Epigenetics

Epigenetics means literally "above the genes” and refers to modifications in gene expression that do not involve changes in the DNA nucleotide sequence. Epigenetic changes may include:

• Chemical modifications (e.g., methylation) to DNA or RNA
• Chemical modifications to DNA associated proteins (e.g., histones)
• Incorporation of histone variants which alter the chromatin conformation
• mRNA degradation by microRNAs (~22 nucleotides-long RNA sequences)

Epigenetic modifications result from environmental influences. Overall, epigenetic changes either enhance or inhibit gene expression without altering the DNA sequence. Phenotypic outcome is dictated by the specific combination of epigenetic modifications.

Epigenetic writers catalyze the addition of chemical groups onto either histone tails or the DNA itself. These modifications are known as epigenetic marks. One such group of epigenetic "writers" is histone methyltransferases, which are further subdivided into lysine methyltransferases and arginine methyltransferases according to their target residue. Protein lysine methyltransferases, also known as PKMTs, catalyze the transfer of a methyl group from the co-factor S-adenosyl methionine (SAM) onto a lysine side chain on the exposed histone tail. Histone lysine methylation can involve the transfer of one, two or three methyl groups onto a histone tail; the degree of methylation is of biological significance since proteins that interact with methylated histones are able to distinguish between mono-, di- and trimethylated lysines. Histone arginine residues may also undergo methylation, in a reaction catalyzed by protein arginine methyltransferases (PRMTs). PRMTs generate either monomethylated or dimethylated arginine residues, where the dimethylation can either occur symmetrically or asymmetrically. The symmetry of the methyl groups added to arginine residues determine the biological effect of the epigenetic modification: asymmetric dimethylation is linked to gene activation whilst symmetric dimethylation is associated with gene repression.

In addition to methyl marks, histone lysine residues may also undergo acetylation through the activity of histone acetyltransferases (HATs). The transfer of an acetyl group from the co-factor acetyl-CoA to lysine residues on histone tails neutralizes the positive charge of lysine; this weakens the affinity of the histone tail for the DNA and reduces chromatin condensation. Since a more relaxed, open chromatin architecture enables the recruitment of transcription factors and polymerases, histone acetylation results in the promotion of gene expression.

Enzymes that catalyze the phosphorylation of histone tails are also important epigenetic "writers". For example, phosphorylation of histone H3 (H3Y41) by JAK2 disrupts binding of the heterochromatin protein HP1α to chromatin, leading to increased DNA accessibility and the transcription of the oncogene lmo2. Other kinases including Haspin, Pim-1, PKC, and ATM/ATR kinases have also been implicated in the phosphorylation of histone proteins and subsequent modification of gene expression.

A further epigenetic mark that alters gene expression is ubiquitination. Lysine residues on histone proteins H2A and H2B can undergo monoubiquitination through the concerted actions of ubiquitin E2 conjugases and ubiquitin E3 ligases. Ubiquitin of histone H2A is associated with gene silencing through the involvement of repressive complexes including Polycomb repressive complex 1 (PRC1). In contrast, histone H2B ubiquitination has been suggested to act as a checkpoint for RNA polymerase activity, providing a stalling mechanism during early transcription elongation for the recruitment of Ctk1 to RNA polymerase II, and has been linked to both gene silencing and gene transcription. A further difference is that histone H2B is a pre-requisite for di- and tri-methylation of histone H3 Lys-9 (H3K9), whereas H2A ubiquitination inhibits histone lysine methylation.

DNA can also undergo methylation through different mechanisms. The addition of a methyl group to a nucleotide by DNA methyltransferases (DNMTs) occurs at the major groove of the DNA double helix, and prevents transcription by blocking the binding of transcription factors and polymerases. DNA methylation has a major involvement in embryonic development, genomic imprinting and the preservation of chromosome stability. There are two known types of DNA methylation - de novo and maintenance methylation. De novo methylation, predominantly carried out by DNA methyltransferases DNMT3A and DNMT3B, catalyzes the addition of methyl groups onto cytosine nucleotides. Since cell replication does not preserve such methylation, maintenance methylation copies these marks from the parent DNA onto the daughter DNA strands. The high affinity of DNMT1 for hemimethylated DNA in vitro suggests that this enzyme is primarily responsible for maintenance DNA methylation in vivo.

