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.T he cytosine bases in a genome can be modified to 5-methylcytosine by adding a methyl group at the 5′ position. This process, called DNA methylation, is conserved from algae to animals and plants (1, 2). DNA methylation can be separated into different types based on the local sequence context. In plants DNA methylation is found at the symmetric CG or CHG (where H = A, C, or T) sites or at nonsymmetric CHH sites. CG and CHG methylation are maintained at high fidelity following DNA replication due to activity of maintenance methyltransferases such as MET1 or chromomethylase (CMT) 3 (3, 4), whereas CHH methylation (mCHH) requires targeting by either domains rearranged methylase 2 (DRM2) or CMT2 (3-6). The DRM2 targeting occurs via RNAdirected DNA methylation (RdDM) and requires the activity of polymerase IV (PolIV) and polymerase V (PolV) complexes (3, 4). There is evidence that recruitment of PolIV and PolV may require the presence of dimethylation of lysine 9 of histone H3 (H3K9me2) or DNA methylation at the targeted genomic regions (7,8). The specific mechanisms that recruit CMT2 are not well characterized but may require specific histone modifications (5, 6).Much of our knowledge of DNA methylation in plants is derived from studies of the model plant Arabidopsis thaliana, which has a relatively small genome and relatively few examples of genes with nearby transposons (36.3%; ref. 9). The maize genome is much more complex, with the majority (85.5%) of genes positioned within 1...

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 (RdDM) in plants is a well-characterized example of RNA interference-related transcriptional gene silencing. To determine the relationships between RdDM and heterochromatin in the repeat-rich maize (Zea mays) genome, we performed whole-genome analyses of several heterochromatic features: dimethylation of lysine 9 and lysine 27 (H3K9me2 and H3K27me2), chromatin accessibility, DNA methylation, and small RNAs; we also analyzed two mutants that affect these processes, mediator of paramutation1 and zea methyltransferase2. The data revealed that the majority of the genome exists in a heterochromatic state defined by inaccessible chromatin that is marked by H3K9me2 and H3K27me2 but that lacks RdDM. The minority of the genome marked by RdDM was predominantly near genes, and its overall chromatin structure appeared more similar to euchromatin than to heterochromatin. These and other data indicate that the densely staining chromatin defined as heterochromatin differs fundamentally from RdDM-targeted chromatin. We propose that small interfering RNAs perform a specialized role in repressing transposons in accessible chromatin environments and that the bulk of heterochromatin is incompatible with small RNA production.

The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin. The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood. We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase). We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 59 and 39 ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints. These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation.

The acidic polysaccharide succinoglycan produced by the rhizobial symbiont Sinorhizobium meliloti 1021 is required for this bacterium to invade the host plant Medicago truncatula and establish a nitrogen-fixing symbiosis. S. meliloti mutants that cannot make succinoglycan cannot initiate invasion structures called infection threads in plant root hairs. S. meliloti exoH mutants that cannot succinylate succinoglycan are also unable to form infection threads, despite the fact that they make large quantities of succinoglycan. Succinoglycan produced by exoH mutants is refractory to cleavage by the glycanases encoded by exoK and exsH, and thus succinoglycan produced by exoH mutants is made only in the high-molecular-weight (HMW) form. One interpretation of the symbiotic defect of exoH mutants is that the low-molecular-weight (LMW) form of succinoglycan is required for infection thread formation. However, our data demonstrate that production of the HMW form of succinoglycan by S. meliloti 1021 is sufficient for invasion of the host M. truncatula and that the LMW form is not required. Here, we show that S. meliloti strains deficient in the exoK- and exsH-encoded glycanases invade M. truncatula and form a productive symbiosis, although they do this with somewhat less efficiency than the wild type. We have also characterized the polysaccharides produced by these double glycanase mutants and determined that they consist of only HMW succinoglycan and no detectable LMW succinoglycan. This demonstrates that LMW succinoglycan is not required for host invasion. These results suggest succinoglycan function is not dependent upon the presence of a small, readily diffusible form.

