Construction and Optimization of a Large Gene Coexpression Network in Maize Using RNA-Seq Data.

With the emergence of massively parallel sequencing, genomewide expression data production has reached an unprecedented level. This abundance of data has greatly facilitated maize research, but may not be amenable to traditional analysis techniques that were optimized for other data types. Using publicly available data, a gene coexpression network (GCN) can be constructed and used for gene function prediction, candidate gene selection, and improving understanding of regulatory pathways. Several GCN studies have been done in maize (Zea mays), mostly using microarray datasets. To build an optimal GCN from plant materials RNA-Seq data, parameters for expression data normalization and network inference were evaluated. A comprehensive evaluation of these two parameters and a ranked aggregation strategy on network performance, using libraries from 1266 maize samples, were conducted. Three normalization methods and 10 inference methods, including six correlation and four mutual information methods, were tested. The three normalization methods had very similar performance. For network inference, correlation methods performed better than mutual information methods at some genes. Increasing sample size also had a positive effect on GCN. Aggregating single networks together resulted in improved performance compared to single networks.

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

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.

Paramutation in evolution, population genetics and breeding.

Paramutation is a fascinating phenomenon in which directed allelic interactions result in heritable changes in the state of an allele. Paramutation has been carefully characterized at a handful of loci but the prevalence of paramutable/paramutagenic alleles is not well characterized within genomes or populations. In order to consider the role of paramutation in evolutionary processes and plant breeding, we focused on several questions. First, what causes certain alleles to become subject to paramutation? While paramutation clearly involves epigenetic regulation it is also true that only certain alleles defined by genetic sequences are able to participate in paramutation. Second, what is the prevalence of paramutation? There are only a handful of well-documented examples of paramutation. However, there is growing evidence that many loci may undergo changes in chromatin state or expression that are similar to changes observed as a result of paramutation. Third, how will paramutation events be inherited in natural or artificial populations? Many factors, including stability of epigenetic state, mating style and ploidy, may influence the prevalence of paramutation states within populations. Developing a clear understanding of the mechanisms and frequency of paramutation in crop plant genomes will facilitate new opportunities in genetic manipulation, and will also enhance plant breeding programs and our understanding of genome evolution.

Accessible DNA and relative depletion of H3K9me2 at maize loci undergoing RNA-directed DNA methylation.

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.

Differential nuclease sensitivity profiling of chromatin reveals biochemical footprints coupled to gene expression and functional DNA elements in maize.

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 5' and 3' 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.

Genetic perturbation of the maize methylome.

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.

Spreading of heterochromatin is limited to specific families of maize retrotransposons.

Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800-1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.

Genome-wide prediction of nucleosome occupancy in maize reveals plant chromatin structural features at genes and other elements at multiple scales.

The nucleosome is a fundamental structural and functional chromatin unit that affects nearly all DNA-templated events in eukaryotic genomes. It is also a biochemical substrate for higher order, cis-acting gene expression codes and the monomeric structural unit for chromatin packaging at multiple scales. To predict the nucleosome landscape of a model plant genome, we used a support vector machine computational algorithm trained on human chromatin to predict the nucleosome occupancy likelihood (NOL) across the maize (Zea mays) genome. Experimentally validated NOL plots provide a novel genomic annotation that highlights gene structures, repetitive elements, and chromosome-scale domains likely to reflect regional gene density. We established a new genome browser (http://www.genomaize.org) for viewing support vector machine-based NOL scores. This annotation provides sequence-based comprehensive coverage across the entire genome, including repetitive genomic regions typically excluded from experimental genomics data. We find that transposable elements often displayed family-specific NOL profiles that included distinct regions, especially near their termini, predicted to have strong affinities for nucleosomes. We examined transcription start site consensus NOL plots for maize gene sets and discovered that most maize genes display a typical +1 nucleosome positioning signal just downstream of the start site but not upstream. This overall lack of a -1 nucleosome positioning signal was also predicted by our method for Arabidopsis (Arabidopsis thaliana) genes and verified by additional analysis of previously published Arabidopsis MNase-Seq data, revealing a general feature of plant promoters. Our study advances plant chromatin research by defining the potential contribution of the DNA sequence to observed nucleosome positioning and provides an invariant baseline annotation against which other genomic data can be compared.

Using iRNA-seq analysis to predict gene expression regulatory level and activity in Zea mays tissues.