Epigenetic reader domains can be thought of as effector proteins that recognize and are recruited to specific epigenetic marks. "Writer" and "eraser" enzymes may also contain such reader domains, leading to the coordination of "read-write" or "read-erase" mechanisms. 

The structure of reader domains typically provides a cavity or surface groove in which to accommodate a specific epigenetic mark. Interactions between the reader domain and the flanking sequence of the modified amino acid also allows the reader domain-containing protein to distinguish between similar epigenetic marks, for example between histone lysine mono-, di- and trimethylation.

Proteins that contain reader domains can be broadly classified into four groups: chromatin architectural proteins, chromatin remodeling enzymes, chromatin modifiers, and adaptor proteins that recruit other machinery involved in gene expression. The first group of these, chromatin architectural proteins, bind to nucleosomes and can either directly induce chromatin compaction or alternatively act as a shield to prevent the binding of proteins involved in RNA transcription. Through self-propagation and oligomerization, these chromatin architectural proteins can exert their inhibitory effect on transcription over a significant length of DNA.

In contrast to chromatin architectural proteins, chromatin remodeling enzymes prompt a more open chromatin architecture; the increased accessibility of chromatin facilitates DNA transcription. This structural shift in chromatin architecture is driven by the energy of ATP hydrolysis. One such example is the yeast chromatin remodeling enzyme complex, RSC, which contains a tandem bromodomain within its Rsc4 subunit that recruits the complex to acetylated lysine residues on histone H3 (H3K14). RSC is involved in a number of cellular processes including nucleosome remodeling, the consequence of which is the promotion of RNA polymerase II recruitment to the underlying DNA, prompting gene transcription.

Aside from chromatin remodeling enzymes and architectural proteins, many other proteins that contain reader domains cannot directly influence chromatin architecture, but instead serve to recruit secondary chromatin modifiers to further modify chromatin or to reverse an existing chromatin modification. The corepressor complex Sin3S is recruited to methylated histone lysine residues through a Sin3 tandem bromodomain. Since histone deacetylases HDAC1, HDAC2 and HDAC3 are also found within the Sin3S complex, the recruitment of this complex prompts a secondary histone modification: histone deacetylation.

The final class of reader domain-containing proteins is adaptor proteins: the principal function of these domains is to recruit factors that are linked to DNA metabolism processes including transcription, DNA damage repair, DNA recombination, DNA replication and RNA processing. Interaction of the BRCT domain of MDC1 - a critical mediator of the DNA damage response - with a phosphorylated serine residue on histone H2AX acts as an adaptor to recruit the histone ubiquitin ligase, RNF8, to double-strand break-flanking chromatin. Subsequent histone ubiquitination itself recruits repair machinery including tumor protein p53 binding protein 1 (TP53BP1).

Epigenetic marks are not necessarily permanent modifications; instead, they can be removed by a group of enzymes known as "erasers" in order to reverse the influence of a given epigenetic mark on gene expression.

Histone acetylation is an important post-translational modification, both for histone and non-histone proteins, and has a comparable impact to phosphorylation on the regulation of cellular processes. Within epigenetics, histone acetylation is an integral mechanism for decreasing chromatin condensation, thereby facilitating gene transcription, but an equally important mechanism is the removal of acetyl groups through the actions of histone deacetylases (HDACs). HDACs can be divided into two groups termed group I and group II; group I, which is further divided into classes I, II and IV, contains zinc-dependent amidohydrolases whereas group II enzymes, also known as class III or SIRTs, are reliant upon nicotinamide adenine dinucleotide (NAD) as a co-factor.

Histone phosphatases can target either phosphorylated serine, threonine or tyrosine residues on histone proteins. Protein Ser/Thr phosphatases PP1, PP2A and PP4, amongst others, have been reported to dephosphorylate histone proteins. For example, the catalytic subunit of PP2A colocalizes with phosphorylated H2AX, a phosphorylated sequence variant of histone protein H2A that is rapidly concentrated within chromatin domains flanking DNA double-strand breaks. Phosphorylated H2AX acts as a docking site for DNA repair proteins, and is released from chromatin once double-strand breaks have been rejoined; this mechanism is thought to involve PP2A.

