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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.

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

Paramutation involves the transfer of a repressive epigenetic mark from a silent allele to an active homolog and, consequently, non-Mendelian inheritance. In tomato, the sulfurea (sulf) paramutation is associated with a high level of CHG hypermethylation in a region overlapping with the transcription start site (TSS) of the SlTAB2 gene that affects chlorophyll synthesis. The CCG subcontext hypermethylation is under-represented at this region relative to CTG or CAG, implicating the CHROMOMETHYLTRANSFERASE3a (CMT3) in paramutation at this locus. Consistent with this interpretation, loss of CMT3 function leads to loss of the sulf chlorosis, the associated CHG hypermethylation, and paramutation. Loss of KRYPTONITE (KYP) histone methyltransferase function has a similar effect linked to reduced H3K9me2 at the promoter region of SlTAB2 and a shift in higher order chromatin structure at this locus. Mutation of the largest subunit of RNA polymerase V (PolV) in contrast does not affect sulf paramutation. These findings indicate the involvement of a CMT3/KYP–dependent feedback loop rather than the PolV-dependent pathway leading to RNA-directed DNA methylation (RdDM) in the maintenance of paramutation.

Paramutation involves the transfer of a repressive epigenetic mark from a silent allele to an active homologue and, consequently, non-Mendelian inheritance. In tomato the sulfurea (sulf) paramutation is associated with a high level of CHG hypermethylation in a region overlapping the transcription start site of the SlTAB2 gene that affects chlorophyll synthesis. The CCG sub-context hypermethylation is under-represented at this region relative to CTG or CAG implicating the CHROMOMETHYLTRANSFERASE3 (CMT3) in paramutation at this locus. Consistent with this interpretation, loss of CMT3 function leads to loss of the sulf chlorosis, the associated CHG hypermethylation and paramutation. Loss of KRYPTONITE (KYP) histone methyl transferase function has a similar effect linked to reduced H3K9me2 at the promoter region of SlTAB2 and a shift in higher order chromatin structure at this locus. Mutation of the largest subunit of RNA polymerase V (PolV) in contrast does not affect sulf paramutation. These findings indicate the involvement of a CMT3/KYP dependent feedback rather than the PolV-dependent pathway leading to RNA directed DNA methylation (RdDM) in the maintenance of paramutation.

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.

Enhancers are important regulators of gene expression during numerous crucial processes including tissue differentiation across development. In plants, their recent molecular characterization revealed their capacity to activate the expression of several target genes through the binding of transcription factors. Nevertheless, identifying these target genes at a genome-wide level remains a challenge, in particular in species with large genomes, where enhancers and target genes can be hundreds of kilobases away. Therefore, the contribution of enhancers to regulatory network is still poorly understood in plants. In this study, we investigate the enhancer-driven regulatory network of two maize tissues at different stages: leaves at seedling stage and husks (bracts) at flowering. Using a systems biology approach, we integrate genomic, epigenomic and transcriptomic data to model the regulatory relationship between transcription factors and their potential target genes. We identify regulatory modules specific to husk and V2-IST, and show that they are involved in distinct functions related to the biology of each tissue. We evidence enhancers exhibiting binding sites for two distinct transcription factor families (DOF and AP2/ERF) that drive the tissue-specificity of gene expression in seedling immature leaf and husk. Analysis of the corresponding enhancer sequences reveals that two different transposable element families (TIR transposon Mutator and MITE Pif/Harbinger) have shaped the regulatory network in each tissue, and that MITEs have provided new transcription factor binding sites that are involved in husk tissue-specificity.SignificanceEnhancers play a major role in regulating tissue-specific gene expression in higher eukaryotes, including angiosperms. While molecular characterization of enhancers has improved over the past years, identifying their target genes at the genome-wide scale remains challenging. Here, we integrate genomic, epigenomic and transcriptomic data to decipher the tissue-specific gene regulatory network controlled by enhancers at two different stages of maize leaf development. Using a systems biology approach, we identify transcription factor families regulating gene tissue-specific expression in husk and seedling leaves, and characterize the enhancers likely to be involved. We show that a large part of maize enhancers is derived from transposable elements, which can provide novel transcription factor binding sites crucial to the regulation of tissue-specific biological functions.

