Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats.

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

RNA-directed DNA methylation mutants reduce histone methylation at the paramutated maize booster1 enhancer.

Paramutation is the transfer of mitotically and meiotically heritable silencing information between two alleles. With paramutation at the maize (Zea mays) booster1 (b1) locus, the low-expressed B' epiallele heritably changes the high-expressed B-I epiallele into B' with 100% frequency. This requires specific tandem repeats and multiple components of the RNA-directed DNA methylation pathway, including the RNA-dependent RNA polymerase (encoded by mediator of paramutation1, mop1), the second-largest subunit of RNA polymerase IV and V (NRP(D/E)2a, encoded by mop2), and the largest subunit of RNA Polymerase IV (NRPD1, encoded by mop3). Mutations in mop genes prevent paramutation and release silencing at the B' epiallele. In this study, we investigated the effect of mutations in mop1, mop2, and mop3 on chromatin structure and DNA methylation at the B' epiallele, and especially the regulatory hepta-repeat 100 kb upstream of the b1 gene. Mutations in mop1 and mop3 resulted in decreased repressive histone modifications H3K9me2 and H3K27me2 at the hepta-repeat. Associated with this decrease were partial activation of the hepta-repeat enhancer function, formation of a multi-loop structure, and elevated b1 expression. In mop2 mutants, which do not show elevated b1 expression, H3K9me2, H3K27me2 and a single-loop structure like in wild-type B' were retained. Surprisingly, high CG and CHG methylation levels at the B' hepta-repeat remained in all three mutants, and CHH methylation was low in both wild type and mutants. Our results raise the possibility of MOP factors mediating RNA-directed histone methylation rather than RNA-directed DNA methylation at the b1 locus.

Cis-regulatory sequences in plants: Their importance, discovery, and future challenges.

The identification and characterization of cis-regulatory DNA sequences and how they function to coordinate responses to developmental and environmental cues is of paramount importance to plant biology. Key to these regulatory processes are cis-regulatory modules (CRMs), which include enhancers and silencers. Despite the extraordinary advances in high-quality sequence assemblies and genome annotations, the identification and understanding of CRMs, and how they regulate gene expression, lag significantly behind. This is especially true for their distinguishing characteristics and activity states. Here, we review the current knowledge on CRMs and breakthrough technologies enabling identification, characterization, and validation of CRMs; we compare the genomic distributions of CRMs with respect to their target genes between different plant species, and discuss the role of transposable elements harboring CRMs in the evolution of gene expression. This is an exciting time to study cis-regulomes in plants; however, significant existing challenges need to be overcome to fully understand and appreciate the role of CRMs in plant biology and in crop improvement.

CHROMOMETHYLTRANSFERASE3/KRYPTONITE maintains the sulfurea paramutation in Solanum lycopersicum.

SignificanceParamutation involves the transfer of a repressive epigenetic mark between silent and active alleles. It is best known from exceptional non-Mendelian inheritance of conspicuous phenotypes in maize but also in other plants and animals. Recent genomic studies, however, indicate that paramutation may be less exceptional. It may be a consequence of wide-cross hybridization and may contribute to quantitative trait variation or unstable phenotypes in crops. Using the sulfurea (sulf) locus in tomato, we demonstrate that a self-reinforcing feedback loop involving DNA- and histone-methyl transferases CHROMOMETHYLTRANSFERASE3 (CMT3) and KRYPTONITE (KYP) is required for paramutation of sulf and that there is a change in chromatin organization. These findings advance the understanding of non-Mendelian inheritance in plants.

Parental DNA Methylation States Are Associated with Heterosis in Epigenetic Hybrids.

Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance; however, it remains unclear whether epigenetic changes are a cause or a consequence of heterosis. Here, we analyze a large panel of over 500 Arabidopsis (Arabidopsis thaliana) epigenetic hybrid plants (epiHybrids), which we derived from near-isogenic but epigenetically divergent parents. This proof-of-principle experimental system allowed us to quantify the contribution of parental methylation differences to heterosis. We measured traits such as leaf area, growth rate, flowering time, main stem branching, rosette branching, and final plant height and observed several strong positive and negative heterotic phenotypes among the epiHybrids. Using an epigenetic quantitative trait locus mapping approach, we were able to identify specific differentially methylated regions in the parental genomes that are associated with hybrid performance. Sequencing of methylomes, transcriptomes, and genomes of selected parent-epiHybrid combinations further showed that these parental differentially methylated regions most likely mediate the remodeling of methylation and transcriptional states at specific loci in the hybrids. Taken together, our data suggest that locus-specific epigenetic divergence between the parental lines can directly or indirectly trigger heterosis in Arabidopsis hybrids independent of genetic changes. These results add to a growing body of evidence that points to epigenetic factors as one of the key determinants of hybrid performance.

