRD4) that are members of BET protein family and play an essential role in transcriptional elongation and cell-cycle regression.  

Typically, histone acetylation is analyzed by radiolabeling or mass spectroscopy, however, with the development of modification-specific antibodies, or antibodies recognizing the acetylated histone tails, it is now possible to detect such modifications at the single-cell level by flow cytometry. Flow cytometry represents a fast, high-throughput means of analyzing large cell populations, providing a significant advantage over single readout systems such as western blotting and immunofluorescence, and enabling the dynamic analysis of distinct cell types within a population to look at heterogeneity in modifications across a population of cells. 

Methylation

Histone proteins can be methylated on the side chains of arginine, lysine, and histidine residues. The most widely studied histone modifications include H3K4, H3K9, H3K27, H3K26, and H3K79. On lysine residues, up to 3 methyl-groups may be attached such that monomethyl, dimethyl, and trimethyl forms may be found, each with distinct functional consequences. For example, H3K9me1 is associated with active gene expression whereas H3K9me3 is associated with gene repression.

Histone methylation is performed by histone lysine methyltransferases (KMTs) resulting in the formation of “euchromatin” structure that is indicative of increased transcriptional activity, or demethylated by the demethylases LSD1, UTX, and JMJD-type enzymes. The orchestration of histone methylase and demethylase activity underlies both the temporal and spatial regulation of gene expression.

Transcription activation or repression is also regulated through DNA methyltransferases. There are three main classes: DNMT1, DNMT3a, and DNMT3b that each maintain specific signatures of DNA methylation that regulate the activity of methyl-binding proteins such as MECP2, MBD1, MBD2, MBD3, MBD4, and Kaiso.  

Numerous studies have now utilized flow cytometry to couple the multiparameter analysis of histone modification (acetylation or methylation) with molecular markers (Fig.2), DNA ploidy, and cell cycle analysis to examine the safety and efficacy of clinical trials for modifiers of these regulatory mechanisms (Ronzino et. al. 2005; Watson et. al. 2013).

Acetylation and methylation are the most frequent epigenetic modification of histone proteins. The modifications are localized at the histone N-terminal tails, in the protein cores, and in the C-terminal regions. So-called enzymatic ‘writers’ add the methyl or acetyl groups, and the proteins that recognize and bind to these regions are referred to as ‘readers.’ Epigenetic changes can be reversed by the activity of ‘erasers’ (Fig. 1).

Fig. 1. Histone modification represents the key mechanism of epigenetic regulation. The genomic DNA is wrapped around histone proteins and packed into chromatin that forms the nucleosome.  This structure mediates the activity of histone post-translational modification.  

Fig. 2. Flow Cytometry analysis of acetylated histone 3. Human PBMCs treated with an HDAC inhibitor (orange signal) or DMSO control (blue signal) were stained intracellularly with ac-Lys9 histone H3 antibody conjugated with Alexa Fluor 647.

Phosphorylation

The phosphorylation of histones is typically associated with cells undergoing apoptosis in response to cell damage, however, there are examples of histone phosphorylation resulting in gene induction and the promotion of changes in chromatin structure.  Mitotic phosphorylation near to methylation or acetylation sites may interfere with the binding of anti-methyl or acetyl-modification specific antibodies, and so it is important to consider cell cycle when designing these types of experiments. A flow cytometry-based diagnostic assay for ataxia telangiectasia (A-T) has been developed using the phosphorylation of H2AX as a biomarker for the disease (Porcedda et. al. 2008)

Ubiquitination

Ubiquitination involves a combination of enzyme systems: ubiquitin-activating [E1s], ubiquitin-conjugating enzymes [E2s], and ubiquitin-protein ligases [E3s]. These work together to coordinate key histone methylation events. 

Citrullination

Citrullination involves the modification of arginine residues on histones, particularly H4 via the enzymatic activity of peptidylarginine deiminases (PADIs). It is believed to play a role in certain autoimmune conditions such as rheumatoid arthritis and multiple sclerosis, as well as a central role in inflammation (Tsourouktsoglou et. al. 2020) and the regulation of pluripotency (Christophorou et. al. 2016)  

ADP-ribosylation

This type of post-translational modification of histone proteins is catalyzed by ADP-ribose transferase (ARTs) and is induced by damage to DNA. The most commonly studied sites for ADP-ribosylation are H1E2 and H1E15, however, there are thought to be more than 30 such histone sites (Karch et. al. 2017)

Epigenetic Related DNA and RNA modifications

Both global and gene-specific DNA methylation is often found in cancer, and methylation patterns have been shown to distinguish tumor types. Global hypomethylation is frequently found in cancer genomes, whereas hypomethylation is associated with CpG islands leading to the silencing of expression of tumor suppressors. 

