Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.BS-Seq | epigenetic profiling | DNA methylation | gene body methylation | UHRF1C ytosine DNA methylation is an epigenetic mark important in many gene regulatory systems, including genomic imprinting, X-chromosome inactivation, silencing of transposons and other repetitive DNA sequences, as well as expression of endogenous genes. Methylation is conserved in most major eukaryotic groups, including many plants, animals, and fungi, although it has been lost from certain model organisms such as the budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans (1-3). DNA methylation can be categorized into three types according to the sequence context of the cytosines, namely CG, CHG, and CHH (H = A, C, or T). CG methylation is maintained by conserved Dnmt1 DNA methyltransferase enzymes. CHH methylation, and, to some extent CHG methylation, is generally maintained by the activity of the conserved Dnmt3 methyltransferases, whereas high levels of CHG methylation seen in the model plant Arabidopsis are maintained by the plant-specific methyltransferase CMT3 (2, 3). Generally speaking, DNA methylation is thought to occur "globally" in vertebrates, with CG sites being heavily methylated genome-wide except for those in CpG islands, whereas invertebrates, plants, and fungi have "mosaic" methylation, characterized by interspersed methylated and unmethylated domains (4). These differences are an interesting starting point for studying divergence in methylation pathways and regulatory mechanisms; however, determining precise genomescale methylation patterns has been a challenge for complex genomes until the recent development of high-throughput sequencing technology. In this paper, we generated shotgun bisulfite sequencing data to profile DNA methylation in eight eukaryotic organisms. These organisms display wide variations in methylati...

Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.

Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson–Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4–5 h on-chip processing time) and library preparation cost (~81¢ per cell) compared to prior microfluidic implementations. We apply this method to measure regulatory variation in peripheral blood mononuclear cells (PBMCs) and show robust, de novo clustering of single cells by hematopoietic cell type.

Related to Ubiquitin (RUB)/Nedd8 is a ubiquitin-like protein that covalently attaches to cullins, a subunit of the SCF (for Skp, Cdc53p/Cul1, and F-box protein) complex, an E3 ubiquitin ligase, and has been shown to be required for robust function of the complex. The effects of reducing protein levels for two Rub proteins, RUB1 and RUB2, were characterized in Arabidopsis thaliana. T-DNA insertional null lines homozygous at a single RUB-encoding locus were analyzed and found to have a wild-type phenotype. A double mutant was never recovered. More than one-quarter of the progeny from the selffertilization of plants with a single functional RUB-encoding gene died as embryos at the two-cell stage. Outcrosses demonstrated reduced inheritance of the null allele from both the male and female parent. Hemigglutinin-tagged forms of RUB1 and RUB2 conjugate to the same cullin protein, CUL1, and produce the same conjugation pattern. To further understand the function of the RUB proteins, a construct designed to produce a double-stranded RUB1 mRNA was introduced into plants, and three lines with reduced levels of RUB1-and RUB2-encoding mRNA and RUB1/2 protein content were analyzed in detail. Mature plants were severely dwarfed, seedlings were insensitive to auxin in root assays, and darkgrown seedlings had a partial triple-response phenotype that was suppressed when seedlings were grown on ethylene perception or synthesis inhibitors. The dsrub lines produced threefold to fivefold more ethylene than the wild type. This study illustrates that RUB1 and RUB2 are genetically and biochemically redundant and demonstrates that RUB1/2 proteins are essential for early embryonic cell divisions and that they regulate diverse processes.

SummaryAppropriate methylation of genomes is essential for gene regulation. Here, we describe the six-member ORTHRUS (ORTH) gene family of Arabidopsis thaliana that plays a role in DNA methylation in vivo. ORTH1) ORTH5 are predicted to encode proteins that contain one plant homeodomain (PHD), two really interesting new gene (RING) domains, and one set ring associated (SRA) domain, whereas ORTHlike-1 encodes a protein with only one RING and SRA domain. cDNAs for ORTH1, ORTH2, ORTH5 and ORTHlike-1 were isolated, and when expressed as glutathione-S-transferase (GST) fusion proteins, were capable of promoting ubiquitylation in vitro with the E2 AtUBC11. ORTH1 promotes ubiquitylation when paired with additional AtUBC8 family members. ORTH1 proteins with substitutions in metal-ligand binding residues in each ORTH1 RING domain individually, and ORTH1 truncation derivatives lacking one or both RING domains, were tested for their ability to catalyze ubiquitylation in vitro. In these assays, either ORTH1 RING domain is capable of promoting ubiquitylation. The PHD alone is not active as an E3 ligase, nor is it required for ligase activity. GFP-ORTH1 and GFP-ORTH2 are nuclear-localized in transgenic Arabidopsis plants. Overexpression of ORTH1 or ORTH2 in Arabidopsis leads to an altered flowering time. Inspection of DNA methylation at FWA and Cen180 repeats revealed hypomethylation when ORTH proteins were overexpressed. Once initiated, a late-flowering phenotype persisted in the absence of the ORTH transgene, consistent with epigenetic effects at FWA. We conclude that ORTH proteins are E3 ligases mediating DNA methylation status in vivo.