Cis-acting determinants of paramutation.

Paramutation is an epigenetic phenomenon whereby in trans communication between homologous sequences leads to meiotically heritable epigenetic changes at one of the alleles. Cis-acting determinants of paramutation are DNA sequences and associated epigenetic modifications that are required for paramutation. Here, we review how characteristics of the underlying DNA sequences determine whether paramutation can occur and how they affect the behavior displayed by the various paramutation phenomena. Paramutation is strongly associated with repeated sequences, especially tandemly repeated sequences. Cis-acting determinants consisting of repeated sequences are consistent with the involvement of RNA-directed DNA methylation (RdDM) in plants and the PIWI-interacting RNA (piRNA) pathway in animals. In the RdDM-based model, siRNAs produced by paramutagenic loci would reinforce the silenced state of paramutagenic loci in cis and initiate transcriptional silencing of paramutable loci in trans. In this review, we discuss how sequence characteristics and epigenetic modifications of cis-acting sequences can trigger the recruitment of silencing machineries.

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.

BIBAC-GW-based vectors for generating reporter lines for site-specific genome editing in planta.

When generating transgenic plants, one of the objectives is to achieve stable expression of the transgene. Transgene silencing can be avoided by single copy integration of the transgene. Binary systems that predominantly result in single copy integrations, such as BIBAC vectors, are also single-copy in E. coli, the organism in which the T-DNA to be delivered to the plant is assembled. Although a low-copy number is important for stable maintenance of large DNA fragments in E. coli, it hampers cloning into the vector due to a low DNA yield. Here we describe BIBAC vectors to which Gateway site-specific recombination sites are added. These sites provide a fast and easy introduction of sequences of interest into any vector. Our Gateway-compatible BIBAC vectors are available with two selectable markers for plants - resistance to Basta (BIBAC-BAR-GW) and DsRed fluorescence in the seed coat (BIBAC-RFP-GW). Using the BIBAC-BAR-GW vector we have generated different fluorescence-based reporter constructs that, when delivered to plant cells, can be used to study and optimize precise, template-dependent site-specific genome editing by CRISPR-Cas9, TALENs or ZFP-nuclease complexes, and oligonucleotide-directed mutagenesis. We have generated 59 reporter lines in A. thaliana with our reporter constructs, and for the lines carrying single T-DNA integrations (32 out of 59) we have determined the integrity of the integrations, their genomic locations and the expression level of the reporters. Similarly to its original counterpart, BIBAC-BAR-GW generates single T-DNA integrations in Arabidopsis with 50% efficiency, and 90% of those are intact. The reporter constructs in the independent transgenic lines exhibit only an up to 3-fold difference in expression level. These features combined with an easy manipulation of the vector due to the added Gateway sites make the BIBAC-GW vectors an attractive tool for generating transgenic plants.

Specific tandem repeats are sufficient for paramutation-induced trans-generational silencing.

Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.

3C Technologies in plants.

Chromosome conformation capture (3C) and 3C-based technology have revolutionized studies on chromosomal interactions and their role in gene regulation and chromosome organization. 3C allows the in vivo identification of physical interactions between chromosomal regions. Such interactions are shown to play a role in various aspects of gene regulation, for example transcriptional activation of genes by remote enhancer sequences, or the silencing by Polycomb-group complexes. The last few years the number of publications involving chromosomal interactions increased significantly. Until now, however, the vast majority of the studies reported are performed in yeast or animal systems. So far, studies on plant systems are extremely limited, possibly due to the plant-specific characteristics that hamper the implementation of the 3C technique. In this paper we provide a plant-specific 3C protocol, optimized for maize tissue, and an extensive discussion on (i) plant-specific adjustments to the protocol, and (ii) solutions to problems that may arise when optimizing the protocol for the tissue or plant of interest. Together, this paper should facilitate the application of 3C technology to plant tissue and stimulate studies on the 3D conformation of chromosomal regions and chromosomes in plants.

The role of DNA methylation, nucleosome occupancy and histone modifications in paramutation.