BackgroundDNA methylation is an important factor in the regulation of gene expression and genome stability. High DNA methylation levels are associated with transcriptional repression. In mammalian systems, unmethylated, low methylated and fully methylated regions (UMRs, LMRs, and FMRs, respectively) can be distinguished. UMRs are associated with proximal regulatory regions, while LMRs are associated with distal regulatory regions. Although DNA methylation is mainly limited to the CG context in mammals, while it occurs in CG, CHG and CHH contexts in plants, UMRs and LMRs were expected to occupy similar genomic sequences in both mammals and plants.ResultsThis study investigated major model and crop plants such as Arabidopsis thaliana, tomato (Solanum lycopersicum), rice (Oryza sativa) and maize (Zea mays), and shows that plant genomes can also be subdivided in UMRs, LMRs and FMRs, but that LMRs are mainly present in the CHG context rather than the CG context. Strikingly, the identified CHG LMRs were enriched in transposable elements rather than regulatory regions. Maize candidate regulatory regions overlapped with UMRs. LMRs were enriched for heterochromatic histone modifications and depleted for DNase accessibility and H3K9 acetylation. CHG LMRs form a distinct, abundant cluster of loci, indicating they have a different role than FMRs.ConclusionsBoth mammalian and plant genomes can be segmented in three distinct classes of loci, UMRs, LMRs and FMRs, indicating similar underlying mechanisms. Unlike in mammals, distal regulatory sequences in plants appear to overlap with UMRs instead of LMRs. Our data indicate that LMRs in plants have a different function than those in mammals.

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.

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.

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.

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.

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.

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.

Background The chromosome conformation capture (3C) technique is a method to study chromatin interactions at specific genomic loci. Initially established for yeast the 3C technique has been adapted to plants in recent years in order to study chromatin interactions and their role in transcriptional gene regulation. As the plant scientific community continues to implement this technology, a discussion on critical controls, validations steps and interpretation of 3C data is essential to fully benefit from 3C in plants. Results Here we assess the reliability and robustness of the 3C technique for the detection of chromatin interactions in Arabidopsis. As a case study, we applied this methodology to the genomic locus of a floral integrator gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and demonstrate the need of several controls and standard validation steps to allow a meaningful interpretation of 3C data. The intricacies of this promising but challenging technique are discussed in depth. Conclusions The 3C technique offers an interesting opportunity to study chromatin interactions at a resolution infeasible by microscopy. However, for interpretation of 3C interaction data and identification of true interactions, 3C technology demands a stringent experimental setup and extreme caution. Electronic supplementary material The online version of this article (10.1186/s13007-017-0251-x) contains supplementary material, which is available to authorized users.

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.

Background While most cells in multicellular organisms carry the same genetic information, in each cell type only a subset of genes is being transcribed. Such differentiation in gene expression depends, for a large part, on the activation and repression of regulatory sequences, including transcriptional enhancers. Transcriptional enhancers can be located tens of kilobases from their target genes, but display characteristic chromatin and DNA features, allowing their identification by genome-wide profiling. Here we show that integration of chromatin characteristics can be applied to predict distal enhancer candidates in Zea mays, thereby providing a basis for a better understanding of gene regulation in this important crop plant. Result To predict transcriptional enhancers in the crop plant maize (Zea mays L. ssp. mays), we integrated available genome-wide DNA methylation data with newly generated maps for chromatin accessibility and histone 3 lysine 9 acetylation (H3K9ac) enrichment in young seedling and husk tissue. Approximately 1500 intergenic regions, displaying low DNA methylation, high chromatin accessibility and H3K9ac enrichment, were classified as enhancer candidates. Based on their chromatin profiles, candidate sequences can be classified into four subcategories. Tissue-specificity of enhancer candidates is defined based on the tissues in which they are identified and putative target genes are assigned based on tissue-specific expression patterns of flanking genes. Conclusions Our method identifies three previously identified distal enhancers in maize, validating the new set of enhancer candidates and enlarging the toolbox for the functional characterization of gene regulation in the highly repetitive maize genome. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1273-4) contains supplementary material, which is available to authorized users.

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.

Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not well understood. Interestingly, there is growing evidence that beside genetic also epigenetic factors contribute to heterosis. Here we used near-isogenic but epigenetically divergent parents to create epigenetic F1 hybrids (epiHybrids) in Arabidopsis, allowing us to quantify the contribution of epigenetics to heterosis. We measured traits such as leaf area (LA), growth rate (GR), flowering time (FT), main stem branching (MSB), rosette branching (RB) and final plant height (HT) and observed several strong positive and negative heterotic phenotypes among the epiHybrids. For LA and HT mainly positive heterosis was observed, while FT and MSB mostly displayed negative heterosis. Heterosis for FT, LA and HT could be associated with several heritable, differentially methylated regions (DMRs) in the parental genomes. These DMRs contain 35 (FT and LA) and 14 (HT) genes, which may underlie the heterotic phenotypes observed. In conclusion, our study indicates that epigenetic divergence can be sufficient to cause heterosis.Author SummaryCrossing two genetically distinct parents generates hybrid offspring. Sometimes hybrids are performing better than their parents in particular traits and this is referred to as heterosis. Hybridization and heterosis are naturally occurring processes and crop breeders intentionally cross genetically different parental lines in order to generate hybrids with maximized traits such as yield or stress tolerance. So far, the mechanisms behind heterosis are not well understood. In this study we focused on the effect of epigenetic variation onto heterosis in hybrids, and for this purpose we created epigenetic hybrids (epiHybrids) by crossing wildtype plants with a selection of genetically very similar but epigenetically divergent lines. An extensive phenotypic analysis of the epiHybrids and their parental lines showed that epigenetic divergence between parental genomes can be a major determinant of heterosis. Importantly, multiple heterotic phenotypes could be associated with meiotically heritable differentially methylated regions (DMRs) in the parental genomes, allowing us to map epigenetic quantitative trait loci (QTLs) for heterosis. Our results indicate that epigenetic variation can contribute to heterosis and suggests that heritable epigenetic variation could be exploited for the improvement of crop traits.

Higher eukaryotes typically contain many different cell types, displaying different cellular functions that are influenced by biotic and abiotic cues. The different functions are characterized by specific gene expression patterns mediated by regulatory sequences such as transcriptional enhancers. Recent genome-wide approaches have identified thousands of enhancers in animals, reviving interest in enhancers in gene regulation. Although the regulatory roles of plant enhancers are as crucial as those in animals, genome-wide approaches have only very recently been applied to plants. Here we review characteristics of enhancers at the DNA and chromatin level in plants and other species, their similarities and differences, and techniques widely used for genome-wide discovery of enhancers in animal systems that can be implemented 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.

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.

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.

Gene regulation in higher eukaryotes frequently involves physical interactions between genomic sequence elements tens of kilobases apart on the same chromosome but can also entail interactions between different chromosomes. Chromosome Conformation Capture (3C) is a powerful tool to identify such interactions. 3C technology is based on formaldehyde crosslinking of chromatin, followed by restriction digestion and intramolecular ligation. Quantitative detection of ligation products by PCR (qPCR; not discussed in this protocol) provides insight into the interaction frequencies between chromosomal fragments and thereby the spatial organization of a genomic region. Detailed 3C protocols have been published for yeast and mammals. However, these protocols cannot simply be transferred to plant tissues. In this paper, we provide a maize-specific 3C protocol and present a general strategy to systematically optimize the protocol for other plants. Once the technique and appropriate controls are established, the 3C procedure (including qPCR) can be completed in 5-7 d.

This work examines the involvement of chromatin looping in the transcriptional regulation of two epialleles of the maize (Zea mays) b1 gene, B-I and B'. These two epialleles are tissue-specifically regulated and are involved in paramutation. B-I and B' are expressed at high and low levels, respectively. A hepta-repeat ∼100 kb upstream of the transcription start site (TSS) is required for both paramutation and high b1 expression. Using chromosome conformation capture, we show that the hepta-repeat physically interacts with the TSS region in a tissue- and expression level–specific manner. Multiple repeats are required to stabilize this interaction. High b1 expression is mediated by a multiloop structure; besides the hepta-repeat, other sequence regions physically interact with the TSS as well, and these interactions are epiallele- and expression level–specific. Formaldehyde-assisted isolation of regulatory elements uncovered multiple interacting regions as potentially regulatory.

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.

Recombination mapping defined a 6-kb region, 100 kb upstream of the transcription start site, that is required for B-I enhancer activity and paramutation—a stable, heritable change in transcription caused by allele interactions in maize (Zea mays). In this region, B-I and B‘ (the only b1 alleles that participate in paramutation) have seven tandem repeats of an 853-bp sequence otherwise unique in the genome; other alleles have one. Examination of recombinant alleles with different numbers of tandem repeats indicates that the repeats are required for both paramutation and enhancer function. The 6-kb region is identical in B-I andB‘, showing that epigenetic mechanisms mediate the stable silencing associated with paramutation. This is the first endogenous gene for which sequences required for paramutation have been defined and examined for methylation and chromatin structure. The tandem repeat sequences are more methylated in B-I (high expressing) relative to B‘ (low expressing), opposite of the typical correlation. Furthermore, the change in repeat methylation follows establishment of the B‘ epigenetic state. B-I has a more open chromatin structure in the repeats relative to B‘. The nuclease hypersensitivity differences developmentally precede transcription, suggesting that the repeat chromatin structure could be the heritable imprint distinguishing the two transcription states.

The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post-transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not sufficient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.

Posttranscriptional silencing of chalcone synthase (Chs) genes in petunia transformants occurs by introducing T-DNAs that contain a promoter-driven or promoterless Chs transgene. With the constructs we used, silencing occurs only by T-DNA loci which are composed of two or more T-DNA copies that are arranged as inverted repeats (IRs). Since we are interested in the mechanism by which these IR loci induce silencing, we have analyzed different IR loci and nonsilencing single-copy (S) T-DNA loci with respect to the expression and methylation of the transgenes residing in these loci. We show that in an IR locus, the transgenes located proximal to the IR center are much more highly methylated than are the distal genes. A strong silencing locus composed of three inverted T-DNAs bearing promoterless Chs transgenes was methylated across the entire locus. The host Chs genes in untransformed plants were moderately methylated, and no change in methylation was detected when the genes were silenced. Run-on transcription assays showed that promoter-driven transgenes located proximal to the center of a particular IR are transcriptionally more repressed than are the distal genes of the same IR locus. Transcription of the promoterless Chs transgenes could not be detected. In the primary transformant, some of the IR loci were detected together with an unlinked S locus. We observed that the methylation and expression characteristics of the transgenes of these S loci were comparable to those of the partner IR loci, suggesting that there has been cross talk between the two types of loci. Despite the similar features, S loci are unable to induce silencing, indicating that the palindromic arrangement of the Chs transgenes in the IR loci is critical for silencing. Since transcriptionally silenced transgenes in IRs can trigger posttranscriptional silencing of the host genes, our data are most consistent with a model of silencing in which the transgenes physically interact with the homologous host gene(s). The interaction may alter epigenetic features other than methylation, thereby impairing the regular production of mRNA.

