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DNA methylation has been utilized for target gene silencing in plants. However, it is not well understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain-of-function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression through DNA methylation, histone H3K27me3 deposition, H3K4me3 demethylation, histone deacetylation, inhibition of RNA polymerase II transcription elongation or Ser-5 dephosphorylation. These proteins also silenced many other genes with different efficacies, and a machine learning model could accurately predict the efficacy of each silencer on the basis of various chromatin features of the target loci. Furthermore, some proteins were also able to target gene silencing when used in a dCas9-SunTag system. These results provide a more comprehensive understanding of epigenetic regulatory pathways in plants and provide an armament of tools for targeted gene manipulation.

Cationic methylpalladium complexes bearing hemilabile bidentate α-amino–pyridines can serve as effective precursors for catalytic alternating copolymerization of norbornene (N) and ethylene (E), under mild conditions. The norbornyl palladium complexes in the formula of {[RHNCH2(o-C6H4N)]Pd(C7H10Me)(NCMe)}(BF4) (R = iPr (2a), tBu (2b), Ph (2c), 2,6-Me2C6H3 (2d), 2,6-iPr2C6H3 (2e)) were synthesized via single insertion of norbornene into the corresponding methylpalladium complexes 1a–1e, respectively. Both square planar methyl and norbornyl palladium complexes exhibit facile equilibria of geometrical isomerization, via sterically-controlled amino decoordination–recoordination of amino–pyridine. Kinetic studies of E-insertion, N-insertion of complexes 1 and 2, and the geometric isomerization reactions have been examined by means of VT-NMR, and found in excellent agreement with the results estimated by DFT calculations. The more facile N-insertion in the cis-isomers, and ready geometric isomerization, cooperatively lead to a new mechanism that accounts for the novel catalytic formation of alternating COC.

Motivation The Full-text index in Minute space (FM-index) is a memory-efficient data structure widely used in bioinformatics for solving the fundamental pattern-matching task of searching for short patterns within a long reference. With the demand for short query patterns, the k -ordered concept has been proposed for FM-indexes. However, few construction algorithms in the state of the art fully exploit this idea to achieve significant speedups in the pan-genome era. Results We introduce the k -ordered induced suffix sorting (kISS) for efficient construction and utilization of k -ordered FM-indexes. We present an algorithmic workflow for building k -ordered suffix arrays, incorporating two novel strategies to improve time and memory efficiency. We also demonstrate the compatibility of integrating k -ordered FM-indexes with locate operations in FMtree. Experiments show that kISS can improve the construction time, and the generated k -ordered suffix array can also be applied to FMtree without any additional in computation or memory usage. Availability and implementation https://github.com/jhhung/kISS. [ABSTRACT FROM AUTHOR]

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.

A new ligand with N8O2 donors containing three potential metalbinding sites (H2L) and its tricopper(II) complex 1 are synthesized. The tricopper species is found to be formed from a hypodentate dicopper(II) complex 2 in basic solutions. Complex 2 may be isolated from the reaction of H2L with a copper source under acidic conditions. Complex 2 can undergo CO2-abstraction to yield an octacopper(II) complex 3. The single crystal structures of complexes 2 and 3 are characterized by X-ray crystallography. [ABSTRACT FROM AUTHOR]

Cationic methylpalladium complexes with hemilabile bidentate ligands of α-amino-pyridines, in the form of {[R1HNCR2H(o-C6H5N)]Pd(Me)(NCMe)}(BF4) (R1= iPr, tBu, Ar R2= H, Me) have been found to be effective precursors for catalytic copolymerization of ethylene and norbornene under mild conditions. The copolymer products exhibit predominant alternating microstructures which are evidenced by NMR and mass spectrometry as well as a kinetic analysis according to the Finman-Ross relationship. [ABSTRACT FROM AUTHOR]