Paramutation is the transfer of epigenetic information between alleles that leads to a heritable change in expression of one of these alleles. Paramutation at the tissue-specifically expressed maize (Zea mays) b1 locus involves the low-expressing B' and high-expressing B-I allele. Combined in the same nucleus, B' heritably changes B-I into B'. A hepta-repeat located 100-kb upstream of the b1 coding region is required for paramutation and for high b1 expression. The role of epigenetic modifications in paramutation is currently not well understood. In this study, we show that the B' hepta-repeat is DNA-hypermethylated in all tissues analyzed. Importantly, combining B' and B-I in one nucleus results in de novo methylation of the B-I repeats early in plant development. These findings indicate a role for hepta-repeat DNA methylation in the establishment and maintenance of the silenced B' state. In contrast, nucleosome occupancy, H3 acetylation, and H3K9 and H3K27 methylation are mainly involved in tissue-specific regulation of the hepta-repeat. Nucleosome depletion and H3 acetylation are tissue-specifically regulated at the B-I hepta-repeat and associated with enhancement of b1 expression. H3K9 and H3K27 methylation are tissue-specifically localized at the B' hepta-repeat and reinforce the silenced B' chromatin state. The B' coding region is H3K27 dimethylated in all tissues analyzed, indicating a role in the maintenance of the silenced B' state. Taken together, these findings provide insight into the mechanisms underlying paramutation and tissue-specific regulation of b1 at the level of chromatin structure.

Analysis of 4C-seq data: A comparison of methods.

The circular chromosome conformation capture technique followed by sequencing (4C-seq) has been used in a number of studies to investigate chromosomal interactions between DNA fragments. Computational pipelines have been developed and published that offer various possibilities of 4C-seq data processing and statistical analysis. Here, we present an overview of four of such pipelines (fourSig, FourCSeq, 4C-ker and w4Cseq) taking into account the most important stages of computations. We provide comparisons of the methods and discuss their advantages and possible weaknesses. We illustrate the results with the use of data obtained for two different species, in a study devoted to vernalization control in Arabidopsis thaliana by the FLOWERING LOCUS C (FLC) gene and to long-range chromatin interactions in mouse embryonic stem cells.

Identification of Key Tissue-Specific, Biological Processes by Integrating Enhancer Information in Maize Gene Regulatory Networks.

Enhancers are key players in the spatio-temporal coordination of gene expression during numerous crucial processes, including tissue differentiation across development. Characterizing the transcription factors (TFs) and genes they connect, and the molecular functions underpinned is important to better characterize developmental processes. In plants, the recent molecular characterization of enhancers revealed their capacity to activate the expression of several target genes. Nevertheless, identifying these target genes at a genome-wide level is challenging, particularly for large-genome species, where enhancers and target genes can be hundreds of kilobases away. Therefore, the contribution of enhancers to plant regulatory networks remains poorly understood. Here, we investigate the enhancer-driven regulatory network of two maize tissues at different stages: leaves at seedling stage (V2-IST) and husks (bracts) at flowering. Using systems biology, we integrate genomic, epigenomic, and transcriptomic data to model the regulatory relationships between TFs and their potential target genes, and identify regulatory modules specific to husk and V2-IST. We show that leaves at the V2-IST stage are characterized by the response to hormones and macromolecules biogenesis and assembly, which are regulated by the BBR/BPC and AP2/ERF TF families, respectively. In contrast, husks are characterized by cell wall modification and response to abiotic stresses, which are, respectively, orchestrated by the C2C2/DOF and AP2/EREB families. Analysis of the corresponding enhancer sequences reveals that two different transposable element families (TIR transposon Mutator and MITE Pif/Harbinger) have shaped part of the regulatory network in each tissue, and that MITEs have provided potential new TF binding sites involved in husk tissue-specificity.

Maize E2F transcription factors. Expression, association to promoters of S-phase genes and interaction with the RBR1 protein in chromatin during seed germination.

For seed germination, it is necessary to restart the cell cycle, a process regulated at multiple levels including transcriptional control, that is executed by the E2F family of transcription factors. We identified 12 genes of the E2F family in maize that are expressed differentially during the first 28 h post imbibition (HAI). E2Fa/b1;1 and E2Fc proteins were characterized as an activator and a putative repressor respectively, both forming heterodimers with DPb2 that bind differentially to consensus E2F response elements in promoters of E2F target genes. Transcripts of target genes for these transcription factors accumulate during germination; in dry seeds E2Fc protein is enriched in the target promoters and is replaced by E2Fa/b1;1 as germination advances. RBR1 is found in the same promoters in non-imbibed and 28 HAI seeds, when DNA replication has concluded, and transcription of the E2F targets should stop. During germination promoters of these target genes seem to be decorated with histone marks related to relaxed chromatin structure. Therefore, E2Fs appear to occupy their target genes in a context of open chromatin, with RBR1 fine tuning the progression between the phases.

3D genome organization: a role for phase separation and loop extrusion?

In eukaryotes, genomic information is encoded in chromosomes, which occupy distinct territories within the nucleus. Inside these territories, chromosomes are folded in a hierarchical set of topological structures, called compartments, topologically associated domains and loops. Phase separation and loop extrusion are the mechanisms indicated to mediate the 3D organization of the genome, and gene activity and epigenetic marks determine the activity level of the formed chromatin domains. The main difference between plants and animals may be the absence of canonical insulator elements in plants. Comparison across plant species indicates that the identification of chromatin domains is affected by genome size, gene density, and the linear distribution of genes and transposable elements.

3C in Maize and Arabidopsis.

With Chromosome Conformation Capture (3C), the relative interaction frequency of one chromosomal fragment with another can be determined. The technique is especially suited for unraveling the 3D organization of specific loci when focusing on aspects such as enhancer-promoter interactions or other topological conformations of the genome. 3C has been extensively used in animal systems, among others providing insight into gene regulation by distant cis-regulatory elements. In recent years, the 3C technique has been applied in plant research. However, the complexity of plant tissues prevents direct application of existing protocols from animals. Here, we describe an adapted protocol suitable for plant tissues, especially Arabidopsis thaliana and Zea mays.

Protocol for Chromatin Immunoprecipitation of Meiotic-Stage-Specific Tomato Anthers.

Interactions occurring between DNA and proteins across the nuclear genome regulate numerous processes, including meiosis. Meiosis ensures genetic variation and balanced segregation of homologous chromosomes. It involves complex DNA-protein interactions across the entire genome to regulate a broad range of processes, including formation and repair of double-strand DNA breaks (DSBs), chromosome compaction, homolog pairing, synapsis, and homologous recombination. The latter meiotic event, meiotic recombination, often occurs at discrete locations in a genome, within a tight time window. The identification of genomic binding sites of meiotic proteins is a major step toward understanding the molecular mechanisms underlying meiotic recombination and provides important information for plant breeding. Collecting meiotic cells from plants is challenging, tedious, and time consuming, since the meiocyte-producing organs, the anthers, are generally small and limited to certain developmental stages of plants. Here we provide a protocol to isolate meiotic-stage-specific anthers and perform ChIP on this material. We have developed a ChIP protocol specifically suited to (1) small amounts of input material and (2) proteins that bind transiently to chromatin and at very low frequency. © 2018 by John Wiley & Sons, Inc.

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2-0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Assessment of heterosis in two Arabidopsis thaliana common-reference mapping populations.

Hybrid vigour, or heterosis, has been of tremendous importance in agriculture for the improvement of both crops and livestock. Notwithstanding large efforts to study the phenomenon of heterosis in the last decades, the identification of common molecular mechanisms underlying hybrid vigour remain rare. Here, we conducted a systematic survey of the degree of heterosis in Arabidopsis thaliana hybrids. For this purpose, two overlapping Arabidopsis hybrid populations were generated by crossing a large collection of naturally occurring accessions to two common reference lines. In these Arabidopsis hybrid populations the range of heterosis for several developmental and yield related traits was examined, and the relationship between them was studied. The traits under study were projected leaf area at 17 days after sowing, flowering time, height of the main inflorescence, number of side branches from the main stem or from the rosette base, total seed yield, seed weight, seed size and the estimated number of seeds per plant. Predominantly positive heterosis was observed for leaf area and height of the main inflorescence, whereas mainly negative heterosis was observed for rosette branching. For the other traits both positive and negative heterosis was observed in roughly equal amounts. For flowering time and seed size only low levels of heterosis were detected. In general the observed heterosis levels were highly trait specific. Furthermore, no correlation was observed between heterosis levels and the genetic distance between the parental lines. Since all selected lines were a part of the Arabidopsis genome wide association (GWA) mapping panel, a genetic mapping approach was applied to identify possible regions harbouring genetic factors causal for heterosis, with separate calculations for additive and dominance effects. Our study showed that the genetic mechanisms underlying heterosis were highly trait specific in our hybrid populations and greatly depended on the genetic background, confirming the elusive character of heterosis.