Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing

Leaf epidermal profiling as a phenotyping tool for DNA methylation mutants

Phenotypic evaluation of epigenetic mutants is mainly based on the analysis of plant growth and morphological features. However, there are cellular level changes that are not visible to the naked eye and require analysis with higher resolution techniques. In this study, we carried out a phenotypic characterisation of several Arabidopsis thaliana hypomethylation mutants by quantitative image analysis combined with flow cytometry. This phenotyping approach permitted identification of abnormalities at the cellular level in mutants with wild-type morphology at the organ level. Morphometry of adaxial leaf epidermis revealed variations in the size and number of pavement cells, and the density and distribution of stomata in the analysed second rosette leaves from the mutants studied. A direct correlation between DNA ploidy status and leaf pavement cell size in wild type and mutant leaves was observed. Recognition of hidden phenotypic variations could facilitate the identification of key genetic loci underlying the phenotypes caused by modifications of DNA methylation. Thus, this study outlines an easy and fast phenotyping strategy that can be used as a reliable tool for characterisation of epigenetic mutants at the cellular level

Epigenetic regulation of sulfur homeostasis in plants

Plants have evolved sophisticated mechanisms for adaptation to fluctuating availability of nutrients in soil. Such mechanisms are of importance for plants to maintain homeostasis of nutrient elements for their development and growth. The molecular mechanisms controlling the homeostasis of nutrient elements at the genetic level have been gradually revealed, including the identification of regulatory factors and transporters responding to nutrient stresses. Recent studies have suggested that such responses are controlled not only by genetic regulation but also by epigenetic regulation. In this review, we present recent studies on the involvement of DNA methylation, histone modifications, and non-coding RNA-mediated gene silencing in the regulation of sulfur homeostasis and the response to sulfur deficiency. We also discuss the potential effect of sulfur-containing metabolites such as S-adenosylmethionine on the maintenance of DNA and histone methylation

Dynamic regulation of DNA demethylation and RNA-directed DNA methylation in Arabidopsis

DNA methylation is an important epigenetic mark present in many eukaryotes, and is involved in many crucial biological processes, such as gene imprinting, regulation of gene expression, and genome stability. Proper genomic DNA methylation patterns are achieved through the concerted action of DNA methylation and demethylation pathways. In the model plant species Arabidopsis thaliana, ROS1 (REPRESSOR OF SILENCING 1) is one of the DNA demethylases and the key component in the demethylation pathway. Dysfunction of ROS1 leads to increase in DNA methylation level at thousands of genomic loci. However, the features of ROS1 targets are not well understood. In the first part of this dissertation, I will describe a study in which we identified and characterized genome-wide ROS1 target loci in Arabidopsis Col-0 and C24 ecotypes. In this study, we showed that ROS1 targets are associated with an enrichment of H3K18ac and H3K27me3, and with depletion of H3K27me1 and H3K9me2. Also we found that ROS1 can prevent the spreading of DNA methylation from highly methylated transposons to their nearby genes. Unexpectedly, we uncovered thousands of previously unidentified RdDM (RNA-directed DNA methylation) targets by analyzing the DNA methylome of ros1/nrpd1 double mutant plants. In addition, we showed that ROS1 also antagonizes RdDM-independent DNA methylation at more than one thousand genomic loci. Our results provide significant insights into the genome-wide effects of both ROS1-mediated active DNA demethylation and RNA-directed DNA methylation, as well as their interactions in plants. In the second part of this dissertation, I will describe another study in which we focused on AGO6 and AGO4, two Argonaute proteins involved in RdDM. AGO6 is generally considered to be redundant with AGO4 in RdDM. However, our genome-wide DNA methylation profiles and immunofluorescence localization analyses showed that redundancy between AGO4 and AGO6 is unexpectedly negligible in the genetic interactions and AGO4 and AGO6 mainly act sequentially in mediating RdDM

Characterization of the expression of transcriptionally silent loci during the plant response against Pseudomonas syringae

