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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 ...

University of Amsterdam

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.

Paramutation: a heritable change in gene expression by allelic interactions in trans.

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.

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

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.

Cis-acting determinants of paramutation.

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.

Plant Enhancers: A Call for Discovery.

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.

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

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.

Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize.

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.

3C in Maize and Arabidopsis.

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 () 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.

Maike Stam

Swammerdam Institute for Life Sciences

University of Amsterdam

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