Alexis Sarazin

Integrative epigenomic mapping defines four main chromatin states in Arabidopsis

Post‐translational modification of histones and DNA methylation are important components of chromatin‐level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ∼90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin‐based regulatory mechanisms in plants.

A Role for RNAi in the Selective Correction of DNA Methylation Defects

DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, which suggests that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis (as in cancer) or meiosis, leading to epiallelic variation. We demonstrated the existence of an efficient mechanism that protects against transgenerational loss of DNA methylation in Arabidopsis. Remethylation is specific to the subset of heavily methylated repeats that are targeted by the RNA interference (RNAi) machinery. This process does not spread into flanking regions, is usually progressive over several generations, and faithfully restores wild-type methylation over target sequences in an RNAi-dependent manner. Our findings suggest an important role for RNAi in protecting genomes against long-term epigenetic defects.

The Arabidopsis miR472-RDR6 Silencing Pathway Modulates PAMP- and Effector-Triggered Immunity through the Post-transcriptional Control of Disease Resistance Genes

RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) is a key RNA silencing factor initially characterized in transgene silencing and virus resistance. This enzyme also contributes to the biosynthesis of endogenous short interfering RNAs (siRNAs) from non-coding RNAs, transposable elements and protein-coding transcripts. One class of protein-coding transcripts that have recently emerged as major sources of RDR6-dependent siRNAs are nucleotide-binding leucine-rich repeat (NB-LRR) proteins, a family of immune-receptors that perceive specific pathogen effector proteins and mount Effector-Triggered Immunity (ETI). Nevertheless, the dynamic post-transcriptional control of NB-LRR transcripts during the plant immune response and the functional relevance of NB-LRRs in signaling events triggered by Pathogen-Associated Molecular Patterns (PAMPs) remain elusive. Here, we show that PTI is constitutive and sensitized in the Arabidopsis rdr6 loss-of-function mutant, implicating RDR6 as a novel negative regulator of PTI. Accordingly, rdr6 mutant exhibits enhanced basal resistance towards a virulent Pseudomonas syringae strain. We further provide evidence that dozens of CC-NB-LRRs (CNLs), including the functionally characterized RPS5 gene, are post-transcriptionally controlled by RDR6 both constitutively and during PTI. These CNL transcripts are also regulated by the Arabidopsis microRNA miR472 and knock-down of this miRNA recapitulates the PTI and basal resistance phenotypes observed in the rdr6 mutant background. Furthermore, both miR472 and rdr6 mutants were more resistant to Pto DC3000 expressing AvrPphB, a bacterial effector recognized by the disease resistance protein RPS5, whereas transgenic plants overexpressing miR472 were more susceptible to this bacterial strain. Finally, we show that the enhanced basal and RPS5-mediated resistance phenotypes observed in the rdr6 mutant are dependent on the proper chaperoning of NB-LRR proteins, and might therefore be due to the enhanced accumulation of CNL proteins whose cognate mRNAs are no longer controlled by RDR6-dependent siRNAs. Altogether, this study supports a model whereby the miR472- and RDR6-mediated silencing pathway represents a key regulatory checkpoint modulating both PTI and ETI responses through the post-transcriptional control of disease resistance genes.

Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis

Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5′ to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.

Features of the Arabidopsis recombination landscape resulting from the combined loss of sequence variation and DNA methylation

The rate of meiotic crossing over (CO) varies considerably along chromosomes, leading to marked distortions between physical and genetic distances. The causes underlying this variation are being unraveled, and DNA sequence and chromatin states have emerged as key factors. However, the extent to which the suppression of COs within the repeat-rich pericentromeric regions of plant and mammalian chromosomes results from their high level of DNA polymorphisms and from their heterochromatic state, notably their dense DNA methylation, remains unknown. Here, we test the combined effect of removing sequence polymorphisms and repeat-associated DNA methylation on the meiotic recombination landscape of an Arabidopsis mapping population. To do so, we use genome-wide DNA methylation data from a large panel of isogenic epigenetic recombinant inbred lines (epiRILs) to derive a recombination map based on 126 meiotically stable, differentially methylated regions covering 81.9% of the genome. We demonstrate that the suppression of COs within pericentromeric regions of chromosomes persists in this experimental setting. Moreover, suppression is reinforced within 3-Mb regions flanking pericentromeric boundaries, and this effect appears to be compensated by increased recombination activity in chromosome arms. A direct comparison with 17 classical Arabidopsis crosses shows that these recombination changes place the epiRILs at the boundary of the range of natural variation but are not severe enough to transgress that boundary significantly. This level of robustness is remarkable, considering that this population represents an extreme with key recombination barriers having been forced to a minimum.

Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.

RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells

At the request of the authors, PLOS Genetics is retracting this publication following an investigation into concerns that were raised regarding the assembly of Fig 4 and S4 Fig, and the statistical analysis used in Fig 2A. The text below has been agreed to by the authors and editors. The corresponding author, Olivier Voinnet, was originally alerted to errors that occurred during the assembly of Fig 4 (panel A) and S4 Fig (panels A and F). These errors have been corrected using the original raw data, and a correction notice was published accordingly. Further analysis of the paper revealed flaws in the interpretation of the transposition data presented in Fig 2A. In the originally submitted version, the L1 copy number was only presented for the DCR P10 and DCR P30 cells, and a T-test performed on the two datasets showed that the L1 copy number was statistically higher in DCR cells than in control cells. During the last stage of the review process, additional datasets were added and a second T-test was then used to establish the statistical analysis published in the final version of the paper. However, it was later realized that T-tests are not appropriate for comparing more than two datasets. At the recommendation of the ETH statistics helpdesk, a suitable Analysis of Variance (ANOVA) test with multiple comparisons was then conducted on the Dcr P30 and Dcr P30 datasets, providing a p-value of 0.0501, which is at the margin of the threshold of significance. The ANOVA test conducted on the Dcr P10 and Dcr P30 datasets revealed a statistically significant p-value of 0.0018. The statistical issue regarding the L1 copy number in DCR versus control ES cells is currently being addressed using a new set of cells and a direct GFP-based transposition assay. This issue will hopefully be clarified in the near future via the submission of an amended study for peer-review. Based on the present uncertainty revealed by the corrected statistical analysis of the L1 copy number—a key element of this paper—and on the previous errors in the figures, the authors have collectively decided to retract this study. Constance Ciaudo and Olivier Voinnet take full responsibility for the mistakes on this paper and wish to apologize. They also wish to state that none of the above-mentioned mistakes involved any of the co-authors from the Curie Institute, whose contributions to the paper were restricted to the bioinformatics analysis of small RNAs (NS, CJC, EB) and the generation of reagents including an ES cell line required for the study (EH, IO). All authors regret the inconvenience caused.

Exploring new models of easiRNA biogenesis

Although silent transposons in plants can be reactivated by stress or during development, their potential deleterious effects are prevented by transposon-derived epigenetically activated small interfering RNAs (easiRNAs). A new study shows how serendipitous interactions between reactivated transposons and endogenous microRNAs might initiate easiRNA biogenesis, establishing an unexpected link between these two classes of silencing small RNAs.

Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays.

Small RNA (sRNA) populations extracted from Arabidopsis plants submitted or not to biotic stress, were reverse-transcribed into cDNAs, and these were subsequently hybridized after labelling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by Massive Parallel Sequence Signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in small RNA populations.

01-P002 Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development. Citation: Ciaudo C, Servant N, Cognat V, Sarazin A, Kieffer E, et al. (2009) Highly Dynamic and Sex-Specific Expression of microRNAs During Early ES Cell Differentiation. PLoS Genet 5(8): e1000620. doi:10.1371/journal.pgen.1000620 Editor: Michael T. McManus, University of California San Francisco, United States of America Received April 21, 2009; Accepted July 29, 2009; Published August 28, 2009 Copyright: 2009 Ciaudo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by a prize from the Schlumberger Foundation for Education and Research awarded to OV and a starting grant from the European Research Council ‘‘Frontiers of RNAi’’ ERC 210890 to OV; and by a grant from the Fondation pour la Recherche Medicale (Equipe FRM), a prize from the Schlumberger Foundation for Education and Research to EH, and by HEROIC (Highthroughput Epigenetic Regulatory Organization in Chromatin), an Integrated Project funded by the European Union under the Framework Programme (LSHG-CT-2005-018883) to EH. CC was supported by a postdoctoral fellowship (Fondation pour la Recherche Medicale). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Edith.Heard@curie.fr (EH); olivier.voinnet@ibmp-ulp.u-strasbg.fr (OV)