Plants regulate gene expression at the transcriptional and post-transcriptional levels to produce a variety of functionally diverse cells and tissues that ensure normal growth, development, and environmental response. Although distinct gene expression patterns have been characterized between different plant tissues, the specific role of transcriptional regulation of tissue-specific expression is not well-characterized in plants. RNA-seq, while widely used to assay for changes in transcript abundance, does not discriminate between differential expression caused by mRNA degradation and active transcription. Recently, the presence of intron sequences in RNA-seq analysis of libraries constructed with total RNA has been found to coincide with genes undergoing active transcription. We have adapted the intron RNA-sequencing analysis to determine genome-wide transcriptional activity in 2 different maize (Zea mays) tissues: husk and V2-inner stem tissue. A total of 5,341 genes were predicted to be transcriptionally differentially expressed between the 2 tissues, including many genes expected to have biological activity relevant to the functional and developmental identity of each tissue. Correlations with transcriptional enhancer and transcription factor activity support the validity of intron RNA-sequencing predictions of transcriptional regulation. A subset of transcription factors was further analyzed using gene regulatory network analysis to determine the possible impact of their activation. The predicted regulatory patterns between these genes were used to model a potential gene regulatory network of transcription factors and regulatory targets.

Computational Analysis of lncRNA from cDNA Sequences.

Based on recent findings, long noncoding (lnc) RNAs represent a potential class of functional molecules within the cell. In this chapter we describe a computational scheme to identify and classify lncRNAs within maize from full-length cDNA sequences to designate subsets of lncRNAs for which biogenesis and regulatory mechanisms may be verified at the bench. We make use of the Coding Potential Calculator and specific Python scripts in our approach.

Direct and Indirect Transcriptional Effects of Abiotic Stress in Zea mays Plants Defective in RNA-Directed DNA Methylation.

Plants respond to abiotic stress stimuli, such as water deprivation, through a hierarchical cascade that includes detection and signaling to mediate transcriptional and physiological changes. The phytohormone abscisic acid (ABA) is well-characterized for its regulatory role in these processes in response to specific environmental cues. ABA-mediated changes in gene expression have been demonstrated to be temporally-dependent, however, the genome-wide timing of these responses are not well-characterized in the agronomically important crop plant Zea mays (maize). ABA-mediated responses are synergistic with other regulatory mechanisms, including the plant-specific RNA-directed DNA methylation (RdDM) epigenetic pathway. Our prior work demonstrated that after relatively long-term ABA induction (8 h), maize plants homozygous for the mop1-1 mutation, defective in a component of the RdDM pathway, exhibit enhanced transcriptional sensitivity to the phytohormone. At this time-point, many hierarchically positioned transcription factors are differentially expressed resulting in primary (direct) and secondary (indirect) transcriptional outcomes. To identify more immediate and direct MOP1-dependent responses to ABA, we conducted a transcriptomic analysis using mop1-1 mutant and wild type plants treated with ABA for 1 h. One h of ABA treatment was sufficient to induce unique categories of differentially expressed genes (DEGs) in mop1-1. A comparative analysis between the two time-points revealed that distinct epigenetically-regulated changes in gene expression occur within the early stages of ABA induction, and that these changes are predicted to influence less immediate, indirect transcriptional responses. Homology with MOP1-dependent siRNAs and a gene regulatory network (GRN) were used to identify putative immediate and indirect targets, respectively. By manipulating two key regulatory networks in a temporal dependent manner, we identified genes and biological processes regulated by RdDM and ABA-mediated stress responses. Consistent with mis-regulation of gene expression, mop1-1 homozygous plants are compromised in their ability to recover from water deprivation. Collectively, these results indicate transcriptionally and physiologically relevant roles for MOP1-mediated regulation of gene expression of plant responses to environmental stress.

Epigenetic Regulation of ABA-Induced Transcriptional Responses in Maize.

Plants are subjected to extreme environmental conditions and must adapt rapidly. The phytohormone abscisic acid (ABA) accumulates during abiotic stress, signaling transcriptional changes that trigger physiological responses. Epigenetic modifications often facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and environmentally-induced expression. In maize (Zea mays), MEDIATOR OF PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent epigenetic pathway that regulates transcriptional silencing of loci genomewide. MOP1 function has been previously correlated with genomic regions adjoining particular types of transposable elements and genic regions, suggesting that this regulatory pathway functions to maintain distinct transcriptional activities within genomic spaces, and that loss of MOP1 may modify the responsiveness of some loci to other regulatory pathways. As critical regulators of gene expression, MOP1 and ABA pathways each regulate specific genes. To determine whether loss of MOP1 impacts ABA-responsive gene expression in maize, mop1-1 and Mop1 homozygous seedlings were subjected to exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes (DEGs) were identified in four pairwise comparisons. Overall, ABA-induced changes in gene expression were enhanced in mop1-1 homozygous plants. The highest number of DEGs were identified in ABA-induced mop1-1 mutants, including many transcription factors; this suggests combinatorial regulatory scenarios including direct and indirect transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a hierarchical, cascading network of responsive genes. Additionally, a modest increase in CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in some genotypes, suggesting that epigenetic variation might influence environmentally-induced transcriptional responses in maize.

