DNA methylation is one of the key epigenetic mechanisms essential for transcriptional regulation, silencing of transposable elements, and genome stabilization. Under physiological conditions, DNA methylation is erased and then established genome-wide during gametogenesis and embryogenesis. De novo DNA methylation by the enzymatic reaction of the de novo DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, occurs during the establishment of DNA methylation patterns specific to each germ cell type or somatic cell type after the erasure. Once cell type-specific DNA methylation patterns are established during embryogenesis, which can extend to early childhood, the maintenance of DNA methyltransferase DNMT1 and its cofactor UHRF1 cooperatively maintain the pattern throughout the individual’s lifetime. Recently, our group found that UHRF1 is also involved in de novo DNA methylation during oogenesis. Moreover, our group has identified two genes, CDCA7 and HELLS, to be the causative genes of ICF syndrome, characterized by hypomethylation of centromeric and pericentromeric repetitive sequences. Because CDCA7/HELLS comprise a chromatin remodeling complex, there are evidently certain regions where chromatin remodeling is required to achieve maintenance of DNA methylation. In this chapter, the current situation with respect to our understanding of de novo and maintenance of DNA methylation mechanisms under physiological conditions in mammals is summarized.
- de novo DNA methylation
- maintenance of DNA methylation
- ICF syndrome
- chromatin remodeling
Methylation at the C5 positions of cytosine (i.e., 5mC) in the CpG context (hereafter called DNA methylation) plays a major role in the transcriptional regulation of gene expression, the silencing of transposable elements (TEs), and genome integrity. The enzymatic activities catalyzing DNA methylation can be classified into two types. One is
De novoDNA methylation
2.1 Timing of physiological
de novoDNA methylation
The erasure of DNA methylation in PGCs is probably the result of a defect in maintenance of DNA methylation, caused by the diminished expression of UHRF1 in the cells . After the demethylation, DNMT3A establishes the methylation pattern in combination with DNMT3L, which itself does not possess enzymatic activity but is indispensable for the activity of DNMT3A [5, 6, 7] in oocytes arrested at an early stage of the first meiotic division or in prospermatogonia arrested at the G1 phase . Although the major role of UHRF1 is in the maintenance of DNA methylation (Section 2.2), our group has recently found that UHRF1 is involved in 25% of the genome-wide
During post-implantation embryogenesis and early childhood, not only DNMT3A but also DNMT3B proves to be essential for establishing the characteristic methylation pattern . These enzymes may work together or independently to establish specific DNA methylation patterns in each cell type. However, it still has to be determined when the establishment of the methylation pattern is completed, although it probably depends on the cell type. The “developmental origins of health and disease” (DOHaD) is a concept that has emerged over the past three decades, linking the risk of diseases in later childhood and adult life with the environmental conditions of the early life, including nutrient availability to the mothers. Accumulating evidence suggests that the environment can change the epigenetic state, including DNA methylation of the fetus and infant, with the state being maintained throughout the lifetime of the individual . A well-known experiment showed that early experience in childhood permanently alters behavior and physiology; interactions between rat mothers and their offspring, including the licking and grooming of the pups by their mother in the first week of life, altered the DNA methylation status of the
2.2 Specification of
de novoDNA methylation sites
The mechanisms underlying the specification of the genomic regions targeted by
During embryogenesis, transcription factors probably define certain transcribed regions in each cell type as only four transcriptional factors (OCT3/4, SOX2, KLF4, and MYC), together known as OSKM or Yamanaka factors, can drive drastic transcriptional change and define epigenetically active regions in differentiated cells, resulting in induced pluripotent stem (iPS) cells . DNMTs can access regions, where the transcription factors are absent, to passively specify regions for DNA methylation (Figure 3). Noncoding RNAs, such as PIWI-interacting RNAs (piRNAs) and long noncoding RNAs (lncRNAs), can also contribute to the specification of regions for DNA methylation (Figure 3). piRNAs are the largest class (26–31 nucleotides) of small noncoding RNA expressed in animal cells, which were first discovered in
3. Maintenance of DNA methylation
3.1 Maintenance of DNA methylation by the DNMT1/UHRF1 complex
Once DNA methylation patterns specific to each cell type are established, the pattern is maintained by the DNMT1/UHRF1 complex throughout the individual’s lifetime . UHRF1 (also known as Np95 or ICBP90) is a multidomain protein, which contains a ubiquitin-like (UBL) domain, a tandem Tudor domain (TTD), a plant homeodomain (PHD) finger, a SET and RING-associated (SRA) domain, and a really interesting new gene (RING) domain. The TTD recognizes di−/tri-methylated H3K9 (H3K9me2/me3) and also LIG1 (LIG1K126me2/me3) [30, 31], the PHD recognizes the unmethylated N-terminus of histone H3 and LIG1 , the SRA domain recognizes hemi-methylated DNA at the replication fork [33, 34, 35], and the RING domain mono-ubiquitylates multiple lysines of histone H3 at K14, K18, and K23 and those of the PCNA-associated factor 15 (PAF15) at K15 and K24 [36, 37, 38, 39]. The UBL domain facilitates both the RING-mediated ubiquitylation and the SRA-mediated recognition of hemi-methylated DNA [40, 41].
