Establishing, maintaining and modifying DNA methylation patterns in plants and animals

Abstract

Cytosine DNA methylation is a stable epigenetic mark that is crucial for diverse biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation. Recent findings in plants and animals have greatly increased our understanding of the pathways used to accurately target, maintain and modify patterns of DNA methylation and have revealed unanticipated mechanistic similarities between these organisms. Key roles have emerged for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases in these processes. Drawing on insights from both plants and animals should deepen our understanding of the regulation and biological significance of DNA methylation.

Figure 1. Cartoon representation of select mouse (Mm), Arabidopsis (At), and Zebrafish (Dr) proteins involved in maintenance methylation, de novo methylation, and demethylation

The MmDnmt3 family and AtDRM2 contain DNA methyltransferase domains (DNA MTase); in AtDRM2 the catalytic motifs are rearranged. The MmDnmt3 proteins also possess a cysteine rich domain that contains a Plant Homeodomain (PHD) zinc finger motif and is referred to as an ATRX-DNMT3-DNMT3L (ADD) domain. MnDnmt3a/b possess a Proline-Tryptophan-Tryptophan-Proline motif (PWWP) that MmDnmt3L lacks. AtDRM2 contains Ubiquitin Associated domains (UBA). MmMIWI2, MmMILI, and AtAGO4 possess a Piwi Argonaute and Zwille domain (PAZ) and a P-element induced wimpy testis domain (PIWI). MmDnmt1 and AtMET1 possess Bromo-Adjacent Homology domains (BAH) and a DNA MTase domain. MmDnmt1 also contains a cysteine rich (CXXC) domain. MmUHRF1 and the AtVIM family contain SET or RING associated (SRA), ring finger E3 ubiquitin protein ligase (RING), and PHD domains. MmUHRF1 also has a Tudor domain and a ubiquitin domain (UBQ). MmLSH1 and AtDDM1 contain DEAD and HELICc helicase domains. AtCMT3 contains a DNA MTase, a Chromatin Organization Modifier (CHROMO) and a BAH domain. AtSUVH4,5, and 6 possess an SRA and a histone methyltransferase domain (Histone MTase). The AtDME/ROS1 family of glycosylases all possesses a helix-hairpin-helix-Gly-Pro-Asp domain (HhH-GDP), a 4Fe-4S cluster (FES), a domain with similarity to histone H1 (H1), and a domain of unknown function (DUF). MmMBD4 contains a HhH-GDP domain and a methyl CpG binding domain (MBD); MmTDG contains a Uracil-DNA glycosylase domain (UDG). Mouse and Zebrafish AID and APOBEC proteins all contain a apolipoprotein B mRNA editing enzyme domain (APOBEC). * indicates activity on additional substrates

Figure 2. Model for RNA directed DNA methylation (RdDM)

Single-stranded RNA transcripts (ssRNA) corresponding to transposons and repeat elements are hypothesized to be generated by Pol IV. CLSY1, a putative chromatin remodeling factor, likely functions early in RdDM, possibly recruiting Pol IV to chromatin or aiding in ssRNA transcript processing. RDR2, an RNA dependant RNA polymerase, is proposed to generate double-stranded RNA (dsRNA) from the ssRNA transcripts. DCL3, a dicer-like protein, is thought to process the dsRNAs into 24nt siRNAs, which are bound by an argonaute protein, AGO4. AGO4 localizes to Cajal bodies and while the function of this association remains unknown, it appears necessary for wild-type levels of RdDM. AGO4 also co-localizes with two Pol V subunits, NRPE1 and NRPE2, and DRM2 at a distinct nuclear foci, the AGO4/NRPD1b(NRPE1) body (not depicted), that may represent a site of active RdDM. Pol V is thought to transcribe intergenic noncoding (IGN) regions throughout the genome. NRPE1 association with chromatin requires another putative chromatin remodeling factor, DRD1, and an SMC domain protein, DMS3. IGN transcripts may serve as a scaffold to recruit AGO4, which interacts with the GW/WG motifs of NRPE1 and SPT5-like, possibly through interactions between AGO4-bound siRNAs and the nascent transcript. An RNA binding protein, IDN2, is proposed to recognize the siRNA/nascent transcript duplex. These associations may aid in targeting DRM2 to genomic loci that produce both 24nt siRNAs and IGN transcripts. Recruitment or retention of DRM2 at such loci may be aided by SUVH9 and SUVH2, two proteins that bind methylated DNA and likely act late in RdDM. Me, DNA methylation.

Figure 3. Model depicting recruitment of the de novo methylation machinery by unmethylated histone 3 lysine 4 (H3K4) tails

The amino-terminal domain of Dnmt3L possesses a cysteine rich domain that interacts with unmethylated H3K4 tails and this interaction is proposed to recruit or activate Dnmt3a2. The carboxy-terminal domains of Dnmt3L and Dnmt3a form a tetrameric complex in which two Dnmt3a proteins interact with each other and are flanked by two Dnmt3L proteins. The Dnmt3a active sites (red stars) are thought to be separated by approximately one helical turn and thus could catalyze methylation (filled circles) on opposite DNA strands ~10bps apart. Once recruited to a specific locus, the Dnmt3L/Dnmt3a tetramer might be able to oligomerize, which could result in an ~10bp periodic pattern of DNA methylation along the same DNA strand.