Pseudomonas syringae es una bacteria en forma de bacilo, Gram-negativa, hemibiotrófa y con flagelos polares, que provoca una amplia variedad de síntomas en plantas, incluyendo manchas necróticas y/o cloróticas foliares y agallas. Pseudomonas syringae sobrevive en las superficies de las hojas como una epífita, antes de entrar en el espacio intercelular a través de aberturas naturales como estomas o heridas, para iniciar el proceso de infección (Hirano and Upper, 2000). P. syringae pv. tomato (en adelante Pto) DC3000, la principal estirpe modelo para el estudio de la interacción de P. syringae con el huésped, es el agente causante de la mancha bacteriana en tomate, y también puede causar enfermedad en la planta modelo Arabidopsis thaliana. Pto posee un sistema de secreción tipo III (T3SS) que es esencial para su patogénesis. El T3SS es un complejo aparato de secreción compuesto de aproximadamente 30 proteínas diferentes. Este sofisticado aparato acopla la secreción de proteínas denominadas efectores,a través de las membranas interna y externa de la bacteria,con la translocación a través de las membranas citoplasmáticas eucariotas, así como a través de la pared celular en el caso del T3SS de bacterias patógenas de plantas (Nguyen et al., 2000). Estudios recientes de la interacción planta-huésped han demostrado que las bacterias fitopatógenas modifican el epigenoma del huésped y que las plantas han desarrollado defensas específicas contra la supresión del silenciamiento transcripcional, orquestada por agentes patógenos. Estos estudios sugieren que la metilación de DNA dirigida por RNA (RdDM) y el silenciamiento génico transcripcional juegan un papel importante en la interacción planta-huésped (Pumplin and Voinnet, 2013). El objetivo de esta Tesis Doctoral ha sido profundizar en el conocimiento de la interacción planta-huésped a nivel epigenético. Para ello se han analizado los cambios que se producen en el estado de metilación del genoma de Arabidopsis y en la activación de loci silenciados transcripcionalmente durante la infección por Pto. Se ha caracterizado la importancia de los determinantes de virulencia de P. syringae y de los distintos mecanismos de defensa en la activación de los loci silenciados transcripcionalmente. Además se ha profundizado en la importancia que tienen los procesos de metilación y desmetilación del DNA en la respuesta de defensa de Arabidopsis frente a P. syringae. Hirano, S.S. and Upper, C.D. (2000) Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae - A pathogen, ice nucleus, and epiphyte. Microbiology and Molecular Biology Reviews 64, 624-653. Nguyen, L., Paulsen, I.T., Tchieu, J., Hueck, C.J. and Saier Jr, M.H. (2000) Phylogenetic analyses of the constituents of Type III protein secretion systems. Journal of Molecular Microbiology and Biotechnology 2, 125-144. Pumplin, N. and Voinnet, O. (2013) RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nature Reviews Microbiology 11, 745-760

MTHFD1 controls DNA methylation in Arabidopsis.

DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases

Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine

Covalent modification by methylation of cytosine residues represents an important epigenetic hallmark. While sequence analysis after bisulphite conversion allows correlative analyses with single-base resolution, functional analysis by interference with DNA methylation is less precise, due to the complexity of methylation enzymes and their targets. A cytidine analogue, 5-azacytidine, is frequently used as an inhibitor of DNA methyltransferases, but its rapid degradation in aqueous solution is problematic for culture periods of longer than a few hours. Application of zebularine, a more stable cytidine analogue with a similar mode of action that is successfully used as a methylation inhibitor in Neurospora and mammalian tumour cell lines, can significantly reduce DNA methylation in plants in a dose-dependent and transient manner independent of sequence context. Demethylation is connected with transcriptional reactivation and partial decondensation of heterochromatin. Zebularine represents a promising new and versatile tool for investigating the role of DNA methylation in plants with regard to transcriptional control, maintenance and formation of (hetero-) chromatin