Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail-DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.

The PWWP domain is a weakly conserved sequence motif found in >60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223-357) has been structurally characterized at 1.8 Å resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Δ218 and Δ369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Δ218 protein, which includes the PWWP domain, binds DNA more strongly than Δ369, which lacks the PWWP domain.Genomic patterns of cytosine methylation at the C5 position play key roles in the organization and control of mammalian chromatin (reviewed in refs 1-3). Mammalian DNA methyltransferases (MTases) all contain a C-terminal catalytic region that is structurally and functionally homologous to bacterial MTases. The eukaryotic DNA MTases have been grouped into three families based on (i) distinctive properties of their N-terminal regions and (ii) intriguing differences in DNA substrate preferences. The first eukaryotic DNA MTase, Dnmt1, contains a large N-terminal regulatory region of ~1,000 amino acids and shows a preference for hemimethylated DNA in vitro [4][5][6] . Dnmt2, a relatively small protein, contains only the MTase domain, and its function is still unclear 7 . Recombinant Dnmt3 acts on unmethylated and hemimethylated DNA at equal rates in vitro 8,9 .The task of dissecting the functional roles of the N-terminal region of the different MTase families is just beginning. These regions vary widely in size and are probably responsible for the diverse biological functions of eukaryotic DNA MTases. These functions include the © 2002 Nature Publishing Group Correspondence should be addressed to X.C. xcheng@emory.edu. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Dnmt3 contains a PWWP domainThe sequences of Dnmt3 orthologs have been determined from human and mouse. They all contain an N-terminal variable region (~280 amino acids in Dnmt3a and ~220 amino acids in Dnmt3b), followed by three stretches of conserved regions: the PWWP motif, six repeats of a CXXC motif and a set of 10 motifs conserved among DNA MTase catalytic regions (Fig. 1a). The PWWP motif 28 was first identified in a gene family related to the hepatomaderived growth factor (HDGF) 29 and WHSC1 genes (Wolf-Hirschhorn Syndrome Candidate) 30 . The corresponding sequence in Dnmt3 is SWWP (Fig. 1b); only the last two positions of the motif...

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.DOI: http://dx.doi.org/10.7554/eLife.17096.001

The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and “transgene clean” targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.

Significance RNA regulation occurs at many levels including processing to mature forms, subcellular localization, and translation. RNA-binding proteins are crucial to direct and regulate these processes. Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins are RNA-binding proteins formed from eight α-helical repeats [Pumilio (PUM) repeats] that recognize specific mRNA sequences. Previous structural studies revealed characteristic curved structures and sequence specificity unique to these classical PUF proteins. We show here that PUM repeats also form different folds with 11 PUM repeats. Moreover, these proteins, exemplified by human Puf-A and yeast Puf6 proteins, recognize double-stranded RNA or DNA without sequence specificity. Interestingly, Puf-A and Puf6 PUM repeats lack specificity for RNA bases yet use residues at conserved positions on topologically equivalent protein surfaces for new nucleic acid recognition modes.

No abstract

Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.