Distinct tissue-specific transcriptional regulation revealed by gene regulatory networks in maize.

BACKGROUND: Transcription factors (TFs) are proteins that can bind to DNA sequences and regulate gene expression. Many TFs are master regulators in cells that contribute to tissue-specific and cell-type-specific gene expression patterns in eukaryotes. Maize has been a model organism for over one hundred years, but little is known about its tissue-specific gene regulation through TFs. In this study, we used a network approach to elucidate gene regulatory networks (GRNs) in four tissues (leaf, root, SAM and seed) in maize. We utilized GENIE3, a machine-learning algorithm combined with large quantity of RNA-Seq expression data to construct four tissue-specific GRNs. Unlike some other techniques, this approach is not limited by high-quality Position Weighed Matrix (PWM), and can therefore predict GRNs for over 2000 TFs in maize. RESULTS: Although many TFs were expressed across multiple tissues, a multi-tiered analysis predicted tissue-specific regulatory functions for many transcription factors. Some well-studied TFs emerged within the four tissue-specific GRNs, and the GRN predictions matched expectations based upon published results for many of these examples. Our GRNs were also validated by ChIP-Seq datasets (KN1, FEA4 and O2). Key TFs were identified for each tissue and matched expectations for key regulators in each tissue, including GO enrichment and identity with known regulatory factors for that tissue. We also found functional modules in each network by clustering analysis with the MCL algorithm. CONCLUSIONS: By combining publicly available genome-wide expression data and network analysis, we can uncover GRNs at tissue-level resolution in maize. Since ChIP-Seq and PWMs are still limited in several model organisms, our study provides a uniform platform that can be adapted to any species with genome-wide expression data to construct GRNs. We also present a publicly available database, maize tissue-specific GRN (mGRN, https://www.bio.fsu.edu/mcginnislab/mgrn/ ), for easy querying. All source code and data are available at Github ( https://github.com/timedreamer/maize_tissue-specific_GRN ).

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.

The Phaeodactylum genome reveals the evolutionary history of diatom genomes.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

An RNA-dependent RNA polymerase is required for paramutation in maize.

Paramutation is an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another. Paramutation at the b1 locus in maize is mediated by unique tandem repeats that communicate in trans to establish and maintain meiotically heritable transcriptional silencing. The mop1 (mediator of paramutation1) gene is required for paramutation, and mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Here we report the map-based cloning of mop1, an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. Nuclear run-on assays reveal that the tandem repeats required for b1 paramutation are transcribed from both strands, but siRNAs were not detected. We propose that the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.

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.

COVID toes: Pernio-like lesions.

More than 40 million people have been infected with the severe acute respiratory syndrome coronavirus 2 since the first infection was reported in December 2019 from Wuhan, China. Multiple reports of cutaneous manifestations of the virus have been described, including a pernio-like eruption, recently termed "COVID toes." We have reviewed the published case series on "COVID toes" in addition to studies identifying possible pathogenic mechanisms behind the eruption.

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.

WITHDRAWN: COVID Toes.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.clindermatol.2021.01.016. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

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.

Use of transgene-induced RNAi to regulate endogenous gene expression.

RNAi can be an effective means to regulate endogenous gene expression in maize and as such represents an important reverse genetics tool. This approach involves designing a transgenic construct that creates a double stranded RNA (dsRNA) upon transcription, and introducing the transgene into maize plants. Transgenic lines bearing such a construct can be generated and characterized that are deficient in the gene of interest. Some variability has been observed in the efficiency of this technique, and there are several important aspects to consider. Herein, a basic protocol for using transgene-induced RNAi in maize is described, and some important considerations that can influence the success of this approach are discussed.

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.