DNA methylation

The most frequent DNA modification involves the covalent binding of a methyl group to the cytosine bases located in CpG dinucleotides (cytosine nucleotide followed by a guanine nucleotide in the linear sequence of bases in 5′→3′ direction linked by a phosphate, 5′-C-phosphate-G-3′). This CpG is underrepresented in the genome, except for the small regions known as CpG islands – regions with a minimum length of 0.5 kb with a higher proportion of CpGs (minimum 55% GC). CpG islands are mainly located in the proximal promoter regions of almost half of all human genes (28), (29). According to the UCSC human version GRCh37/hg19, 28 691 CpG islands are scattered throughout the genome. Interestingly, chromosome 19 is the one with the highest number of CpG islands (n=2541) while chromosome Y harbors the lowest number (n=181). A feasible explanation for this may lie in the fact that chromosome 19 is rich in biological and evolutionary significance with the highest gene density of all human chromosomes. In addition, the GC content of the chromosome is unusually high (48% vs. 41% of the median whole human genome) with two-thirds of genes having at least one CpG island (30). In contrast, chromosome Y has evolved by a gene loss leading to a paucity of genes, which correlates with the paucity of CpG islands (31).

DNA methylation is the major modification of eukaryotic genomes related to downregulation of gene expression. The overall methylation grade of CpGs in mammals can fluctuate at different developmental stages or under different pathological conditions. CpGs are mostly unmethylated at the early stages of development, allowing the expression of a particular gene if the appropriate transcription factors are present and the chromatin structure is accessible (32). When development progresses, DNA methylation helps to restrict embryonic lineages and prevent regression to an undifferentiated state (33). In non-embryonic cells, around 80% of the CpG residues that do not form a CpG island are methylated (34), while CpG islands are mostly unmethylated at all times (28).

In some pathological conditions, such as human cancers, two epigenetic events have been observed (35): global hypomethylation of the CpG residues that do not form a CpG island is linked to genomic instability, which may take the form of activation of mitotic recombination or activation of transposons (36), (37); and hypermethylation of promoter areas, especially CpG islands, associated with the corresponding silencing of tumor suppressor genes (38), (39). miRNA genes have been frequently found in the cancer-associated genomic regions (40) and can play two opposing roles in this disease, either as oncogenes or as tumor suppressors (24). Several studies have shown that tumor suppressor miRNAs, such as miR-203, can be silenced by aberrant methylation of CpG islands adjacent to their promoters (41), (42). miR-203 can be inactivated in human tumors by both genetic and epigenetic mechanisms. In hematopoietic malignancies, including chronic myelogenous leukemia, its methylation enhances ABL1 and BCR-ABL1 oncogene expression and its re-expression dramatically reduces the proliferation of tumor cells in an ABL1-dependent manner (43).

The frequency of human miRNA gene methylation is nearly one order of magnitude higher than that of the protein-encoding genes (44). To further shed light on this fact, we depicted the genomic distribution of human miRNAs (obtained from miRbase v21) (45) throughout the chromosomes and merged this chromosome map with the CpG island genomic density using karyoploteR (46) (Figure 1A). The results revealed the hotspots for miRNA methylation in chromosomes 1, 2, 9, 11, 16, 17 and 19, which are in accordance with a study by Kunej et al. (47). Among these chromosomes, chromosome 19 stands for which has the highest number of miRNAs overlapping a CpG island (n=19). Remarkably, using regioneR (48), which assesses the relation between genomic regions using a permutation test, we observed that a higher proportion of miRNA genes than expected by chance is embedded in CpG islands susceptible to methylation (189 of 1881; z-score=46.13; number of permutations=1000; p-value=0.0009, Figure 1B and C, Table 1 and shown in red in Figure 1A), which might explain why certain miRNAs are more prone to be epigenetically regulated by methylation than others. Furuta et al. (49) studied the methylation grade of CpG islands close to 39 miRNA genes in hepatocellular carcinoma cell lines and found that miR-124-1, miR-124-3, miR-203 and miR-375, which were completely embedded in a CpG island, underwent methylation-mediated silencing (Figure 2A).

Figure 1:

MicroRNAs overlapping CpG islands.