Ethylene polymerization catalyzed by Ni(II) complexes that bear new bidentate ligands with a functional hybrid of amine and imine has been studied. A class of new α-aminoaldimines and their nickel complexes [R1R2NCMe2CH=N(2,6-R32C6H3)]NiBr2 (R1 = R2 = Me, R3 = Me (Ni-1b); R3 = iPr (Ni-1c); 1 = R2 = Et, R3 = H (Ni-2a); Me (Ni-2b); iPr (Ni-2c); R1 = R2 = nPr, R3 = iPr (Ni-3c); (R1R2) = c-C3H6 R3 = tPr (Ni-4c); (R1R2) = c-C4H8, R3 = iPr (Ni-5c)) were synthesized. The molecular structures of six nickel complexes were determined by X-ray crystallography, showing distorted tetrahedral configurations. The SQUID data of Ni-1c confirms its ground state of triplet spin. Using methylaluminoxanes (MAO) as the activator, the nickel complexes are found to catalyze ethylene polymerization under moderate pressure and ambient temperature. The activity reaches to 106 g PE mol Ni-1 h-1, and increases with the ethylene pressure in the range of 14-28 bar. The highly branching PE products have Mn ∼ 105 with PDI < 2. The amine and imine functionalities demonstrate independent control to the polymerization reactions, wherein the activity appears to be facilitated by using the catalysts installed with bulky imino substituents as well as with less sterically hindered amino substituents. This is ascribed to the C2 unsymmetric coordination in the square planar resting state in which the bulky polymer chain prefers cis to the imine and the small ethylene monomer is cis to the amine. [ABSTRACT FROM AUTHOR]

New α-aminoaldimines with the formula of Et2NCMe2CHNR (R = iPr, tBu, Ph) and their dichloro or diacetato complexes of Ni, Pd, Pt are prepared and structurally characterized. A nickel complex is in a distorted tetrahedral configuration, and the Pd and Pt complexes (4–6) are of square planar form. The α-aminoaldimines can chelate to the metal in a C2-unsymmetric bidentate motif through the hetero functionalities of amine and imine, which show comparable transinfluence. Square planar organometallic palladium derivatives bearing α-aminoaldimines, including Pd–methyl, Pd–acetyl, and Pd–(η2-acetylnorboryl) (7–10), are also synthesized. The latter two species are a result of CO-insertion into Pd–methyl complexes and ensuing norbornene-insertion, respectively. The geometrical isomerism is found in the transconfiguration in most studied cases. Such a stereoselectivity results from the thermodynamic stability governed predominantly by steric control. The stereoselectivity is also supported by DFT calculations. [ABSTRACT FROM AUTHOR]

A new ligand with N8O2 donors containing three potential metalbinding sites (H2L) and its tricopper(II) complex 1 are synthesized. The tricopper species is found to be formed from a hypodentate dicopper(II) complex 2 in basic solutions. Complex 2 may be isolated from the reaction of H2L with a copper source under acidic conditions. Complex 2 can undergo CO2-abstraction to yield an octacopper(II) complex 3. The single crystal structures of complexes 2 and 3 are characterized by X-ray crystallography. [ABSTRACT FROM AUTHOR]

Introduction: The radiofrequency ablation (RFA) of liver cancer is a desirable treatment option, as it is minimally invasive. An accurate numerical simulation can greatly help physicians better plan their surgical protocols. Previously, the displacement current in the RFA process was considered negligible, and therefore RFA simulation was modeled as a direct current (DC) system instead of an alternating current (AC) system. Our study investigated the hypothesis that the displacement current in the RFA process should not always be considered negligible., Methods: AC measurements of ex vivo bovine liver ablation were performed, and numerical simulations were also conducted to test the hypothesis that the relative permittivity would significantly decrease after the liver tissue reached a high temperature., Results: The displacement current was observed to be a sizable fraction of the conduction current, especially before the onset of the first pause. The simulation results indicated that the relative permittivity is likely to decrease to several hundred or lower at elevated temperatures., Conclusions: Our study results suggest that the DC model may be inadequate, especially before the first roll-off and that additional information could be available during RFA treatment by considering the AC nature of RFA, which could lead to improved numerical simulation. Additional measurements of tissue parameters are needed to reach the full potential of the AC model for further development of ablation control.