Regulation and function of DNA methylation in plants and animals

Abstract

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review.

Figure 1

The RNA-directed DNA methylation pathway in plants. In transposons and other DNA repeat regions, aberrant single-stranded RNAs are proposed to be produced by DNA-dependent RNA polymerase IV (Pol IV). The chromatin remodeling protein CLSY may facilitate Pol IV transcription. RNA-dependent RNA polymerase RDR2 converts the aberrant single-stranded RNAs to double-stranded RNAs, which are then cleaved into 24-nt siRNAs by the Dicer-like protein DCL3. The 24-nt siRNAs are bound by an ARGONAUTE protein AGO4, AGO6, or AGO9. In intergenic non-coding (IGN) regions, DNA-dependent RNA polymerase V (Pol V) generates single-stranded scaffold RNA transcripts. Generation of Pol V RNA transcripts requires RDM4/DMS4, DRD1, DMS3, and RDM1. RDM1 may bind single-stranded methylated DNA and help recruit Pol V and Pol II to appropriate chromatin regions. DRD1, DMS3, and RDM1 form a stable protein complex, named DDR. KTF1 is an RNA-binding protein, which tethers AGO4 to nascent Pol V or Pol II RNA transcripts to form the RNA-directed DNA methylation effector complex. IDN2 may stabilize the base-pairing between the nascent scaffold transcripts and 24-nt siRNAs. The effector complex directs the de novo DNA methyltransferase DRM2 to specific chromatin regions to catalyze new DNA methylation.

Figure 2

De novo DNA methylation in the mammalian germline. Two Piwi proteins, MILI and MIWI2, are required for piRNA (Piwi-interacting RNA) generation. The piRNAs are generated in fetal male gonads, and play important roles in silencing transposons by causing DNA methylation. The primary piRNAs are bound with cytoplasmic MILI, which cleaves antisense transposon transcripts. The secondary piRNAs are bound with MIWI2, which cleaves sense transposon transcripts. The sense transposon transcripts produce primary piRNAs with 5′ uridine (U), whereas antisense transposon transcripts produce secondary piRNAs with an adenine (A) at position 10. Generation of piRNAs also require Tudor domain-containing (TDRD) proteins, mouse VASA homolog (MVH), and putative DExD-box helicase MOV10L1. The interaction between Piwi proteins and TDRD proteins is essential for generation of piRNAs. TDRD9-MIWI2 and TDRD1-MILI are two conserved complexes that generate primary piRNAs and secondary piRNAs, respectively. MVH and MOV10L1 are required for the activity of both MILI and MIWI2 in piRNA generation and de novo DNA methylation. DNMT3L interacts with unmethylated H3K4, and recruits DNMT3A to specific genomic regions for DNA methylation. The histone H3K4 demethylase KDM1B catalyzes demethylation of H3K4, by which it promotes de novo DNA methylation.

Figure 3

Active DNA demethylation and its function in plants. The plant 5-methylcytosine DNA glycosylases ROS1, DME, DML2, and DML3 function as active DNA demethylases. (A) ROS1 was discovered by screening for repressor of silencing in Arabidopsis plants expressing the RD29A promoter-driven luciferase reporter gene. ROS1 prevents transgene silencing that is caused by RNA-directed DNA methylation. ROS1 also functions to prevent over-methylation and alleviate the silencing of some endogenous genes and transposons. ROS3 is an RNA-binding protein that may direct ROS1 to specific genome targets. (B) DME is preferentially expressed in endosperms, and is responsible for genome-wide DNA demethylation and gene imprinting. Genome-wide DNA demethylation activates transposons and other repetitive DNA sequences, leading to the enhanced production of siRNAs in endosperms. These siRNAs might be transported into embryos, and contribute to DNA hypermethylation, to ensure genome stability in embryos. Black and white circles represent methylated and unmethylated cytosines, respectively.

Figure 4

Dynamic changes in DNA methylation during mouse development. Shortly after fertilization, the paternal genome undergoes active demethylation, and the maternal genome is passively demethylated during subsequent cleavage divisions. After implantation, the embryo undergoes de novo methylation that establishes a new methylation pattern. Imprinted genes escape the waves of demethylation and de novo methylation during embryogenesis. Genome-wide demethylation and de novo methylation also occur in the male and female germ cells during gametogenesis, which are critical for the establishment of genomic imprinting. DNMTs and other regulatory factors involved in these processes are indicated.