The removal of ubiquitin groups from histone lysine residues is catalyzed by proteases known as deubiquitinating enzymes (DUBs). These can be further categorized into groups including ubiquitin-specific proteases (USPs) and Jab1/MPN domain-associated metalloisopeptidase (JAMM) domain proteins. Both USP and JAMM family members have been shown to target histone proteins H2A and H2B, where they regulate transcription, DNA repair, gene expression and cell cycle progression. Compared to other histone modifications, the functions of histone ubiquitinaton are less well understood, yet increasing evidence points to an important role for this epigenetic modification in the DNA damage response.

The first histone demethylase to be discovered was lysine-specific demethylase 1 (LSD1), also known as KDM1. LSD1 contains an amino oxidase domain which binds the co-factor, flavin adenine dinucleotide (FAD), crucial for demethylation. A further family of lysine demethylases have since be identified, termed Jumonji C domain-containing demethylases (JMJD). The demethylases do not require FAD as a co-factor but instead are dependent on Fe²⁺/2-oxoglutarate (2-OG) for catalysis. As yet, only one enzyme with arginine demethylase activity has been identified, JMJD6, which is a 2-OG-dependent JmjC demethylase.

In contrast to the well-defined mechanisms of the removal of epigenetic marks from histones, the mechanism by which methyl groups are removed from nucleotides remains poorly understood. What is known, however, is that DNA demethylation can occur both actively and passively. Passive DNA demethylation involves the failure of maintenance DNMTs to methylate newly synthesized DNA strands during mitosis, whilst the molecular machinery that catalyzes active DNA demethylation occurs is yet to be elucidated. Since demethylation of DNA is crucial for processes such as epigenetic reprogramming in germ cells, further research into the mechanisms of active DNA demethylation may identify novel targets in stem cell research.

Epigenetic mechanisms have a key role in embryonic development; the genome of pluripotent cells is generally highly methylated while the process of differentiation is associated with a loss of DNA-methylation marks. Additionally, pluripotent cells generally have a greater incidence of chromatin in an open state, which is determined by specific histone modifications, while differentiated cells are more enriched in condensed chromatin. Epigenetic mechanisms have been also recently recognized to regulate autophagy, a homeostatic process controlling cellular components.

Abnormal frequency or location of epigenetic marks, often due to the aberrant function of DNA- or histone-modifying enzymes, has been associated with various disease states including cancer and neurodegeneration. The dysregulation of these epigenetic modifications has been shown to result in oncogenesis and cancer progression. The cell cycle, as well as proliferation and metastasis can be regulated by histone modification, DNA methylation and chromatin remodeling. For example, hypermethylation of tumor-suppressor genes has been identified as a pro-tumorigenic aberrant epigenetic mechanisms.

Neurodegenerative conditions are generally associated with aging. Environmental factors, such as smoking and diet, promote changes in epigenetic markers over time potentially leading to neuronal death. For example Parkinson's disease is associated with inhibition of histone acetylation and inhibitors of histone deacetylase (HDAC) reduce neurotoxicity. HDAC inhibitors also prevent the amyloid β-induced hyper-phosphorylation of tau protein in Alzheimer's disease and the expression of genes that are responsible for cognition loss, while in Huntington's disease, HDAC inhibitors are also beneficial.

In rheumatoid arthritis (RA) a number of epigenetic modifications have been identified as having pathogenic importance. Synovial fibroblasts in RA display a pattern of DNA methylation that is distinct from healthy fibroblasts. Similarly, CD4+ T cell hypomethylation in RA patients has been shown to result in dysregulated cell function. In T_reg cells, DNA methylation of promoter and enhancers of key genes can also inhibit the ability of these cells to suppress the ongoing inflammation.

Read the blog: The Epigenetics of Depression: How Psychological Stress Alters DNA