DNA Methylation – Introduction

Epigenetics is defined as heritable changes in gene activity and expression that occur without alteration in DNA sequence. Histone modifications have been shown to function as a code that coordinates the recruitment of effector proteins, and regulates chromatin structure, gene expression, and genome stability. DNA methylation is a biochemical process where a methyl group is added to the cytosine DNA nucleotides. Maintenance of DNA methylation by DNA methyltransferases is essential for normal mammalian development and long-term transcriptional silencing. The conversion of 5-methylcytosine to 5-hydroxymetyhylcytosine by ten–eleven translocation enzymes (TETs) is the initial step of active DNA demethylation. Many diseases have been associated with differentially methylated regions, and demethylating agents or methyl donors are evaluated as epigenetic therapies.

Keywords

epigenetics; histone modifications; DNA methylation; DNA methyltransferases; methyl-binding domain proteins; TET enzymes; DNA hydroxymethylation; nutriepigenomics

Outline

1.1 Epigenetics

In this first chapter we introduce the concept of epigenetics, especially the members of the DNA methylation machinery. Epigenetics is the study of cellular and physiological traits that are not caused by changes in the DNA sequence. Epigenetics describes the study of stable, long-term alterations in the transcriptional potential of a cell, but also can induce transient changes. Some of those alterations are heritable. For example, during embryogenesis, totipotent stem cells become the various pluripotent cell lines of the embryo, which in turn become fully differentiated cells. This process is regulated by epigenetics [1].

1.2 Histone Modifications

Histones are the core protein components of chromatin complexes and they provide the structural backbone around which DNA wraps at regular intervals, generating chromatin. The nucleosome represents the first level of chromatin organization and is composed of two of each of histones H2A, H2B, H3, and H4, assembled in an octameric core with DNA tightly wrapped around the octamer [2]. The first epigenetic mechanism is the post-translational modification of the amino acids that make up histone proteins. If the amino acids in the chain are changed, the shape of the histone might be modified. DNA is not completely unwound during replication, and thus it is possible that the modified histones may be carried into each new copy of the DNA. Once there, these histones may act as templates, initiating shaping of the surrounding new histones in the new manner. By altering the shape of the histones around them, these modified histones ensure that a lineage-specific transcription program is maintained after cell division. Although histone modifications occur throughout the entire sequence, the histone tails are particularly highly modified. These modifications include acetylation, methylation, ubiquitylation, phosphorylation, sumoylation, ribosylation, and citrullination, of which acetylation and methylation are the most highly studied.

1.2.1 Histone Acetylation and Deacetylation

Histone modifications are linked to essentially every cellular process requiring DNA access, including transcription, replication, and repair. Histone acetylation is carried out by enzymes called histone acetyltransferases (HATs) that are responsible for adding acetyl groups to lysine residues on histone tails, while histone deacetylases (HDACs) are those that remove acetyl groups from acetylated lysines [3,4]. For example, acetylation of the K14 and K9 lysines of the tail of histone H3 by HATs is generally related to transcriptional competence. The presence of acetylated lysine on histone tails leads to a relaxed chromatin state that promotes transcriptional activation of selected genes; in contrast, deacetylation of lysine residues leads to chromatin compaction and transcriptional inactivation [5].

1.2.2 Histone Methylation

Histone methylation is a process by which methyl groups are transferred to amino acids of histones. Depending on the target site, methylation can modify histones so that different portions of chromatin are activated or inactivated. In most cases methylation and demethylation of histones turn the genes in DNA “off” and “on,” respectively, either by loosening or encompassing their tails, thereby allowing transcription factors to access or blocking them from accessing the DNA. Trimethylation of histone H3 at lysine 4 (H3K4me3) is an active mark for transcription [6]. However, dimethylation of histone H3 at lysine 9 (H3K9me2) is a signal for transcriptional silencing [7]. Histone methyltransferases (e.g., SUV4-20H1/KMT5B, SUV4-20H2/KMT5C, ATXR5) or demethylases (UTX/KDM6A, JMJD3/KDM6B, JMJD2D/KDM4D) actively add or remove various methylation marks in a cell type-specific and context-dependent way [8,9]. SET domain lysine methyltransferases (SUVAs/KMTs) catalyze the site- and state-specific methylation of lysine residues in histone and non-histone substrates [10]. These modifications play fundamental roles in transcriptional regulation, heterochromatin formation, X chromosome inactivation, and DNA damage response, and have been implicated in the epigenetic regulation of cell identity and fate.

1.3 DNA Methylation

The second epigenetic mechanism is the addition of methyl groups to the DNA, mostly at CpG sites, to convert cytosine to 5-methylcytosine (5-mC). 5-mC performs much like a regular cytosine, pairing with a guanine in double-stranded DNA. Transcription of most protein coding genes in mammals is initiated at promoters rich in CG sequences, where cytosine is positioned next to a guanine nucleotide linked by a phosphate called a CpG site. Such short stretches of CpG-dense DNA are known as CpG islands. In the human genome, 60–80% of 28 million CpG dinucleotides are methylated [11]. The chromatin structure adjacent to CpG island promoters facilitates transcription, while methylated CpG islands impart a tight compaction to chromatin that prevents onset of transcription and, therefore, gene expression. Some areas of the genome are methylated more heavily than others, and highly methylated areas tend to be less transcriptionally active. Methylation of cytosines can also persist from the germline of one of the parents into the zygote, marking the chromosome as being inherited from one parent or the other; this is called genetic imprinting [12]. DNA methylation frequently occurs in repeated sequences, and helps to suppress the expression and mobility of transposable elements such as LINE-1 [13]. DNA methylation is associated with histone modifications, particularly the absence of histone H3 lysine 4 methylation (H3K4me0) and the presence of H3 lysine 9 dimethylation (H3K9me2) [14].

1.3.1 DNA Methyltransferases

DNA methylation patterns are known to be established and modified in response to environmental factors by a complex interplay of at least three independent DNA methyltransferases (DNMTs): DNMT1, DNMT3A, and DNMT3B [15]. These catalyze the methyl group transfer from S-adenosyl-L-methionine to cytosine bases on the DNA [16].

By preferentially modifying hemimethylated DNA, DNA methyltransferase 1 (DNMT1) transfers patterns of methylation to a newly synthesized strand after DNA replication; it is therefore often referred to as the “maintenance” methyltransferase [17]. DNMT1 is essential for proper embryonic development, imprinting, and X-inactivation [18].

DNMT3 is a family of DNA methyltransferases that can methylate hemimethylated and unmethylated CpG at the same rate. The architecture of DNMT3 enzymes is similar to that of DNMT1, with a regulatory region attached to a catalytic domain [19]. There are three known members of the DNMT3 family: DNMT3A, 3B, and 3L. DNMT3A and 3B can mediate methylation-independent gene repression, while DNMT3A can co-localize with heterochromatin protein (HP1) [20] and methyl-CpG-domain binding proteins (MBDs). They can also interact with DNMT1, which might be a co-operative event during DNA methylation. DNMT3L contains DNA methyltransferase motifs and is required for establishing maternal genomic imprints, despite being catalytically inactive. DNMT3L is expressed during gametogenesis when genomic imprinting takes place, but also plays a role in stem cell biology [21].

1.4 Methyl-Binding Domain Proteins

DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene; second, and likely more important, methylated DNA may be bound by proteins known as MBDs [22]. MBDs then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins...