Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis.

Chromosomes form 3D structures that are critical to the regulation of cellular and genetic processes. Here, we present a study of global chromatin interaction patterns in Arabidopsis thaliana. Our genome-wide approach confirmed interactions that were previously observed by other methods as well as uncovered long-range interactions such as those among small heterochromatic regions embedded in euchromatic arms. We also found that interactions are correlated with various epigenetic marks that are localized in active or silenced chromatin. Arabidopsis chromosomes do not contain large local interactive domains that resemble the topological domains described in animals but, instead, contain relatively small interactive regions scattered around the genome that contain H3K27me3 or H3K9me2. We generated interaction maps in mutants that are defective in specific epigenetic pathways and found altered interaction patterns that correlate with changes in the epigenome. These analyses provide further insights into molecular mechanisms of epigenetic regulation of the genome.

Large-scale heterochromatin remodeling linked to overreplication-associated DNA damage.

Previously, we have shown that loss of the histone 3 lysine 27 (H3K27) monomethyltransferases ARABIDOPSIS TRITHORAX-RELATED 5 (ATXR5) and ATXR6 (ATXR6) results in the overreplication of heterochromatin. Here we show that the overreplication results in DNA damage and extensive chromocenter remodeling into unique structures we have named "overreplication-associated centers" (RACs). RACs have a highly ordered structure with an outer layer of condensed heterochromatin, an inner layer enriched in the histone variant H2AX, and a low-density core containing foci of phosphorylated H2AX (a marker of double-strand breaks) and the DNA-repair enzyme RAD51. atxr5,6 mutants are strongly affected by mutations in DNA repair, such as ATM and ATR. Because of its dense packaging and repetitive DNA sequence, heterochromatin is a challenging environment in which to repair DNA damage. Previous work in animals has shown that heterochromatic breaks are translocated out of the heterochromatic domain for repair. Our results show that atxr5,6 mutants use a variation on this strategy for repairing heterochromatic DNA damage. Rather than being moved to adjacent euchromatic regions, as in animals, heterochromatin undergoes large-scale remodeling to create a compartment with low chromatin density.

Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production.

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.

Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency.

Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases AID and APOBEC1 can deaminate 5-methylcytosine in vitro and in Escherichia coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulphite next generation sequencing in wild-type and AID-deficient mouse PGCs at embryonic day (E)13.5. Wild-type PGCs revealed marked genome-wide erasure of methylation to a level below that of methylation deficient (Np95(-/-), also called Uhrf1(-/-)) embryonic stem cells, with female PGCs being less methylated than male ones. By contrast, AID-deficient PGCs were up to three times more methylated than wild-type ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in AID-deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. AID deficiency interferes with genome-wide erasure of DNA methylation patterns, indicating that AID has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.

Relationship between nucleosome positioning and DNA methylation.

Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.

Evolutionary insights from de novo transcriptome assembly and SNP discovery in California white oaks.

BACKGROUND: Reference transcriptomes provide valuable resources for understanding evolution within and among species. We de novo assembled and annotated a reference transcriptome for Quercus lobata and Q. garryana and identified single-nucleotide polymorphisms (SNPs) to provide resources for forest genomicists studying this ecologically and economically important genus. We further performed preliminary analyses of genes important in interspecific divergent (positive) selection that might explain ecological differences among species, estimating rates of nonsynonymous to synonymous substitutions (d N/d S) and Fay and Wu's H. Functional classes of genes were tested for unusually high d N/d S or low H consistent with divergent positive selection. RESULTS: Our draft transcriptome is among the most complete for oaks, including 83,644 contigs (23,329 ≥ 1 kbp), 14,898 complete and 13,778 partial gene models, and functional annotations for 9,431 Arabidopsis orthologs and 19,365 contigs with Pfam hits. We identified 1.7 million possible sequence variants including 1.1 million high-quality diallelic SNPs - among the largest sets identified in any tree. 11 of 18 functional categories with significantly elevated d N/d S are involved in disease response, including 50+ genes with d N/d S > 1. Other high-d N/d S genes are involved in biotic response, flowering and growth, or regulatory processes. In contrast, median d N/d S was low (0.22), suggesting that purifying selection influences most genes. No functional categories have unusually low H. CONCLUSIONS: These results offer preliminary support for the hypothesis that divergent selection at pathogen resistance are important factors in species divergence in these hybridizing California oaks. Our transcriptome provides a solid foundation for future studies of gene expression, natural selection, and speciation in Quercus.

Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.

Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences. Recent genomic studies in Arabidopsis thaliana have revealed that many endogenous genes are methylated either within their promoters or within their transcribed regions, and that gene methylation is highly correlated with transcription levels. However, plants have different types of methylation controlled by different genetic pathways, and detailed information on the methylation status of each cytosine in any given genome is lacking. To this end, we generated a map at single-base-pair resolution of methylated cytosines for Arabidopsis, by combining bisulphite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyser and Solexa sequencing technology. This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genome-wide scale within specific sequence contexts. Here we describe methylation on previously inaccessible components of the genome and analyse the DNA methylation sequence composition and distribution. We also describe the effect of various DNA methylation mutants on genome-wide methylation patterns, and demonstrate that our newly developed library construction and computational methods can be applied to large genomes such as that of mouse.