Summary To induce post-transcriptional silencing of flower pigmentation genes by homologous sense transgenes in transgenic petunias, it is not necessary for the transgenes to be highly transcribed. Even promoterless transgenes can induce silencing. Here it is shown that in these cases silencing is mediated by multimeric transgene/T-DNA loci in which the T-DNAs are arranged as inverted repeats (IRs). With the transgene constructs used, monomeric T-DNA loci are unable to confer silencing even though they modulate IR-induced silencing. IRsfs with the silencing sequences proximal to the centre (IRc) induce a more severe silencing than IRs with these sequences distal to the centre (IRn). Somatic reversion of silencing, as observed in a side branch of one of the chalcone synthase (Chs) transformants, was associated with a deletion of the IR locus from L1 cells, the meristematic cell layer that expresses the endogenous Chs genes in the flower corolla. Taken together, these data indicate that the post-transcriptional silencing mechanism can be activated by inverted transgene repeats. It is also shown that a silent IR UidA—ChsA locus silences the expression of a monomeric 35S promoter-driven UidA—ChsA transgene only in corollas where the endogenous Chs genes are highly transcribed. These results are consistent with a model in which an IR, by virtue of its palindromic sequence organization, is able to promote the production of aberrant RNAs from the endogenous homologs as a result of ectopic pairing.

In genetically modified plants, the introduced transgenes are sometimes not expressed. They can be silenced. Transgenes can also cause the silencing of endogenous plant genes if they are sufficiently homologous, a phenomenon known as co-suppression. Silencing occurs transcriptionally and post-transcriptionally but silencing of endogenous genes seems predominantly post-transcriptional. If viral transgenes are introduced and silenced, the post-transcriptional process also prevents homologous RNA viruses from accumulating; this is a means of generating virus-resistant plants. A major goal of current research is to dissect the mechanism(s) of these sequence-homology-dependent gene silencing phenomena. Various factors seem to play a role, including DNA methylation, transgene copy number and the repetitiveness of the transgene insert, transgene expression level, possible production of aberrant RNAs, and ectopic DNA–DNA interactions. The causal relationship between these factors and the link between transcriptional and post-transcriptional silencing is not always clear. In this review we discuss various observations associated with gene silencing and attempt to relate them.

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., Author Summary Paramutation is a fascinating process in which genes communicate to efficiently establish changes in their expression that are stably transmitted to future generations without any changes in DNA sequences. While paramutation was first described in the 1950s and extensively studied through the 1960s, its underlying mechanism remained mysterious for many years. Over the past ten years paramutation at the b1 locus in maize was shown to require transcribed, non-coding tandem repeats located 100 kb upstream of b1. These repeats generate small RNAs, and mutations in multiple genes mediating small RNA silencing at the transcriptional level prevent paramutation. While underlying mechanisms are shared, current models for RNA-mediated transcriptional silencing that are based on experiments with S. pombe and Arabidopsis do not explain many aspects of paramutation. In this manuscript we used a transgenic approach to demonstrate that the b1 non-coding tandem repeats are sufficient to send and respond to the paramutation signals and that this occurs even when the repeats are not at their normal chromosomal location.

Chimaeric PCaMV35S cry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5′ splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

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.

Epigenetic gene regulation involves the stable propagation of gene activity states through mitotic, and sometimes even meiotic, cell divisions without changes in DNA sequence. Paramutation is an epigenetic phenomenon involving changes in gene expression that are stably transmitted through mitosis as well as meiosis. These heritable changes are mediated by in trans interactions between homologous DNA sequences on different chromosomes. During these in trans interactions, epigenetic information is transferred from one allele of a gene to another allele of the same gene, resulting in a change in gene expression. Although paramutation was initially discovered in plants, it has recently been observed in mammals as well, suggesting that the mechanisms underlying paramutation might be evolutionarily conserved. Recent findings point to a crucial role for small RNAs in the paramutation process. In mice, small RNAs appear sufficient to induce paramutation, whereas in maize, it seems not to be the only player in the process. In this review, potential mechanisms are discussed in relation to the various paramutation phenomena.

Background Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality. Results We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches. Conclusion Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.

Paramutation is the result of heritable changes in gene expression that occur upon interaction between alleles. Whereas Mendelian rules, together with the concept of genetic transmission via the DNA sequence, can account for most inheritance in sexually propagating organisms,paramutation-like phenomena challenge the exclusiveness of Mendelian inheritance. Most paramutation-like phenomena have been observed in plants but there is increasing evidence for its occurrence in other organisms, including mammals. Our knowledge of the underlying mechanisms, which might involve RNA silencing, physical pairing of homologous chromosomal regions or both, is still limited. Here, we discuss the characteristics of different paramutation-like interactions in the light of arguments supporting each of these alternative mechanisms.(c) 2005 Elsevier Ltd.

Paramutation is a widespread epigenetic phenomenon that was first described in pea and then extensively studied in maize, whereby combining two specific alleles results in a heritable change in the expression of one of the alleles. Far from being restricted to endogenous plant genes, paramutation-like interactions have been described in several kingdoms, in which they can occur between homologous transgenes or between transgenes and homologous endogenous genes at allelic or non-allelic positions. In this review, we discuss potential mechanisms underlying paramutation, compare paramutation to several other trans-sensing phenomena, and speculate on the potential roles and evolutionary implications of these intriguing homology-sensing mechanisms.

This chapter focuses on the paramutation at two maize loci, b1 and p1. The chapter describes the recent experiments that have identified cis-acting regions required for paramutation at these two loci and compares the similarities and differences in the results. A model for the mechanism of paramutation is presented that incorporates these recent findings and discusses the possible functions for paramutation. Paramutation-like interactions can occur between two homologous transgenes, or a transgene and a homologous endogenous gene. At b1 , paramutation is fully penetrant and the resulting B ’ alleles are extremely stable, independent of how the alleles are maintained (homozygous or heterozygous with B-I or neutral alleles). Similar to the b1 paramutation, the paramutable P1-rr (red pericarp and red cob) allele can change spontaneously to a lower expression state, P1-pr (patterned pericarp and red cob). Models that attempt to explain paramutation must account for various features. Certain enhancer sequences are associated with the sequences required for paramutation in two different genes. The high-expressing alleles can be silenced spontaneously.

Introduction of antisense and sense transgenes into plants has been widely used to generate mutant phenotypes. Besides their use in fundamental research to determine gene function, antisense and sense transgenes are currently used to modify and improve crop plants. Since 1988, a large number of transgenic plants containing antisense and sense genes have been generated (see Van Blokland et al. 1993 for an overview). Despite the success of these approaches the mechanisms by which antisense and sense genes suppress gene activity are still poorly understood.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under control of the cauliflower (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers due to an inappropriate cell type-specificity of the 35S promoter within this organ. By inserting the anther box, a sequence homology found in other genes expressed in anthers, the expression pattern driven by this promoter was modified. Using this modified 35S promoter to drive antisense chs gene expression inhibition of anther pigmentation was realized. Transformants with white anthers are male sterile due to a defect in pollen development, indicating that flavonoids may have an essential function in microsporogenesis. Hence, blocking the synthesis of natural compounds during anther development provides a novel way to introduce nuclear male sterility.

Paramutation is an interaction between alleles that leads to a heritable change in the expression of one allele. In B′/B-I plants, B-I (high transcription) always changes to B′ (low transcription). The new B′ allele retains the low expression state in the next generation and paramutates B-I at a frequency of 100%. Comparisons of the structure and expression of B′ with that of a closely related allele that does not participate in paramutation demonstrated that transcription from the same promoter-proximal sequences is not sufficient for paramutation. Fine-structure recombination mapping localized sequences required for B′ expression and paramutation. The entire 110 kb upstream of the B′ transcription start site was cloned and sequenced and the recombination breakpoints were determined for 12 recombinant alleles. Sequences required for expression and paramutation mapped to distinct regions, 8.5-49 kb and 93-106 kb upstream of the B′ transcription start site, respectively. Sequencing and DNA blot analyses indicate that the B′ region required for paramutation is mostly unique or low copy in the maize genome. These results represent the first example of long-distance regulatory elements in plants and demonstrate that paramutation is mediated by long-distance cis and trans interactions.