DNA Methylation

DNA methylation is the classic and probably best studied epigenetic modification. Thereby, methyl marks are added to the cytosine ring of cytosine-phosphatidyl-guanine (CpG) dinucleotides. In addition, recently also non-CpG, but CHG or CHH, where H stands for A, C, or T, DNA methylation has been found to occur.³ This type of DNA methylation seems to be important mainly in pluripotent stem cells and disappears during differentiation. CpG methylation of DNA preferably is found at sites with a high percentage of CpGs, so-called CpG islands. Recently however, differences in the methylation pattern between tissues were found to occur frequently not at CpG islands, but at regions with lower CpG density that lie around 2 kb upstream or downstream of CpG islands, so-called CpG island shores.⁴ At both locations, DNA methylation leads to transcriptional repression.

During embryonic development, de novo methylation marks are established by DNA methyltransferase (DNMT) 3a and 3b. During somatic cell replication, methylation marks are stable and are maintained by DNMT1. Gene silencing by DNA methylation is crucial for the formation of heterochromatin. It promotes stable silencing of repetitive sequences such as alu sequences and retrotransposons, genomic imprinting, X chromosome inactivation in female mammals, and tissue-specific gene expression. However, in recent years it has become clear that DNA methylation is more dynamic than was previously assumed. Global genomic demethylation is seen in the paternal pronucleus shortly after fertilization, and gene-specific demethylation has been observed.⁵ Promoters of estrogen-responsive genes, for instance, are periodically methylated and demethylated depending on ligand binding of the estrogen receptor α.^6,7 Methylation marks are believed to be removed passively (e.g., DNMT1 does not restore methyl marks after replication) or actively. Different mechanisms have been proposed to account for active DNA demethylation; however, as yet definite experimental proof in mammalian cells exists for none of them.⁸ In short, conceivable mechanisms for active DNA demethylation include enzymatic removal of the methyl group, excision and substitution of 5-meC by C, oxidative demethylation thereby converting 5-meC to 5-hydroxymethylcytosine, and demethylation by S-adenosylmethionine (SAM) domains, forming a 5-meC radical.

DNA methylation can directly interfere with binding sites of transcription factors, thereby inhibiting induction of gene expression, or transcriptional repressors can bind to methylated DNA, blocking transcription. By their presence, they inhibit the binding of transcription factors and recruit histone deacetylases, which change the histone code and further block gene expression (Figure 22-1).

Figure 22-1 DNA methylation. Generally, DNA gets methylated at the pyrimidine ring of the cytosine. The methyl mark leads to changes in the chromatin structure and recruits effector molecules. A, adenine; C, cytosine; G, guanine; T, thymine.

Histone Modifications

Within the nucleus, DNA is packaged around an octamer comprising homodimers of four different histone proteins: H2A, H2B, H3, and H4. This so-called nucleosome is stabilized and kept in place by H1. Each histone protein has an N-terminal tail, which is the main target of a variety of histone modifications. Amino acids in the histone tails can be acetylated, methylated, phosphorylated, ubiquitinated, SUMOylated, ADP ribosylated, and deiminated (Figure 22-2). Not all of these modifications are equally well studied, and detailed knowledge about their regulation and interactions is still missing. The sum of the different histone modifications at a distinct region of the genome is called the histone code. It is generally believed that the histone code can influence transcriptional activity directly by affecting chromatin structure, thereby making it more or less accessible for transcription factors and indirectly leading to the attraction of effector molecules that in turn recruit and stabilize the transcription machinery.⁹ For most histone modifications, it is yet impossible to predict whether they enhance or restrict transcriptional activity and how this will influence the biologic output. Also, more and more accumulating evidence indicates that the different histone modifications are strongly interconnected and can enforce or counteract each other. Nevertheless, some modifications can clearly be associated with transcriptionally active or inactive chromatin (Table 22-1).

Figure 22-2 Histone modifications. The different histones get modified at specific amino acids in their tails. These modifications can change the chromatin structure or can lead to the binding of effector molecules. K, lysine; R, arginine; S, serine; T, threonine. Modifications: Ac, acetylation; Cit, citrullination; Me, methylation; P, phosphorylation; SUMO, SUMOylation; Ub, ubiquitination.

Table 22-1 Epigenetic Modifications

Epigenetics and Noncoding RNAs

Noncoding RNAs (ncRNAs) are transcribed RNA molecules that do not translate into protein. Long known ncRNAs include transfer RNA (tRNA) and ribosomal RNA (rRNA). More interesting with regard to epigenetic modifications, however, are the more recently discovered long ncRNAs such as the Xist (X inactive specific transcript) transcript and small ncRNAs such as microRNAs (miRs). In female cells, Xist is transcribed from the later inactivated X chromosome (Xi). It specifically binds to Xi and promotes its silencing by histone ubiquitination, methylation, and loss of acetylation.³⁰ Recently, another long ncRNA named Air has been shown to interact with a specific set of genes and to recruit the chromatin-modifying machinery to these loci.³¹ In plants and yeast, multiple examples of interaction between ncRNAs and epigenetic chromatin modifications have been found.^32–34 Overall these data suggest that recognition of complementary DNA sequences by ncRNAs might guide epigenetic modifications to specific genomic loci.

MicroRNAs are small ncRNAs that are encoded in intergenic regions and overlapping introns or exons of other genes.³⁵ The primary miR transcript (pri-miR) containing the information of a single miR or an miR cluster is further processed by an enzymatic complex that includes the RNase III enzyme Drosha. After formation of a hairpin, the precursor miR (pre-miR) is exported to the cytoplasm (Figure 22-3). Pre-miRs are further cleaved by the RNase III enzyme Dicer and are released as short duplexes, on average 22 oligonucleotides long, which constitute the mature miR. From this duplex, two mature miRs can be formed. However, dependent on the thermodynamic stability of each end, in some cases one of the strands is fast degraded; this is usually called the miR star (*) or passenger strand.³⁶ The other strand, termed the guide strand, is more stable and biologically active. In other cases, both strands are equally stable, and the miR from the 5′ end is referred to as 5p, whereas the miR from the 3′ end is denominated 3p. After incorporation into the RISC (RNA-induced silencing complex), mature miRs bind to the 3′ untranslated region of their target mRNA and modulate their expression by degradation or by inhibition of protein translation. The system is highly promiscuous, so that one miR can modulate expression of multiple proteins, and expression of one protein is influenced by a variety of different miRs. Mainly the miR sequence from base 2 to base 8 or 9 from the 5′ end, the so-called seed region, plays a crucial role in target recognition.³⁷ In addition, the position of the complementary sequence on the gene and the presence of other miR target sites influence the repressive action of miRs.³⁸ The expression of around 30% of all genes is believed to be regulated via miRs.³⁹

Figure 22-3 MicroRNA biogenesis. MicroRNAs are coded in exonic, intronic, or intergenic regions of the genome. They are transcribed by RNA polymerase II individually or in clusters. Drosha and Dicer are RNase III enzymes that further process the primary transcript (pri-miR) and the precursor miR (pre-miR), respectively. The resulting miR-Duplex is loaded onto the RNA-induced silencing complex (RISC), where the separated strands bind their target mRNA and induce its degradation or inhibit its translation.