Jawon Song

Epigenetic and Genetic Influences on DNA Methylation Variation in Maize Populations

This study examines the DNA methylation patterns of a diverse set of maize lines to show that many methylation variants are associated with local genetic variation, some of which may be due to specific transposon insertion variation. These results provide insight into how DNA methylation varies within a crop and highlight the complex nature of genetic and epigenetic influences on DNA methylation. DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

Genomic Distribution of H3K9me2 and DNA Methylation in a Maize Genome

DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.

Genetic Perturbation of the Maize Methylome

Genetic analyses of maize genes in DNA methylation pathways reveal differences between maize and Arabidopsis, including evidence that DNA methylation is required for growth and development in maize. DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.

RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

Significance RNA-directed DNA methylation (RdDM) provides a system for targeting DNA methylation to asymmetric CHH (H = A, C, or T) sites. This RdDM activity is often considered a mechanism for transcriptional silencing of transposons. However, many of the RdDM targets in the maize genome are located near genes or regulatory elements. We find that the regions of elevated CHH methylation, termed mCHH islands, are the boundaries between highly methylated (CG, CHG), silenced chromatin and more active chromatin. Analysis of RdDM mutants suggests that the function of the boundary is to promote and reinforce silencing of the transposable elements located near genes rather than to protect the euchromatic state of the genes. The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.

Examining the Causes and Consequences of Context-Specific Differential DNA Methylation in Maize

Natural variation of DNA methylation in maize is evident among five diverse maize inbred lines. DNA methylation is a stable modification of chromatin that can contribute to epigenetic variation through the regulation of genes or transposons. Profiling of DNA methylation in five maize (Zea mays) inbred lines found that while DNA methylation levels for more than 99% of the analyzed genomic regions are similar, there are still 5,000 to 20,000 context-specific differentially methylated regions (DMRs) between any two genotypes. The analysis of identical-by-state genomic regions that have limited genetic variation provided evidence that DMRs can occur without local sequence variation, but they are less common than in regions with genetic variation. Characterization of the sequence specificity of DMRs, location of DMRs relative to genes and transposons, and patterns of DNA methylation in regions flanking DMRs reveals a distinct subset of DMRs. Transcriptome profiling of the same tissue revealed that only approximately 20% of genes with qualitative (on-off) differences in gene expression are associated with DMRs, and there is little evidence for association of DMRs with genes that show quantitative differences in gene expression. We also identify a set of genes that may represent cryptic information that is silenced by DNA methylation in the reference B73 genome. Many of these genes exhibit natural variation in other genotypes, suggesting the potential for selection to act upon existing epigenetic natural variation. This study provides insights into the origin and influences of DMRs in a crop species with a complex genome organization.

Apyrase Suppression Raises Extracellular ATP Levels and Induces Gene Expression and Cell Wall Changes Characteristic of Stress Responses

Suppressing the expression of two apyrase genes raises extracellular ATP levels and induces gene expression, growth, and cell wall changes characteristic of stress responses, thus implicating extracellular nucleotides as early signals linking biotic and abiotic stresses to growth inhibition. Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.

CpG island-mediated global gene regulatory modes in mouse embryonic stem cells

Both transcriptional and epigenetic regulations are fundamental for the control of eukaryotic gene expression. Here we perform a compendium analysis of >200 large sequencing data sets to elucidate the regulatory logic of global gene expression programs in mouse embryonic stem (ES) cells. We define four major classes of DNA-binding proteins (Core, PRC, MYC and CTCF) based on their target co-occupancy, and discover reciprocal regulation between the MYC and PRC classes for the activity of nearly all genes under the control of the CpG island (CGI)-containing promoters. This CGI-dependent regulatory mode explains the functional segregation between CGI-containing and CGI-less genes during early development. By defining active enhancers based on the co-occupancy of the Core class, we further demonstrate their additive roles in CGI-containing gene expression and cell type-specific roles in CGI-less gene expression. Altogether, our analyses provide novel insights into previously unknown CGI-dependent global gene regulatory modes.

Repliscan: a tool for classifying replication timing regions

BackgroundReplication timing experiments that use label incorporation and high throughput sequencing produce peaked data similar to ChIP-Seq experiments. However, the differences in experimental design, coverage density, and possible results make traditional ChIP-Seq analysis methods inappropriate for use with replication timing.ResultsTo accurately detect and classify regions of replication across the genome, we present Repliscan. Repliscan robustly normalizes, automatically removes outlying and uninformative data points, and classifies Repli-seq signals into discrete combinations of replication signatures. The quality control steps and self-fitting methods make Repliscan generally applicable and more robust than previous methods that classify regions based on thresholds.ConclusionsRepliscan is simple and effective to use on organisms with different genome sizes. Even with analysis window sizes as small as 1 kilobase, reliable profiles can be generated with as little as 2.4x coverage.

Implications of CpG islands on chromosomal architectures and modes of global gene regulation

Abstract CpG islands (CGIs) have long been implicated in the regulation of vertebrate gene expression. However, the involvement of CGIs in chromosomal architectures and associated gene expression regulations has not yet been thoroughly explored. By combining large-scale integrative data analyses and experimental validations, we show that CGIs clearly reconcile two competing models explaining nuclear gene localizations. We first identify CGI-containing (CGI+) and CGI-less (CGI−) genes are non-randomly clustered within the genome, which reflects CGI-dependent spatial gene segregation in the nucleus and corresponding gene regulatory modes. Regardless of their transcriptional activities, CGI+ genes are mainly located at the nuclear center and encounter frequent long-range chromosomal interactions. Meanwhile, nuclear peripheral CGI− genes forming heterochromatin are activated and internalized into the nuclear center by local enhancer–promoter interactions. Our findings demonstrate the crucial implications of CGIs on chromosomal architectures and gene positioning, linking the critical importance of CGIs in determining distinct mechanisms of global gene regulation in three-dimensional space in the nucleus.

Genomic analysis of the DNA replication timing program during mitotic S phase in maize (Zea mays L.) root tips

The time during S phase at which different maize DNA sequences replicate reveals a complex temporal program influenced by genomic features, transcriptional activity, and chromatin structure. All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the “Repli-seq” assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.