DNA methylation editing by CRISPR-guided excision of 5-methylcytosine

Tools for active targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involve TET-mediated oxidation of 5- methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPRassociated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing

A DNA 3′ Phosphatase Functions in Active DNA Demethylation in Arabidopsis

DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′ phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and colocalize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway

Desmetilación activa del ADN: un mecanismo epigenético para la reactivación de genes silenciados

Los mecanismos de control epigenético son esenciales para una regulación estable de los patrones de expresión génica y desempeñan un papel central en los ciclos de vida de animales y plantas. La metilación de la citosina en el carbono 5 del anillo de pirimidina (5-meC) es una marca epigenética estable, pero reversible, que promueve el silenciamiento génico transcripcional. Comprender cómo se regula el estado de metilación del genoma a nivel global o local requiere una definición de los procesos enzimáticos que metilan y desmetilan el ADN. Sin embargo, aunque las enzimas responsables del establecimiento y mantenimiento de la metilación de ADN han sido bien caracterizadas, los mecanismos de desmetilación no se conocen con exactitud. Nuestro grupo, junto con otros, ha obtenido datos genéticos y bioquímicos que sugieren que dos proteínas de Arabidopsis con dominio ADN glicosilasa (ROS1 y DME) actúan como ADN desmetilasas capaces de activar la expresión de genes previamente silenciados. Nuestros resultados previos indican que ROS1 y DME catalizan la liberación de 5-meC del ADN mediante un mecanismo ADN glicosilasa. Estos resultados sugieren que una de las funciones de ROS1 y DME es iniciar el borrado de 5-meC mediante un proceso de escisión de bases y proporcionan una importante evidencia bioquímica a favor de la existencia de una ruta de desmetilación activa en plantas. En la actualidad, nuestro grupo de investigación se concentra en caracterizar funcionalmente este novedoso mecanismo de control epigenético mediante una aproximación multidisciplinar que combina metodologías del campo de la bioquímica, la genética y la biofísica. Este estudio suministrará una información esencial para entender los mecanismos responsables de la reprogramación epigenética en el núcleo celular, con aplicaciones potenciales en biotecnología y biomedicin

Dynamics of coherently pumped lasers with linearly polarized pump and generated fields

The influence of light polarization on the dynamics of an optically pumped single-mode laser with a homogeneously broadened four-level medium is theoretically investigated in detail. Pump and laser fields with either parallel or crossed linear polarizations are considered, as are typical in far-infrared-laser experiments. Numerical simulations reveal dramatically different dynamic behaviors for these two polarization configurations. The analysis of the model equations allows us to find the physical origin of both behaviors. In particular, the crossed-polarization configuration is shown to be effective in decoupling the pump and laser fields, thus allowing for the appearance of Lorenz-type dynamics

DNA Damage in Plant Herbarium Tissue

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4–3.8% of fresh control DNA and 1.0–1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens

The C-terminal domain of Arabidopsis ROS1 DNA demethylase interacts with histone H3 and is required for DNA binding and catalytic activity.

Active DNA demethylation plays an important role in controlling methylation patterns in eukaryotes. In plants, the DEMETER-LIKE (DML) family of 5-methylcytosine DNA glycosylases initiates DNA demethylation through a base excision repair pathway. However, it is poorly understood how these DNA demethylases are recruited to their target loci and the role that histone marks play in this process. Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) is a representative enzyme of the DML family, whose members are uniquely characterized by a basic amino-terminal domain mediating nonspecific binding to DNA, a discontinuous catalytic domain, and a conserved carboxy-terminal domain of unknown function. Here, we show that ROS1 interacts with the N-terminal tail of H3 through its C-terminal domain. Importantly, phosphorylation at H3 Ser28, but not Ser10, abrogates ROS1 interaction with H3. Conserved residues at the C-terminal domain are not only required for H3 interaction, but also for efficient DNA binding and catalytic activity. Our findings suggest that the C-terminal domain of ROS1 may function as a histone reader module involved in recruitment of the DNA demethylase activity to specific genomic regions