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.

Conservation and divergence of methylation patterning in plants and animals.

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.

High-quality genome and methylomes illustrate features underlying evolutionary success of oaks.

The genus Quercus, which emerged ∼55 million years ago during globally warm temperatures, diversified into ∼450 extant species. We present a high-quality de novo genome assembly of a California endemic oak, Quercus lobata, revealing features consistent with oak evolutionary success. Effective population size remained large throughout history despite declining since early Miocene. Analysis of 39,373 mapped protein-coding genes outlined copious duplications consistent with genetic and phenotypic diversity, both by retention of genes created during the ancient γ whole genome hexaploid duplication event and by tandem duplication within families, including numerous resistance genes and a very large block of duplicated DUF247 genes, which have been found to be associated with self-incompatibility in grasses. An additional surprising finding is that subcontext-specific patterns of DNA methylation associated with transposable elements reveal broadly-distributed heterochromatin in intergenic regions, similar to grasses. Collectively, these features promote genetic and phenotypic variation that would facilitate adaptability to changing environments.

Algal Functional Annotation Tool: a web-based analysis suite to functionally interpret large gene lists using integrated annotation and expression data.

BACKGROUND: Progress in genome sequencing is proceeding at an exponential pace, and several new algal genomes are becoming available every year. One of the challenges facing the community is the association of protein sequences encoded in the genomes with biological function. While most genome assembly projects generate annotations for predicted protein sequences, they are usually limited and integrate functional terms from a limited number of databases. Another challenge is the use of annotations to interpret large lists of 'interesting' genes generated by genome-scale datasets. Previously, these gene lists had to be analyzed across several independent biological databases, often on a gene-by-gene basis. In contrast, several annotation databases, such as DAVID, integrate data from multiple functional databases and reveal underlying biological themes of large gene lists. While several such databases have been constructed for animals, none is currently available for the study of algae. Due to renewed interest in algae as potential sources of biofuels and the emergence of multiple algal genome sequences, a significant need has arisen for such a database to process the growing compendiums of algal genomic data. DESCRIPTION: The Algal Functional Annotation Tool is a web-based comprehensive analysis suite integrating annotation data from several pathway, ontology, and protein family databases. The current version provides annotation for the model alga Chlamydomonas reinhardtii, and in the future will include additional genomes. The site allows users to interpret large gene lists by identifying associated functional terms, and their enrichment. Additionally, expression data for several experimental conditions were compiled and analyzed to provide an expression-based enrichment search. A tool to search for functionally-related genes based on gene expression across these conditions is also provided. Other features include dynamic visualization of genes on KEGG pathway maps and batch gene identifier conversion. CONCLUSIONS: The Algal Functional Annotation Tool aims to provide an integrated data-mining environment for algal genomics by combining data from multiple annotation databases into a centralized tool. This site is designed to expedite the process of functional annotation and the interpretation of gene lists, such as those derived from high-throughput RNA-seq experiments. The tool is publicly available at http://pathways.mcdb.ucla.edu.

Copy number variation influences gene expression and metabolic traits in mice.

Copy number variants (CNVs) are genomic segments which are duplicated or deleted among different individuals. CNVs have been implicated in both Mendelian and complex traits, including immune and behavioral disorders, but the study of the mechanisms by which CNVs influence gene expression and clinical phenotypes in humans is complicated by the limited access to tissues and by population heterogeneity. We now report studies of the effect of 19 CNVs on gene expression and metabolic traits in a mouse intercross between strains C57BL/6J and C3H/HeJ. We found that 83% of genes predicted to occur within CNVs were differentially expressed. The expression of most CNV genes was correlated with copy number, but we also observed evidence that gene expression was altered in genes flanking CNVs, suggesting that CNVs may contain regulatory elements for these genes. Several CNVs mapped to hotspots, genomic regions influencing expression of tens or hundreds of genes. Several metabolic traits including cholesterol, triglycerides, glucose and body weight mapped to three CNVs in the genome, in mouse chromosomes 1, 4 and 17. Predicted CNV genes, such as Itlna, Defcr-1, Trim12 and Trim34 were highly correlated with these traits. Our results suggest that CNVs have a significant impact on gene expression and that CNVs may be playing a role in the mechanisms underlying metabolic traits in mice.

BS Seeker: precise mapping for bisulfite sequencing.

