Fundamental to epigenetic regulation is the intricate organization of each cell’s genome into chromatin (Chap. 456). The fundamental unit of the packaging into chromatin is the nucleosome, consisting of 147 base pairs of DNA wrapped around an octamer of 8 histone proteins (two copies of each of the four core histone proteins: H2A, H2B, H3, and H4). The level of compaction of this chromatin structure determines the accessibility of the DNA strand to transcription factors, the DNA repair machinery, and other DNA-binding entities. Thus, compaction has a profound influence on gene expression levels and on local DNA mutation rates. Open regions of chromatin (euchromatin) tend to be transcriptionally active, whereas compacted chromatin (heterochromatin) tends to be transcriptionally repressed.
Histones include the four core histones, which are the most abundant and most frequently found throughout the genome, and variant histones of H2A, H2B, and H3. The structure of core and variant histones include amino- and carboxyl-terminal “tails,” which are extended and unstructured, and highly conserved globular domains. The x-ray crystal structure of the nucleosome particle has illuminated the dynamic alterations of chromatin by an astounding range of regulatory mechanisms, summarized below.
The three main processes that regulate chromatin compaction, and thus access to the DNA template, include direct methylation modifications of the DNA strand itself, post-translational modifications of histones, and remodeling of nucleosomes to alter their location and composition with variant histones (Fig. 471-1). The major modification of DNA is cytosine methylation of CpG dinucleotides (5-mC), associated with gene repression and catalyzed by the DNMT1, DNMT3A, and DNMT3B enzymes. DNMT3A and 3B catalyze the addition of methyl groups on unmethylated DNA de novo at CpG dinucleotides that are typically located throughout transcribed genes and in intergenic regions, but lacking at promoters, while DNMT1 is critical for the maintenance of the methylation state after DNA replication and after transcription during the S phase of the cell cycle. To further alter and reverse methylation, the TET enzymes (TET1–3) progressively oxidize 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), which are unable to be recognized by DNMT1 but can be removed by additional enzymes. Hence, these are mechanisms to passively lose 5-mC following DNA replication, or to actively remove 5-mC, both returning to unmethylated cytosine.
Epigenetic pathways influence multiple physiologic and disease pathways. As depicted in the center of the illustration, epigenetics refers to the chemical modifications of DNA and histones, which influence chromatin structure, gene expression, and susceptibility to mutations. These molecular pathways, in turn, play important roles in development, cancer, metabolism, aging, neural function, and behavior, and in the immune system.
Histone post-translational modifications (hPTMs) are rich sources of diverse signaling to, and marking of, the chromatin template, including at least 60 different covalent chemical modifications on the histone N- and C-terminal tails and within the globular domains. The hPTMs are added (written) and removed (erased) by enzymes, and also serve as sequence- and PTM-specific binding surfaces for effector proteins and complexes (readers) to carry out a wide range of downstream actions including transcription, replication, and DNA repair and recombination. One key point is that the staggering numbers of writers, erasers, and readers provide unlimited potential for diagnostic and therapeutic pharmacologic discovery.
Throughout this chapter, we focus on histone methylation and acetylation, the most abundant and the most well-studied hPTMs (Fig. 471-1), although several others, such as serine/threonine/tyrosine phosphorylation, lysine ubiquitination, lysine SUMOylation, and lysine ADP-ribosylation also play important roles in epigenetic regulation. For instance, histone phosphorylation targets histone H2A at Ser139 (γH2A.X), which marks DNA double-strand breaks immediately following damage and is critical for the recruitment of the DNA repair machinery. Histone mono-ubiquitination functions similarly to other hPTMs, in signaling and marking the chromatin template, in particular serving to mark the initiation region or elongation of transcribed genes for future rounds of transcription, whereas histone SUMOylation plays a role in transcriptional repression. Poly-ubiquitination serves to tag proteins for degradation by the proteasome, and dysfunction in this system may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease. ADP-ribosylation involves a class of enzymes, the poly-ADP-ribose polymerases (PARPs), which transfer ADP-ribose units from NAD+ to a variety of nuclear proteins. This PARylation alters the chromatin environment through the recruitment and modification of chromatin-associated proteins. In general, future studies of the wealth of types and functions of hPTMs will enhance our understanding of these chromatin-based mechanisms and processes and will illuminate new opportunities and targets for therapies.
In contrast, there is extensive understanding of histone lysine acetyltransferases (KATs) and methyltransferases (KMTs). KATs, previously known as HATs, were among the first identified histone modification enzymes. They attach acetyl groups on the lysine residues of histone tails and other proteins, resulting in both a novel side chain (acetyl-lysine) and an increase in negative charge (from positive charged lysine to neutral acetyl-lysine). This alteration results in loosening of chromatin structure to become more permissive to the binding of transcription factors, and it also creates a novel binding surface for the association of reader proteins. Acetylation on core histones, such as lysine 9 on histone H3 (H3K9ac) or lysine 27 (H3K27ac), is typically associated with transcriptional activation. Acetylation is very dynamic and can be rapidly removed by histone deacetylases (HDACs), of which there are multiple classes, including HDACs and sirtuins (NAD-dependent deacetylases), which return the lysine to unmodified ground state.
Methylation of histone tails by KMTs provides more nuanced regulation, in that particular methylated lysines are associated with transcriptional activation (e.g., H3K4me3, H3K36me3, H3K79me3), transcriptional repression (e.g., H3K27me3), or DNA repeat and centromeric silencing (e.g., H3K9me3). The output is strictly determined by effector protein binding, as methylation of lysine does not alter side chain electrostatic charge. Lysine methylation is a more stable chemical modification than is acetylation, and, while demethylases are identified for some of the specific methylated sites (H3K4, H3K9, H3K36, H3K27), it is provocative that H3K79 and H4K20 demethylases have not yet been discovered.
Frequently coordinating with histone modification enzymes are nucleosome remodeling enzymes, which use the energy derived from the hydrolysis of ATP to reposition and remove nucleosomes along the DNA template, and to exchange core histones and variant histones (including variants that are located at the transcriptional initiation sites [H2AZ] and over the transcribed genes [H3.3]). These complexes activate or repress transcription. The SWI/SNF family creates nucleosome-free regions for transcriptional activation, the ISWI family evenly spaces nucleosomes to repress transcription, and the INO80 family exchanges H2A with H2AZ at transcription start sites to poise transcriptional activation. Other remodeling complexes play key roles in the DNA damage response and apoptosis, among additional genomic processes.
Because multiple enzymes redundantly write, erase, and recognize many of the hPTMs, there is great complexity and the potential for fine-tuning of gene regulation. While extensive knowledge gaps remain to fully explicate mechanisms of chromatin regulation, epigenetics has become a fully established discipline within biomedical research. In the coming years, it is likely that the basic understanding of these processes will be harnessed for further betterment of human health.