Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome.

The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements. It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial. We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems. In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes. The representation of unique maize sequences not found in plant reference genomes is also greatly enriched. This demonstrates that repeats, and not genes, are the primary targets of methylation in maize. The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required.

Dicer promotes genome stability via the bromodomain transcriptional co-activator BRD4.

RNA interference is required for post-transcriptional silencing, but also has additional roles in transcriptional silencing of centromeres and genome stability. However, these roles have been controversial in mammals. Strikingly, we found that Dicer-deficient embryonic stem cells have strong proliferation and chromosome segregation defects as well as increased transcription of centromeric satellite repeats, which triggers the interferon response. We conducted a CRISPR-Cas9 genetic screen to restore viability and identified transcriptional activators, histone H3K9 methyltransferases, and chromosome segregation factors as suppressors, resembling Dicer suppressors identified in independent screens in fission yeast. The strongest suppressors were mutations in the transcriptional co-activator Brd4, which reversed the strand-specific transcription of major satellite repeats suppressing the interferon response, and in the histone acetyltransferase Elp3. We show that identical mutations in the second bromodomain of Brd4 rescue Dicer-dependent silencing and chromosome segregation defects in both mammalian cells and fission yeast. This remarkable conservation demonstrates that RNA interference has an ancient role in transcriptional silencing and in particular of satellite repeats, which is essential for cell cycle progression and proper chromosome segregation. Our results have pharmacological implications for cancer and autoimmune diseases characterized by unregulated transcription of satellite repeats.

DNA methylation and epigenetic inheritance in plants and filamentous fungi.

Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. We consider the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation.

Arabidopsis thaliana DNA methylation mutants.

Three DNA hypomethylation mutants of the flowering plant Arabidopsis thaliana were isolated by screening mutagenized populations for plants containing centromeric repetitive DNA arrays susceptible to digestion by a restriction endonuclease that was sensitive to methylated cytosines. The mutations are recessive, and at least two are alleles of a single locus, designated DDM1 (for decrease in DNA methylation). Amounts of 5-methylcytosine were reduced over 70 percent in ddm1 mutants. Despite this reduction in DNA methylation levels, ddm1 mutants developed normally and exhibited no striking morphological phenotypes. However, the ddm1 mutations are associated with a segregation distortion phenotype. The ddm1 mutations were used to demonstrate that de novo DNA methylation in vivo is slow.

Gene trap tagging of PROLIFERA, an essential MCM2-3-5-like gene in Arabidopsis.

Gene trap transposon mutagenesis can identify essential genes whose functions in later development are obscured by an early lethal phenotype. In higher plants, many genes are required for haploid gametophyte viability, so that the phenotypic effects of their disruption cannot be readily observed in the diploid plant body. The PROLIFERA (PRL) gene, identified by gene trap transposon mutagenesis in Arabidopsis, is required for megaga-metophyte and embryo development. Reporter gene expression patterns revealed that PRL was expressed in dividing cells throughout the plant. PRL is related to the MCM2-3-5 family of yeast genes that are required for the initiation of DNA replication.

Genetic definition and sequence analysis of Arabidopsis centromeres.

High-precision genetic mapping was used to define the regions that contain centromere functions on each natural chromosome in Arabidopsis thaliana. These regions exhibited dramatic recombinational repression and contained complex DNA surrounding large arrays of 180-base pair repeats. Unexpectedly, the DNA within the centromeres was not merely structural but also encoded several expressed genes. The regions flanking the centromeres were densely populated by repetitive elements yet experienced normal levels of recombination. The genetically defined centromeres were well conserved among Arabidopsis ecotypes but displayed limited sequence homology between different chromosomes, excluding repetitive DNA. This investigation provides a platform for dissecting the role of individual sequences in centromeres in higher eukaryotes.

DNA methylation in eukaryotes.