DNA Methylation is central to many cellular processes, including chromosome and chromatin structure and stability, X-chromosome inactivation, and the regulation of transcription, particularly through embryonic development. Methylomics is the study of the methylome or identification of methylated cytosine in the genome. Within the genome are so-called CpG islands consisting of 500-200bp of CpG rich areas. Methylation of these CpG islands at the cytosine residue at the 5 position results in the formation of 5-methlycytosine (5mC) and this has been implicated in gene silencing. 

5mC may be converted to 5-hydroxymethylcytosine [5hmC], 5-formylcyosine [5fC] and 5-carboxylcytosine [5caC] by Ten-Eleven Translocation [TET1-3] enzymes. The formation of 5hmC residues is associated with active gene expression, particularly around embryonic development, and brain maturation. However, it is the finding that they may be involved in tumor development that has now made them a focus of epigenetic research particularly in relation to hematopoietic malignancies including AML, MPD, MDS, and CMML.

RNA modifications also include methylation, the most common of which is N6-methyladenosine [m6A) particularly within mRNA. m6A methylation is a dynamic process that is coordinated by methyltransferases and demethylases and these have now become targets for therapeutic intervention for cancers such as AML. m6A RNA modifications have been shown to play key roles in RNA transcription, processing, translation, and metabolism, with implications for chronic diseases including obesity, infertility, and cancer.

Polycomb and its regulation of Chromatin Remodeling

Polycomb Group (PcG) are transcriptional regulatory proteins that selectively repress gene expression during embryonic development by forming complexes known as Polycomb Repressive Complexes (PRCs) that regulate chromatin structure and maintain it in a transcriptionally inactive formation. These protein complexes also induce the modification of histone by interacting with histone methyltransferases and demethylases to enhance transcriptional silencing. 

Adaptation of Flow Cytometry for Epigenetic Studies.

One of the first studies to adopt reagents for ChIP for flow cytometry applications was by Obier and Muller, and although this program was successful, there was a need to validate each of the antibodies used in order to optimize staining of the fixed samples. In recent years, a larger number of monoclonal antibodies have been developed that target the specific reader, writer, and eraser proteins, polycomb repressive complex protein subunits, as well as antibodies that directly target the epigenetic modification sites on histones.  In fact, in 2010, The National Institute of Health supported major investment into the development of novel epigenetic reagents through a series of SBIR grants to companies such as New England Biolabs. This investment has supported research into this field of medicine, and now many companies have developed innovative probes and monoclonal conjugates to identify key epigenetic markers. 

There are now a growing number of studies utilizing flow cytometry to interrogate chromatin structure. This new and exciting application of flow cytometry offers several advantages over traditional Western Blot and ELISA since it enables quantitative, high-throughput analysis of multiple intranuclear and cell surface markers at a single-cell level. So, although it currently is not possible to display loci-specific chromatin structures, it can be applied to disclose subpopulations of cells with distinct chromatin modifications and sort these out of samples for further analysis by ChIP for example. In this regard, chromatin flow cytometry represents a potent means of gaining insights into the epigenetic mechanisms underlying disease development and progression as well a normal cell differentiation (Obier and Müller, 2007). 

Epigenetic profiling has been further advanced by the adoption of cytometry by Time of Flight (EpiTOF) that enables the analysis of a broad array of global histone modifications, PcG activities, and chromatin structures, within different cell types. These types of complex studies are helping us to understand the links between epigenetic profiles and cellular phenotype and function (Cheung, P. et. al. 2018).

Importance of Epigenetics in the Immune System

Epigenetic reprogramming is now emerging as a critical determinant of the immune response and establishment of memory. Innate immunity represents the first line of host defense and although often thought of as primitive and generic; however, we are now finding out that epigenetic histone modifications play a central role in orchestrating broad immunological protection by in-printing innate immune cells such as monocytes/macrophages and natural killer cells, in the process described as trained immunity. The involvement of these cell types in chronic disorders such as insulin resistance, diabetes mellitus, and rheumatoid arthritis opens the door to novel therapeutic approaches to reverse the epigenetic programming of these cells that promote these chronic conditions. Inhibition of either glycolysis or mTOR signaling has been shown to disrupt the epigenetic changes associated with trained immunity, and the fluctuation of key metabolite levels (e.g. α-ketoglutarate, succinate, fumarate, NAD+, and acetyl-CoA) has been implicated in the continual adjustment of gene expression through epigenetic control of the chromatin landscape (van der Heijden et. al. 2018).