BACKGROUND: Bisulfite sequencing using next generation sequencers yields genome-wide measurements of DNA methylation at single nucleotide resolution. Traditional aligners are not designed for mapping bisulfite-treated reads, where the unmethylated Cs are converted to Ts. We have developed BS Seeker, an approach that converts the genome to a three-letter alphabet and uses Bowtie to align bisulfite-treated reads to a reference genome. It uses sequence tags to reduce mapping ambiguity. Post-processing of the alignments removes non-unique and low-quality mappings. RESULTS: We tested our aligner on synthetic data, a bisulfite-converted Arabidopsis library, and human libraries generated from two different experimental protocols. We evaluated the performance of our approach and compared it to other bisulfite aligners. The results demonstrate that among the aligners tested, BS Seeker is more versatile and faster. When mapping to the human genome, BS Seeker generates alignments significantly faster than RMAP and BSMAP. Furthermore, BS Seeker is the only alignment tool that can explicitly account for tags which are generated by certain library construction protocols. CONCLUSIONS: BS Seeker provides fast and accurate mapping of bisulfite-converted reads. It can work with BS reads generated from the two different experimental protocols, and is able to efficiently map reads to large mammalian genomes. The Python program is freely available at http://pellegrini.mcdb.ucla.edu/BS_Seeker/BS_Seeker.html.

Tissue-Specific Transcriptomes Reveal Gene Expression Trajectories in Two Maturing Skin Epithelial Layers in Zebrafish Embryos.

Epithelial cells are the building blocks of many organs, including skin. The vertebrate skin initially consists of two epithelial layers, the outer periderm and inner basal cell layers, which have distinct properties, functions, and fates. The embryonic periderm ultimately disappears during development, whereas basal cells proliferate to form the mature, stratified epidermis. Although much is known about mechanisms of homeostasis in mature skin, relatively little is known about the two cell types in pre-stratification skin. To define the similarities and distinctions between periderm and basal skin epithelial cells, we purified them from zebrafish at early development stages and deeply profiled their gene expression. These analyses identified groups of genes whose tissue enrichment changed at each stage, defining gene flow dynamics of maturing vertebrate epithelia. At each of 52 and 72 hr post-fertilization (hpf), more than 60% of genes enriched in skin cells were similarly expressed in both layers, indicating that they were common epithelial genes, but many others were enriched in one layer or the other. Both expected and novel genes were enriched in periderm and basal cell layers. Genes encoding extracellular matrix, junctional, cytoskeletal, and signaling proteins were prominent among those distinguishing the two epithelial cell types. In situ hybridization and BAC transgenes confirmed our expression data and provided new tools to study zebrafish skin. Collectively, these data provide a resource for studying common and distinguishing features of maturing epithelia.

Modeling the regulatory network of histone acetylation in Saccharomyces cerevisiae.

Acetylation of histones plays an important role in regulating transcription. Histone acetylation is mediated partly by the recruitment of specific histone acetyltransferases (HATs) and deacetylases (HDACs) to genomic loci by transcription factors, resulting in modulation of gene expression. Although several specific interactions between transcription factors and HATs and HDACs have been elaborated in Saccharomyces cerevisiae, the full regulatory network remains uncharacterized. We have utilized a linear regression of optimized sigmoidal functions to correlate transcription factor binding patterns to the acetylation profiles of 11 lysines in the four core histones measured at all S. cerevisiae promoters. The resulting associations are combined with large-scale protein-protein interaction data sets to generate a comprehensive model that relates recruitment of specific HDACs and HATs to transcription factors and their target genes and the resulting effects on individual lysines. This model provides a broad and detailed view of the regulatory network, describing which transcription factors are most significant in regulating acetylation of specific lysines at defined promoters. We validate the model, both computationally and experimentally, to demonstrate that it yields accurate predictions of these regulatory mechanisms.

Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype.

BACKGROUND: Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids. RESULTS: We sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction, and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches. CONCLUSIONS: Functional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.

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.

Utilizing logical relationships in genomic data to decipher cellular processes.

The wealth of available genomic data has spawned a corresponding interest in computational methods that can impart biological meaning and context to these experiments. Traditional computational methods have drawn relationships between pairs of proteins or genes based on notions of equality or similarity between their patterns of occurrence or behavior. For example, two genes displaying similar variation in expression, over a number of experiments, may be predicted to be functionally related. We have introduced a natural extension of these approaches, instead identifying logical relationships involving triplets of proteins. Triplets provide for various discrete kinds of logic relationships, leading to detailed inferences about biological associations. For instance, a protein C might be encoded within an organism if, and only if, two other proteins A and B are also both encoded within the organism, thus suggesting that gene C is functionally related to genes A and B. The method has been applied fruitfully to both phylogenetic and microarray expression data, and has been used to associate logical combinations of protein activity with disease state phenotypes, revealing previously unknown ternary relationships among proteins, and illustrating the inherent complexities that arise in biological data.

MORC family ATPases required for heterochromatin condensation and gene silencing.

Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause derepression of DNA-methylated genes and TEs but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, which are predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes.

Identification of genes required for de novo DNA methylation in Arabidopsis.

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1, and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.