Figure 4. DNA methylation changes during plant development

Diagram of an Arabidopsis (A) flower, and (B) the male and female gametophytes. Male gametogenesis occurs in stamens (St) and generates tricellular pollen grains that contain a vegetative nucleus (VN) and two sperm cells (SC). Female gametogenesis occurs in ovules (Ov) and produces a multicellular gametophyte with three antipodal cells (AC), two synergid cells (SC), one egg cell (EC) and a diploid (2n) central cell nucleus (CCN). (upper) Model showing transposon reactivation and siRNAs production specifically in the VN. These siRNAs may travel to the SCs to reinforce transposon silencing. (lower) Model showing siRNAs in the CCN, which may arise as a consequence of global demethylation. These siRNAs may travel to the EC and reinforce silencing. Reinforced silencing in the sperm and egg cells could account for the observed hypermethylation of the embryo. (C) Fertilization of the EC and CCN generate the embryo and endosperm, respectively. The embryo will give rise to the mature Arabidopsis plant while the endosperm is a terminally differentiated tissue. Imprinting is observed the endosperm, which nourishes the embryo, and is thus analogous to the placenta in mammals, where imprinting also occurs. In plants, maternal imprinting results from demethylation in the CCN by the DME glycosylase, which likely accounts for the observed hypomethylation in this tissue. After fertilization, the unmethylated (open circles) maternal alleles (♀) are expressed in the endosperm, while the paternal allele (♂) is methylated (closed circles) and silent.

Figure 5. piRNAs and male gametogenesis

(A) Ping-pong model In mammals, as in flies, piRNAs are proposed to arise via a ping-pong amplification cycle that produces primary piRNAs with a 5’ uridine (U) and secondary piRNAs with an adenine (A) at position 10. In mammals, transposon transcripts, mainly sense oriented, are the presumed substrates for primary piRNA production. Cleavage of these transcripts produces primary piRNAs proposed to preferentially associate with cytoplasmic Mili. Mili, bound with sense piRNAs, cleaves antisense transcripts, producing secondary piRNAs that preferentially associate with cytoplasmic and nuclear Miwi2. Nuclear Miwi2, bound with antisense piRNAs, cleaves sense transposon transcripts, producing more primary piRNAs. piRNA complexes are also proposed to guide DNA methylation to homologous genomic loci by potentially interacting with nascent transposon transcripts and directly or indirect recruiting de novo methyltransferases, possibly Dnmt3a/3L complexes. Mili and Miwi2 contain symmetrical dimethylarginines (Me-ARG) and interact with tudor domain-containing (Tdrd) proteins. (B) Model for transposon silencing during male gametogenesis. Genome wide demethylation (E10.5-E12.5) in primordial germ cells (PGC) erases DNA imprints and could briefly reactivate transposons. De novo methylation and paternal imprinting are observed in testes from E14.5 through birth. Consistent with prior transposon expression, piRNAs bound to Mili and Miwi2 (expressed by E12.5 and E15.5, respectively) are enriched for transposon sequences during this time period and are proposed to facilitate targeted reestablishment of DNA methylation at transposons. Although less studied, PIWI argonautes are expressed in female germ cells of flies, mammals, frogs and silkworms and piRNAs are present in fly and silkworm, ovaries and in frog oocytes.

Figure 6. Maintenance of DNA methylation in plants and mammals

(A) Model depicting maintenance of CG methylation during replication. Dnmt1 is proposed to be recruited to replication foci through interactions with UHRF1, an SRA domain protein that specifically interacts with hemi-methylated DNA, and with PCNA. Once recruited, Dnmt1 functions to maintain methylation patterns by restoring the hemi-methylated DNA to a fully methylated state. In plants, MET1, and the VIM family of SRA domain proteins, homologs of Dnmt1 and UHRF1, respectively, likely function in a similar manner to maintain CG methylation patterns. Black and Gray circles represent methylated and unmethylated cytosines respectively. (B) Model depicting maintenance of CHG methylation in plants. A reinforcing loop of DNA and histone methylation is proposed to maintain CHG methylation in plants. The CMT3 DNA methyltransferase maintains methylation in the CHG context, which is recognized by the SRA domain of the KYP/SUVH4 histone methyltransferase. KYP catalyzes H3K9 dimethylation (H3K9me2), a modification that is required for the maintenance of CHG methylation, and the chromodomain of CMT3 binds methylated histone 3 (H3) tails.

Figure 7. Active DNA Demethylation via DNA glycosylase activity and Base Excision Repair

In Arabidopsis (green shaded proteins) methylated (CH₃) cytosine (C) bases are removed by the DEMETER (DME)/ROS1 family of bifunctional 5-methylcytosine glycosylases. First, the methylated cytosine base is released by cleavage of the N-glycosidic bond, generating an abasic site. Next the phosphodiester linkage is broken both 3’ and 5’ of the abasic site through apyrimidic (AP) lysase activity, generating a single nucleotide gap in the DNA. The DNA is then proposed to be repaired by unknown DNA polymerase and ligase activities, resulting in a net loss of cytosine methylation. In Zebrafish and mammals, no efficient 5-methylcytosine glycoslases have been identified. However, in Zebrafish (blue shaded proteins), it has been proposed that the AID and APOBEC family of deaminases first convert methylated cytosines into thymines (T), generating thymine/guanine (G) (T:G) mismatches. Then, these mismatches could be recognized by the MBD4 glycosylase, resulting in removal of the thymine base and generation of an abasic site. Unlike the DME/ROS1 glycosylases, MBD4 is a monfunctional DNA glycosylase, thus another unidentified protein is likely required to provide the AP lyase activity in order to remove the sugar ring to generate a single nucleotide gap. As in Arabidopsis, this substrate is proposed to be repaired by unidentified DNA polymerase and ligase activities.