Clonal expansion resulting in immunological memory is a hallmark of the adaptive immune response mediated through antigen-specific T cells and B-cells.  T cell activation and differentiation into effector cells is accompanied by widespread epigenetic modifications and marked changes in gene expression. NK cells have similarly acquired the ability to generate memory through clonal expansion and share many common epigenetic signatures with the underlying signature of antigen-experienced memory CD8+ T cells (Lau et. al. 2018).  Unraveling the molecular mechanisms that underlie immunological memory has huge implications for the design of targeted vaccines. 

The establishment of T-cell memory is one of the most studied epigenetic systems of the immune system. Memory T cells exhibit an ability to rapidly recall a response and undergo proliferation and functional differentiation upon activation; this ability is mediated through epigenetic imprinting that confers constitutive and inducible gene expression associated with this antigen response. Interestingly, effector T-cells and memory T-cells display changes in DNA methylation profiles compared with that of naïve T-cells, and this methylation is mediated through the activity of methyltransferase Dnmt3a (Youngblood, B. et. al. 2017). T-cell epigenetic memory has been shown to persist for many years and provide the foundation for long-lasting immunity after infection or vaccination. 

Significantly, the epigenetic state of T-cells also appears to contribute to reduce tumor immunity as well as autoimmunity, and so understanding the epigenetic gene regulatory pathways opens the possibility to novel and innovative therapeutic interventions.

Final Thoughts

The emerging field of epigenetics is remarkably complex, involving a number of different binding proteins and enzymatic processes that regulate the chromatin landscape and dictate gene expression. 

With the increasing availability of monoclonal antibodies targeting specific epigenetic modulations, flow cytometry is providing valuable clinical insights by providing a means of resolving stages of immune cell differentiation based on cell surface phenotypes and global epigenetic modifications. Thanks in part to these studies, we are now beginning to understand the functional significance of genetic variants to immune system function and the epigenetic interactions that are involved in modulating this activity. 

References:

Porcedda, P., et. al. (2008) A rapid flow cytometry test based on histone H2AX phosphorylation for the sensitive and specific diagnosis of ataxia telangiectasia. Cytometry Part A. https://doi.org/10.1002/cyto.a.20566

Tsourouktsoglou, T. et. al. (2020) Histone, DNA, and Citrullination Promote Neutrophil Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4. Cell Reports vol. 31 (5) 107602

Christophorou, M. et. al. (2014) Citrullination regulates pluripotency and Histone H1 binding to chromatin. Nature 507(7490) p. 104-8.

Ronzini, S., et. al. (2005) New method to detect Histone acetylation levels by flow cytometry. Watson, M., et. al. (2013) The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytometry. Cytometry Part A. doi.org/10.1002/cyto.a.22344

Obier, N. and Müller, A. M. (2007) Chromatin Flow Cytometry Identifies Changes in Epigenetic Cell States. Cells Tissues Organs DOI: 10.1159/000240247

Cheunga, P. et. al. (2018) Single-cell epigenetics- Chromatin modification atlas unveiled by mass cytometry. Clinical Immunology vol. 196. p40-48. 

Van der Heijden, C. D. C. C. et. al. (2018) Epigenetics and Trained Immunity. Antioxidants and Redox Signaling. 29(11): 1023-1040.

Youngblood, B., et. al. (2017) Effector CD8 T-cells dedifferentiate into long-lived memory cells. Nature 552 p.404-9. 

Lau, C. M. et. al. (2018) Epigenetic control of innate and adaptive immune memory, Nat. Immunol. PMID: 30082830 PMCID: PMC6225771 DOI: 10.1038/s41590-018-0176-1

Karch, K. R. et. al. (2017) The nucleosome surface is the main target of histone ADP-ribosylation in response to DNA damage. Mol. Biosyst. 13(12): p2660-71.

Watson, M. et. al. (2013) The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytometry. Cytometry part A vol. 85 (1). P.78-87  https://doi.org/10.1002/cyto.a.22344

Dr. Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Torrey Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.