Transcriptionally silenced transgenes in maize are activated by three mutations defective in paramutation.

Plants with mutations in one of three maize genes, mop1, rmr1, and rmr2, are defective in paramutation, an allele-specific interaction that leads to meiotically heritable chromatin changes. Experiments reported here demonstrate that these genes are required to maintain the transcriptional silencing of two different transgenes, suggesting that paramutation and transcriptional silencing of transgenes share mechanisms. We hypothesize that the transgenes are silenced through an RNA-directed chromatin mechanism, because mop1 encodes an RNA-dependent RNA polymerase. In all the mutants, DNA methylation was reduced in the active transgenes relative to the silent transgenes at all of the CNG sites monitored within the transgene promoter. However, asymmetrical methylation persisted at one site within the reactivated transgene in the rmr1-1 mutant. With that one mutant, rmr1-1, the transgene was efficiently resilenced upon outcrossing to reintroduce the wild-type protein. In contrast, with the mop1-1 and rmr2-1 mutants, the transgene remained active in a subset of progeny even after the wild-type proteins were reintroduced by outcrossing. Interestingly, this immunity to silencing increased as the generations progressed, consistent with a heritable chromatin state being formed at the transgene in plants carrying the mop1-1 and rmr2-1 mutations that becomes more resistant to silencing in subsequent generations.

Altered nucleosome positions in maize haplotypes and mutants of a subset of SWI/SNF-like proteins.

Chromatin remodelers alter DNA-histone interactions in eukaryotic organisms and have been well characterized in yeast and Arabidopsis. While there are maize proteins with similar domains as known remodelers, the ability of the maize proteins to alter nucleosome position has not been reported. Mutant alleles of several maize proteins (RMR1, CHR101, CHR106, CHR127, and CHR156) with similar functional domains to known chromatin remodelers were identified. Altered gene expression of Chr101, Chr106, Chr127, and Chr156 was demonstrated in plants homozygous for the mutant alleles. These mutant genotypes were subjected to nucleosome position analysis to determine whether misregulation of putative maize chromatin proteins would lead to altered DNA-histone interactions. Nucleosome position changes were observed in plants homozygous for chr101, chr106, chr127, and chr156 mutant alleles, suggesting that CHR101, CHR106, CHR127, and CHR156 may affect chromatin structure. The role of RNA polymerases in altering DNA-histone interactions was also tested. Changes in nucleosome position were demonstrated in homozygous mop2-1 individuals. These changes were demonstrated at the b1 tandem repeats and at newly identified loci. Additionally, differential DNA-histone interactions and altered gene expression of putative chromatin remodelers were demonstrated between different maize haplotypes.

Computational Analysis of LncRNA from cDNA Sequences.

Based on recent findings, long noncoding (lnc) RNAs represent a potential class of functional molecules within the cell. In this chapter we describe a computational scheme to identify and classify lncRNAs within maize from full-length cDNA sequences to designate subsets of lncRNAs for which biogenesis and regulatory mechanisms may be verified at the bench. We make use of the Coding Potential Calculator and specific Python scripts in our approach.

Surgical management of leiomyosarcoma of the mediastinum.

Leiomyosarcoma of the mediastinum is sufficiently rare; therefore therapeutic approaches are not well defined. Current therapy primarily rests with the thoracic surgeon. We report a single case of a large leiomyosarcoma of the mediastinum invading the heart and right lung. Our diagnostic and therapeutic approach to this patient is presented and the pertinent literature is reviewed. We conclude that little advancement has been made with regard to adjuvant therapies for leiomyosarcoma of the mediastinum, and the only opportunity for cure remains with full surgical extirpation.

Totally endoscopic robotic thymectomy for myasthenia gravis.

The current recommendations for treating myasthenia gravis include surgical thymectomy for patients between puberty and 60 years of age. This is a report of a new method for surgical thymectomy using the robotic da Vinci surgical system for a totally endoscopic approach. This new procedure combines the potential advantages of minimally invasive methods with the efficacy of open procedures.

Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes--mop1-1, Mop2-1, and mop3-1--suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation.

Chromatin structure and gene expression changes associated with loss of MOP1 activity in Zea mays.

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.

Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.