Recent advances have expanded our understanding of the processes underlying the establishment, maintenance, and elaboration of DNA methylation patterns in eukaryotes. The functional significance of DNA methylation is sought in a comparison of results on a variety of epigenetic phenomena in different eukaryotes. The recent development of DNA methylation mutants in mice, Neurospora, and Arabadopsis will allow traditional genetic dissection to be applied to long-standing problems regarding the function and regulation of eukaryotic DNA methylation. Although methylation appears to be important for maintenance of different epigenetic states, the mechanism that establishes these states is likely to involve additional processes.

RNA interference is essential for cellular quiescence.

Quiescent cells play a predominant role in most organisms. Here we identify RNA interference (RNAi) as a major requirement for quiescence (G0 phase of the cell cycle) in Schizosaccharomyces pombe RNAi mutants lose viability at G0 entry and are unable to maintain long-term quiescence. We identified suppressors of G0 defects in cells lacking Dicer (dcr1Δ), which mapped to genes involved in chromosome segregation, RNA polymerase-associated factors, and heterochromatin formation. We propose a model in which RNAi promotes the release of RNA polymerase in cycling and quiescent cells: (i) RNA polymerase II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G0 entry, and (ii) RNA polymerase I release prevents heterochromatin formation at ribosomal DNA during quiescence maintenance. Our model may account for the codependency of RNAi and histone H3 lysine 9 methylation throughout eukaryotic evolution.

A diffusion model for the coordination of DNA replication in Schizosaccharomyces pombe.

The locations of proteins and epigenetic marks on the chromosomal DNA sequence are believed to demarcate the eukaryotic genome into distinct structural and functional domains that contribute to gene regulation and genome organization. However, how these proteins and epigenetic marks are organized in three dimensions remains unknown. Recent advances in proximity-ligation methodologies and high resolution microscopy have begun to expand our understanding of these spatial relationships. Here we use polymer models to examine the spatial organization of epigenetic marks, euchromatin and heterochromatin, and origins of replication within the Schizosaccharomyces pombe genome. These models incorporate data from microscopy and proximity-ligation experiments that inform on the positions of certain elements and contacts within and between chromosomes. Our results show a striking degree of compartmentalization of epigenetic and genomic features and lead to the proposal of a diffusion based mechanism, centred on the spindle pole body, for the coordination of DNA replication in S. pombe.

Duplication and suppression of chloroplast protein translocation genes in maize.

The HCF106 (high chlorophyll fluorescence) gene of maize encodes a chloroplast membrane protein required for translocation of a subset of proteins across the thylakoid membrane. Mutations in HCF106 caused by the insertion of Robertson's Mutator transposable elements have been mapped to chromosome 2S. Here we show that there is a closely related homolog of HCF106 encoded elsewhere in the maize genome (HCF106c) that can partially compensate for these mutations. This homolog maps on chromosome 10L and is part of the most recent set of segmental duplications in the maize genome. Triple mutants that are disrupted in both the HCF106 and Sec-dependent protein translocation pathways provide evidence that they act independently. The HCF106c gene accounts for a previously reported exception to the correlation between epigenetic suppression of hcf106 and methylation of Mutator transposons. We also demonstrate that insertions of Robertson's Mutator elements into either introns or promoters can lead to mutations whose phenotypes are suppressed in the absence of Mu activity, while alleles with insertions in both positions are not suppressed. The implications of these observations are discussed.

Reprogramming the epigenome in Arabidopsis pollen.

Epigenetic reprogramming in Arabidopsis thaliana occurs in developing pollen. The male gametophyte is derived from haploid microspores via two postmeiotic cell divisions to give rise to the gametes (sperm cells, SC) and the vegetative cell (VC). The purification of individual cell types during pollen development coupled with genome-wide DNA methylation analysis and small RNA sequencing has revealed a dynamic regulation of the epigenome during gametogenesis. Interestingly, imprinted loci and previously identified variable epialleles are hypermethylated in the germline; however, their stability after fertilization appears to require targeted demethylation in the neighboring vegetative cell nucleus, possibly by releasing mobile small RNAs that reinforce transcriptional gene silencing and DNA methylation in the gametes. These results have led to a new model for the establishment and transgenerational maintenance of epigenetic marks in flowering plants.