(A) MicroRNAs and CpG island distribution on different chromosomes. Chromosomes are represented as horizontal rectangles, in which red rectangles indicate the centromeres. Green density graph shows the distribution of CpG islands above chromosomes (UCSC human version GRCh37/hg19). Gray bars projected above the chromosomes indicate the genomic positions of the 1881 miRNAs (miRbase v21), 189 of which are depicted by red bars to highlight the miRNAs whose genomic position overlaps a CpG island. (B) Permutation test analysis (RegioneR, Bioconductor) showing association between miRNAs and CpG islands. The overlapPerm Test function was used for calculation with the following parameters: per.chromosome=FALSE and count.once=FALSE. (C) Local z-score graph to evaluate the strength of the association. The graph shows that the observed association is highly dependent on the exact position.

Figure 2:

Summary of the different mechanisms involved in the methylation-dependent downregulation of the miRNA genes described in several cell lines, murine models and tumor tissues.

(A) miRNA gene overlapping a CpG island. (B) miRNA with a promoter region enriched in CpG residues susceptible to methylation. (C) miRNA cluster with a CpG island 2 kb upstream of its transcription start site (TSS). (D) Intronic miRNA whose transcription is regulated by the CpG island located in the promoter of the host gene. (E) Intronic miRNA whose transcription is independent of the host gene but is dependent on its own independent promoter. (F) miRNA whose expression is regulated by a distant enhancer that can be methylated. (eDMR, enhancer differential methylated region).

miRNAs can be classified into two broad categories according to their genomic region: intergenic and intragenic (50), (51). Intergenic miRNAs (Figure 2A–C) are located in the regions between annotated genes, while intragenic miRNAs (Figure 2D and E) are located within exons (exonic miRNAs) or introns (intronic miRNAs) of the protein-coding genes. In humans, according to the miRIAD database (52), a total of 1157 (61.5%) miRNAs are intragenic (169 exonic and 988 intronic), while 724 (38.5%) are intergenic.

The transcription of intergenic miRNAs is independent of coding genes as they are transcribed mostly by RNApol III (50), (51). They have their own transcription regulatory elements, including promoter, transcription start site (TSS) and terminator signals. Three patterns of epigenetic regulation of intergenic miRNAs have been observed. Firstly, as described above, a miRNA gene can directly overlap a CpG island, as is the case of miR-203 (49) (Figure 2A). Secondly, a miRNA can have a promoter region that is enriched in CpG residues but is not a CpG island, such as miR-34a, whose expression levels are modulated by methylation of the CpG residues located in the region comprising up to 2.5 kb of its TSS in its promoter (53) (Figure 2B). Finally, a miRNA can have a CpG island upstream of its TSS, such as the miR-17-92 cluster located in Chr13, which has a CpG island ~2 kb upstream of its TSS (54) (Figure 2C). Most of the CpG islands that are located close to the miRNA’s TSS are involved in their transcription regulation. However, the maximum distance to the TSS from where a CpG island can regulate the miRNA transcription has not yet been defined and probably varies according to the size of each miRNA promoter. Our group performed an in silico analysis in order to determine the distances between pri-miRNAs and their nearest CpG islands in a range of distances from 1 to 10 000 bp (Figure 3). Like most authors, we considered only the CpG islands located at maximum 2000 bp away from the TSS of the analyzed gene. Among 1881 miRNAs analyzed, 116 had a CpG island within 1000 pb upstream of their promoters, 51 (44%) of which were intergenic miRNAs, and 80 miRNAs had a CpG island within 1001–2000 pb, 23 (29%) of which were intergenic.

Figure 3:

Number of miRNAs with an upstream CpG island located close to the gene.

A range of distances (1–10 000 bp) is shown and the number of intragenic and intergenic miRNAs is indicated for each distance.

Conversely, intragenic miRNAs, located in the same DNA strand and coexpressed with their host genes, are usually transcribed by RNApol II (55) (Figure 2D). However, some studies have found a low level of correlation between the expression of a miRNA and that of its host gene (56), (57), which is not what might be expected if their transcription is regulated by the same promoter. A possible explanation, as proposed by Ozsolak et al. (58) and Monteys et al. (59), could be that a considerable percentage of intronic miRNAs [~35%, using miRBasev9 (58) and v12.0 (59)] are associated with Pol II or Pol III promoters that could drive transcription independently of their host gene promoter (Figure 2E). More recently, Marsico et al. (60), using their ProMIRNA tool (http://promirna.molgen.mpg.de), detected up to 50% of intragenic miRNAs regulated by their own independent promoters (mirBase v 18.1). These authors also performed an analysis to identify the unique features of intronic miRNA promoters, such as miR-126 (Figure 2E). The results showed that (1) the CpG content changed according to the promoter type, where the intronic independent miRNA promoters were less enriched in CpGs than the host gene promoters; (2) intronic promoters were usually narrow and enriched in TATA box elements; and (3) intronic promoters tended to be regulated by a different set of transcription factors enriched in tissue-specific master regulators. The authors concluded that the intronic independent miRNA promoters may modulate miRNA expression in a tissue or cellular state-specific manner and that the observed differences in the regulatory elements point toward a different evolution of the two promoter classes.