42 43 44 45 Acknowledgments 46 3 We are grateful to Nathan Springer for providing the SeqCap DNA methylation data. 47 This work was funded by the National Science Foundation funds to K.M.M (CMB-48 035919) and start-up funds from the University of Washington Bothell School of STEM 49 to T.F.M. P.A.C. was supported by a grant from NSF (IOS-1802848). 50 51 Significance statement 52 MOP1-dependent gene expression changes in response to ABA were identified as 53 having synergistic and combinatorial direct and indirect effects. Overlapping regulatory 54 networks were uncovered, reinforcing the idea that epigenetic regulation is crucial to 55 plant response and adaptation to abiotic stress. 56 57 58 4 ABSTRACT 59 60 Plants are subjected to extreme environmental conditions and must adapt rapidly. The 61 phytohormone abscisic acid (ABA) accumulates during abiotic stress, signaling 62 transcriptional changes that trigger physiological responses. Epigenetic modifications 63 often facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and 64 environmentally-induced expression. In maize (Zea mays), MEDIATOR OF 65 PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent 66 epigenetic pathway that regulates transcriptional silencing of loci genomewide. MOP1 67 function has been previously correlated with genomic regions adjoining particular types 68 of transposable elements and genic regions, suggesting that this regulatory pathway 69 functions to maintain distinct transcriptional activities within genomic spaces, and that 70 loss of MOP1 may modify the responsiveness of some loci to other regulatory 71 pathways. As critical regulators of gene expression, MOP1 and ABA pathways each 72 regulate specific genes. To determine whether loss of MOP1 impacts ABA-responsive 73 gene expression in maize, mop1-1 and Mop1 homozygous seedlings were subjected to 74 exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes 75 (DEGs) were identified in four pairwise comparisons. Overall, ABA-induced changes in 76 gene expression were enhanced in mop1-1 homozygous plants. The highest number of 77 DEGs were identified in ABA-induced mop1-1 mutants, including many transcription 78 factors; this suggests combinatorial regulatory scenarios including direct and indirect 79 transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a 80 hierarchical, cascading network of responsive genes. Additionally, a modest increase in 81 5 CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in 82 some genotypes, suggesting that epigenetic variation might influence environmentally-83 induced transcriptional responses in maize. 84 85 INTRODUCTION 86 87As sessile organisms, plants must adapt rapidly to fluctuating and often extreme abiotic 88 stress conditions that negatively impact crop productivity and yield, such as water 89 deprivation/drought, high salinity, nutrient deficiency and extreme temperatures. In 90 addition to its role in plant develop...

Though the mechanisms governing nuclear organization are not well understood, it is apparent that epigenetic modifications coordinately modulate chromatin organization as well as transcription. In maize, MEDIATOR OF PARAMUTATION1 (MOP1) is required for 24 nt siRNA-mediated epigenetic regulation and transcriptional gene silencing via a putative Pol IV- RdDM pathway. To elucidate the mechanisms of nuclear chromatin organization, we investigated the relationship between chromatin structure and transcription in response to loss of MOP1 function. We used a microarray based micrococcal nuclease sensitivity assay to identify genome-wide changes in chromatin structure in mop1-1 immature ears and observed an increase in chromatin accessibility at chromosome arms associated with loss of MOP1 function. Within the many genes misregulated in mop1 mutants, we identified one subset likely to be direct targets of epigenetic transcriptional silencing via Pol-IV RdDM. We found that target specificity for MOP1-mediated RdDM activity is governed by multiple signals that include accumulation of 24 nt siRNAs and the presence of specific classes of gene-proximal transposons, but neither of these attributes alone is sufficient to predict transcriptional misregulation in mop1-1 homozygous mutants. Our results suggest a role for MOP1 in regulation of higher-order chromatin organization where loss of MOP1 activity at a subset of loci triggers a broader cascade of transcriptional consequences and genome-wide changes in chromatin structure.

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