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.

Summary DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here we report the identification of a new RdDM effector, RDM3/KTF1. Loss-of-function mutations in RDM3/KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci, and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA silencing effector complexes.

DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.histone modifications | small RNA | gene silencing | transposon D NA cytosine methylation is a conserved epigenetic mark that plays important roles in maintaining genome stability, transcriptional gene silencing, and developmental regulation (1, 2). In plants, DNA methylation occurs in three sequence contexts: CG, CHG, and CHH (H = A, C, T). CG and CHG methylation are symmetric in sequence and are maintained through a semiconservative mechanism that requires the DNA methyltransferases (METHYLTRANSFERASE 1) (MET1) and CHROMOMETHYLASE 3 (CMT3), respectively. In contrast, the asymmetric CHH methylation needs to be established during each cell cycle (1, 2). In plants a 24-nt small interfering RNA (siRNA)-dependent DNA methylation pathway is involved in recruiting the de novo DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) and is responsible for DNA methylation at many transposable elements and repetitive sequences (3, 4).Two plant-specific homologs of RNA polymerase II play important roles in the RNA-directed DNA methylation (RdDM) pathway (5). RNA polymerase IV presumably initiates 24-nt siRNA biogenesis by specifically transcribing RdDM target loci to produce single-stranded RNA (ssRNA) transcripts, which serve as templates for RNA-dependent RNA polymerase 2 (RDR2) to generate double-stranded RNAs (dsRNAs). CLASSY 1 (CLSY1), a putative ATP-dependent chromatin remodeling protein, is prop...

SUMMARY DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. Twenty-four-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ~20 to 60 nt in length, often having same 5’ ends but differing in 3’ ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3’-5’ exonucleases. Our findings suggest an alternative route for siRNA biogenesis: precursor transcripts are bound by AGO4 and subsequently subjected to 3’-5’ exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

We report that a neuron-specific isoform of LSD1, LSD1n, resulting from an alternative splicing event, acquires a novel substrate specificity targeting histone H4 K20 methylation, both in vitro and in vivo. Selective genetic ablation of LSD1n leads to deficits in spatial learning and memory, revealing the functional importance of LSD1n in the regulation of neuronal activity-regulated transcription in a fashion indispensable for long-term memory formation. LSD1n occupies neuronal gene enhancers, promoters and transcribed coding regions, and is required for transcription initiation and elongation steps in response to neuronal activity, indicating the crucial role of H4K20 methylation in coordinating gene transcription with neuronal function. This study reveals that the alternative splicing of LSD1 in neurons, associated with altered substrate specificity, serves as an underlying mechanism acquired by neurons to achieve more precise control of gene expression in the complex processes underlying learning and memory.