Although most studies analyzing the role of methylation in miRNA regulation have focused on promoter regions and CpG islands, there is some evidence that methylation of distant regions of the genome, such as enhancers, can also modulate miRNA expression, as is the case of miR-200a/b and miR-9-1 (61) (Figure 2F). miR-9-1, in uterine and head and neck cancers, and miR-200a/b, in breast and colon cancer, respectively, are upregulated as a consequence of the hypomethylation of an enhancer differential methylated region (eDMR). In both cases, their functions involve E-cadherin and the epithelial-mesenchymal transition that is crucial for metastasis (62), (63). Methylation has the same effect on enhancers as on CpG islands, inhibiting their activity. Recently, it has been observed that CpG islands located far from any known transcript, known as orphan CpG islands, can display features of active enhancers with the presence of chromatin features H3K4me1 and H3K27Ac (64).

DNA hydroxymethylation

DNA hydroxymethylation is a recently discovered modification of CpG dinucleotides that involves the addition of a hydroxyl group on 5-methylcytosines (5mCs) to produce 5-hydroxymethylcytosine (5hmCs) (65), (66). This modification, mediated by the TET family of proteins (Tet1, Tet2, Tet3), is an intermediate step in the process of active demethylation of 5mCs, and as such, it is responsible for enhancing transcriptional efficiency (67) and is essential in a range of biological processes, such as embryonic development, stem cell function and cancer formation (68), (69).

The 5hmC marks are found at reduced levels (~1%) compared to the 5mC levels (4–5%) in the human genome. However, like 5mC alterations, the 5hmC patterns undergo considerable changes linked to genome instability (70), (71) across several forms of human cancers (72). Recent studies revealed that 5hmC is consistently found at significantly reduced levels (more than 50% reduction, p≤0.01) in various solid tumors (73), (74), (75). Recently, using a mouse model, Pan et al. (76) studied the role of hydroxymethylation in neurophysiological processes and provided the first evidence that miRNAs can be regulated by hydroxymethylation of their promoters. Working with a mouse model of formalin-induced acute inflammatory pain, they observed a significant increase in 5hmC and a decrease in 5mC in the miR-365-3p promoter that resulted in the enhancement of miR-365-3p transcription and in the subsequent increase of miR-365-3p expression (Figure 4). The TET-mediated hydroxymethylation of miR-365-3p played a key role in the regulation of nociceptive behavior through targeting the potassium channel KCNH2. This epigenetic modification in the nociceptive pathway contributes to pain processes and analgesia response. To date, the effect of hydroxymethylation of miRNAs has not been explored in humans, but given the findings in mouse models, it seems to warrant investigation in humans as well.

Figure 4:

Hydroxymethylation (hm) of the miR-365-1 promoter enhances its transcription in mice.

When the promoter is unmethylated, the miRNA shows a baseline transcription that can be inhibited by the methylation (m) of the promoter by DNMT enzymes. The intermediate step in the demethylation of the promoter, mediated by the TET enzymes, produces a hm of the promoter that leads to enhanced transcription of the miRNA. DNMT, DNA methyl transferase; TSS, transcription start site.

PTM of histones

Repression of gene transcription can be associated with PTM in the amino terminal coils of histones, which leads to chromatin modifications impacting gene expression, usually by altering the chromatin structure. Chromatin is integrated by nucleosomes, which consist of DNA wrapped twice around a histone octamer, each containing two copies of four highly conserved histones (H2A, H2B, H3, H4), which are susceptible to PTMs, including methylation, acetylation, phosphorylation, ubiquitination, biotinylation, sumoylation and ADP-ribosylation (77), (78). In fact, the most well-studied types of modifications are lysine acetylation and methylation. While lysine acetylation can relax the chromatin structure and enhance transcription activation, lysine methylation can cause different effects on gene expression, depending on the positions and degree of methylation. Methylation at H3K4, K36 and K79 is associated with gene activation, whereas methylation at H3K9, H3K27 and H4K20 correlates with transcriptional repression (77), (78).