Current consensus has it that the process of maintenance of DNA methylation operates as follows. After DNA replication, UHRF1 directly recognizes hemi-methylated DNA and mono-ubiquitylates histone H3K14, K18, and K23, to recruit DNMT1 to the hemi-methylation sites. Then, DNMT1 recognizes two of the three ubiquitylated histone lysine residues through the replication foci targeting sequence (RFTS) domain and methylates the nascent strand in hemi-methylated DNA, resulting in the maintenance of the methylation patterns (Figure 4). Immediately prior to the methylation of hemi-methylated DNA by DNMT1, it has been reported that the deubiquitylation of histones by ubiquitin specific peptidase 7 (USP7) is required . DNA ligase 1 (LIG1), which is critical for the joining together of Okazaki fragments , is also involved in this process . Euchromatic histone lysine methyltransferase 2 (EHMT2, also called G9a) and EHMT1 (also called GLP) methylate K126 of LIG1. UHRF1 recognizes the methylated LIG1, and this interaction facilitates the recruitment of UHRF1 to DNA replication sites. Since LIG1 is indispensable for completing the lagging strand synthesis, the interaction between UHRF1 and LIG1 may be especially important for maintenance of DNA methylation of the strand (Figure 4).
3.2 Maintenance of DNA methylation by the CDCA7/HELLS chromatin remodeling complex
The cell division cycle-associated 7 (CDCA7)/helicase lymphoid-specific (HELLS) chromatin remodeling complex is also involved in maintenance of DNA methylation. Recently, an international group including us identified
Patients with the ICF syndrome are classified into two groups . One group includes ICF syndrome type-1 (ICF1), which shows DNA hypomethylation only at the pericentromeric repeats. A causative gene for this group is
A recent study revealed that, in addition to centromeric and pericentromeric repeats, DNA methylation levels of other heterochromatic late-replicating regions are affected in ICF2, ICF3, and ICF4 patients, though not in ICF1 patients . As
Using human embryonic kidney 293 cells, our group reported that
3.3 Maintenance of DNA methylation by the proteins associated with multi-locus imprint disorder
It is reported that mutations in genes encoding zinc finger protein 57 (ZFP57) and components of subcortical maternal complex (SCMC), including NLRP2, NLRP5, NLRP7, PADI6, OOEP, and TLE6, cause the multi-locus imprint disorder, which exhibits DNA hypomethylation at multiple imprinting control regions (ICRs) [58, 59, 60, 61]. Since the hypomethylation is observed in both paternally and maternally methylated ICRs, these factors are thought to be involved in maintenance of DNA methylation against genome-wide DNA demethylation in preimplantation embryos (Figure 1). Mutations in
I identified UHRF1 as a novel methyl-CpG binding protein in 2004 by biotin-avidin pulldown assay using biotin-labeled methylated DNA mixed with nuclear extracts and subsequent mass spectrometric analysis [64, 65]. Since then, an understanding of the mechanism by which maintenance of DNA methylation is achieved has quickly expanded and deepened, progress that I would never have imagined at that time. When the involvement of UHRF1 in maintenance of DNA methylation was reported , the recognition of hemi-methylated DNA by UHRF1 was reported [32, 34, 35], and the ubiquitylation of histone H3 by UHRF1 was reported , each time I felt that the mechanism of maintenance of DNA methylation had been resolved. However, the mechanism is more complicated than expected, and more factors could still be involved to assist the DNMT1/UHRF1 complex, depending on context such as replication timing, replication strand, and higher-order chromatin structure. We still cannot take our eyes off advances in this field.
This work was supported by JSPS KAKENHI Grant number JP18K06961. I would like to thank Enago (www.enago.jp) for the English language review.
Conflict of interest
The author has declared that no conflict of interest exists.
I would like to thank Drs. Kyohei Arita (Yokohama City University), Jafar Sharif (RIKEN), and Atsuya Nishiyama (the University of Tokyo) for involving me in the UHRF1 community and discussing many interesting topics. I would like to thank Dr. Hisato Kobayashi (Nara Medical University) for notifying me of a possible role of LTR-retrotransposons in oocytes. I also would like to thank Profs. Hiroyuki Sasaki (Kyushu University) and Hironori Funabiki (Rockefeller University) for deepening my insights into epigenetics and chromatins. Finally, I would like to thank Prof. Yusuke Nakamura (Japanese Foundation for Cancer Research) for encouraging me with challenging experiments, which resulted in the identification of UHRF1, when I was a PhD student.