Germline reprogramming of heterochromatin in plants.

Heterochromatin is composed of transposable elements (TEs) and other repeats and was once considered to be a wasteland of redundant genetic material and potentially harmful TE. Therefore, the reprogramming of heterochromatin and subsequent reactivation of TE in the immature seed and pollen is paradoxical in plants. Recent studies have shown that reactivation of TE occurs specifically in germline companion cells, the vegetative nucleus (VN) in pollen (Slotkin et al. 2009) and the endosperm in seed (Gehring et al. 2009). In the ovule, ARGONAUTE 9 (AGO9) not only has a role in silencing TE in the egg cell but also in preventing the formation of multiple asexual gametophytes (Olmedo-Monfil et al. 2010). We propose that reprogramming of heterochromatin in germline companion cells reveals TE in a controlled manner to expose them within the germline and, by the production of small interfering RNA (siRNA), ensures TE silencing in the next generation. We also propose that the mechanisms evolved to silence TE may actually promote sexual reproduction by inhibiting the formation of asexual gametes.

Darwin's "Abominable Mystery": the role of RNA interference in the evolution of flowering plants.

Darwin was famously concerned that the sudden appearance and rapid diversification of flowering plants in the mid-Cretaceous could not have occurred by gradual change. Here, we review our attempts to resolve the relationships among the major seed plant groups, i.e., cycads, ginkgo, conifers, gnetophytes, and flowering plants, and to provide a pipeline in which these relationships can be used as a platform for identifying genes of functional importance in plant diversification. Using complete gene sets and unigenes from 16 plant species, genes with positive partitioned Bremer support at major nodes were used to identify overrepresented gene ontology (GO) terms. Posttranscriptional silencing via RNA interference (RNAi) was overrepresented at several major nodes, including between monocots and dicots during early angiosperm divergence. One of these genes, RNA-dependent RNA polymerase 6, is required for the biogenesis of trans-acting small interfering RNA (tasiRNA), confers heteroblasty and organ polarity, and restricts maternal specification of the germline. Processing of small RNA and transfer between neighboring cells underlies these roles and may have contributed to distinct mutant phenotypes in plants, and in particular in the early split of the monocots and eudicots.

Epigenetic inheritance and reprogramming in plants and fission yeast.

Plants and fission yeast exhibit a wealth of epigenetic phenomena, including transposon regulation, heterochromatic silencing, and gene imprinting. They provide excellent model organisms to address the question of how epigenetic information is propagated to daughter cells. We have addressed the questions of establishment, maintenance, and inheritance of heterochromatic silencing using the fission yeast Schizosaccharomyces pombe and the plant Arabidopsis thaliana by using a variety of genetic and genomic approaches. We present here results showing the cell cycle dependence of RNA in fission yeast RNA interference (RNAi), which is required for proper transcriptional silencing of the centromeric heterochromatin, and that this process occurs during S phase, allowing for precise copying and reestablishment of heterochromatic histone modifications following DNA replication and cell division. We also show that in plants, cells in culture and male germ-line cells undergo massive epigenomic changes correlated with the appearance of a novel class of 21-nucleotide small interfering RNA (siRNA) from transcriptionally reactivated transposable elements (TEs) following loss of heterochromatic DNA and histone methylation. We propose a model for the role of deliberate TE reactivation in germ-line companion cells as part of a developmental mechanism for first revealing and then silencing TEs via small RNA, which may contribute to reprogramming during early development in plants and animals.

Slicing and spreading of heterochromatic silencing by RNA interference.