The EpimiR database (79) is a comprehensive repository that includes all histone modifications affecting miRNA expression in different species, including humans, described until 2013. Table 2 lists all miRNAs whose expression is regulated by lysine acetylation or methylation, based on EpimiR information (‘high confidence’ criterion) and a review of the literature (January 2013–June 2017; references are shown in Supplementary File 1). Table 2 includes well-known oncogenic miRNAs, such as miR-21, which has been shown to play an oncogenic role in most tumors by blocking cell differentiation and by promoting proliferation. For example, Zhou et al. (80) demonstrated that RBP2, a histone 3 lysine 4 (H3K4) demethylase, downregulated miR-21 expression by reducing H3K4 trimethylation at the proximal promoter region of miR-21 in chronic myeloid leukemia cells. Table 2 also includes tumor suppressor miRNAs, such as miR-34a, which is known to inhibit cell proliferation and induce cell cycle arrest, apoptosis and senescence, as well as to increase chemosensitivity. EZH2, a histone 3 lysine 27 (H3K27) methyltransferase, is a major player in the silencing of miR-34a in pancreatic ductal adenocarcinoma. Inhibition of EZH2 upregulated miR-34a expression in pancreatic ductal adenocarcinoma cells, while EZH2 overexpression repressed miR-34a expression. EZH2 was specifically guided to the promoter region by HOTAIR, a long non-coding RNA, to catalyze H3K27 trimethylation of the miR-34a promoter. The authors concluded that the identification of HOTAIR-guided miR-34a silencing opened a new avenue in miR-34a-oriented therapy against pancreatic ductal adenocarcinoma (81).

Table 2:

Different post-translational modifications (PTMs) of histones affecting miRNA genes and their subsequent effect on gene expression.

PTM	Effect on miRNA expression	miRNAs
H3ac		let-7a-1, let-7d, let-7f-1, miR-9-1,miR-10a, miR-10b, miR-17, miR-18a,miR-19a, miR-19b, miR-20a, miR-22, miR-23a, miR-26a, miR-30a, miR-92, miR-124a, miR-126,miR-129-2, miR-130b, miR-137,miR-199a-2,miR-200a, miR-205,miR-223, miR-376a, miR-512-5p
H4ac		let-7a-1, let-7d, let-7f-1, miR-23a, miR-26a, miR-29, miR-30a, miR-124a, miR-223
H3K4ac		miR-29b
H3K9ac		miR-21, miR-29b, miR-132, miR-139, miR-212,miR-224, miR-452
H3K14ac		miR-21, miR-224, miR-452
H3K4me2		miR-15a, miR-16, miR-29b, miR-132
H3K4me3		let-7a-1, let-7d, let-7e, let-7f-1,miR-10a, miR-10b, miR-17, miR-18a, miR-19a,miR-19b, miR-20a, miR-21,miR-23a, miR-26a, miR-30a, miR-31, miR-92a-1, miR-150, miR-193b, miR-199a-2, miR-200b/200a/429, miR-200c/141, miR-205, miR-223, miR-365, miR-451
H3K9me2		miR-9-3, miR-96, miR-182, miR-183, miR-199a-2, miR-205, miR-212, miR-302, miR-302c
H3K9me3		miR-124a, miR-329, miR-622
H3K27me3		let-7b, miR-9, miR-10a, miR-10b, miR-21, miR-22, miR-23a, miR-26a, miR-27a, miR-29, miR-31, miR-34a, miR-34b/c, miR-101, miR-124a, miR-139, miR-143, miR-150, miR-181a/b,miR-193b, miR-199b, miR-200b/200a/429, miR-200c/141, miR-205, miR-212, miR-223, miR-329, miR-365, miR-429, miR-449a,miR-449b, miR-451, miR-568, miR-622,miR-1275

1. All modifications included in this table are in lysines.

2. K, lysine; ac, acetylation; me, methylation; me2, dimethylation; me3, trimethylation.

These findings highlight the relevance of the transcriptional regulation of miRNAs mediated by changes of the chromatin states through modifications in the methylation/acetylation patterns of amino terminal coils of histones. Taken together, they suggest new avenues of research on drugs targeting genes involved in the PTM of histones that could affect miRNA expression.