RNA interference (RNAi) can mediate gene silencing posttranscriptionally by target RNA cleavage, or transcriptionally by chromatin and DNA modification. Argonaute is an essential component of the RNAi machinery that displays endonucleolytic activity guided by bound small RNAs. This slicing activity has recently been shown to be required for gene silencing and spreading of histone modifications characteristic of heterochromatin in Schizosaccharomyces pombe. Argonaute proteins with catalytic and nucleic acid binding capacities are found to function in RNAi within both the plant and animal kingdoms. Here we review the requirement of slicing for silencing and spreading in S. pombe, plants, and humans.

Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets.

Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5 mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RARalpha, BMP7 and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples.

Weeding out the genes: the Arabidopsis genome project.

The Arabidopsis genome sequence is scheduled for completion at the end of this year (December 2000). It will be the first higher plant genome to be sequenced, and will allow a detailed comparison with bacterial, yeast and animal genomes. Already, two of the five chromosomes have been sequenced, and we have had our first glimpse of higher eukaryotic centromeres, and the structure of heterochromatin. The implications for understanding plant gene function, genome structure and genome organization are profound. In this review, the lessons learned for future genome projects are reviewed as well as a summary of the initial findings in Arabidopsis.

Functional genomics: probing plant gene function and expression with transposons.

Transposable elements provide a convenient and flexible means to disrupt plant genes, so allowing their function to be assessed. By engineering transposons to carry reporter genes and regulatory signals, the expression of target genes can be monitored and to some extent manipulated. Two strategies for using transposons to assess gene function are outlined here: First, the PCR can be used to identify plants that carry insertions into specific genes from among pools of heavily mutagenized individuals (site-selected transposon mutagenesis). This method requires that high copy transposons be used and that a relatively large number of reactions be performed to identify insertions into genes of interest. Second, a large library of plants, each carrying a unique insertion, can be generated. Each insertion site then can be amplified and sequenced systematically. These two methods have been demonstrated in maize, Arabidopsis, and other plant species, and the relative merits of each are discussed in the context of plant genome research.

An unusual wheat insertion sequence (WIS1) lies upstream of an alpha-amylase gene in hexaploid wheat, and carries a "minisatellite" array.

Comparison of the 5' flanking regions of three alpha-amylase genes from chromosome 6B of hexaploid wheat by heteroduplex and sequence analysis revealed the presence of a 1.6 kb stem-loop insertion sequence (WIS1) in one of them. Polymorphism among hexaploid wheat varieties suggests the relatively recent insertion/excision of this sequence from its present position. The complete sequence of the stem-loop insertion shows that it has many of the features found in transposable elements, including target site duplication and terminal inverted repeats. One unusual feature is a tandem array of direct repeats comprising a wheat "minisatellite" sequence. Both the insertion sequence and the minisatellite are found at multiple locations in the wheat genome, but the functional significance of their association in WIS1 is unknown. The minisatellite arrays share a common core structure, and long arrays are polymorphic between different hexaploid varieties.

Inactivation of maize transposon Mu suppresses a mutant phenotype by activating an outward-reading promoter near the end of Mu1.

We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon. When the Mu transposon system is in an active phase, hcf106 conditions a nonphotosynthetic, pale green phenotype. However, when the Mu system is inactive (a state correlated with hypermethylation of Mu elements), the plant adopts a normal phenotype despite the continued presence of the transposon within the gene. The molecular mechanisms that mediate this suppression of the mutant phenotype have now been investigated. We show here that the Mu element responsible for the hcf106 lesion lies within sequences encoding the 5'-untranslated leader of the Hcf106 mRNA. When the Mu transposon system is active, this insertion interferes with the accumulation of mRNA from the hcf106 allele. However, when Mu is inactive, mRNA similar in size and abundance to that transcribed from the normal allele accumulates. These transcripts initiate at many sites throughout a 70-base-pair region, within and immediately downstream of the Mu1 insertion. Thus, an unusual promoter spanning the downstream junction between Mu1 and Hcf106 substitutes for the normal Hcf106 promoter but only when Mu is inactive. The pattern of mRNA accumulation in different organs and in response to light suggests that the activity of this promoter is conditional not only upon the phase of Mu activity, but also upon signals that regulate the normal Hcf106 promoter.