Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots.

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips.

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.

Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.

Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

In vivo mapping of arabidopsis scaffold/matrix attachment regions reveals link to nucleosome-disfavoring poly(dA:dT) tracts.

Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function.

A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development.

The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation.

Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state.

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.

Dynamic localization of the DNA replication proteins MCM5 and MCM7 in plants.

Genome integrity in eukaryotes depends on licensing mechanisms that prevent loading of the minichromosome maintenance complex (MCM2-7) onto replicated DNA during S phase. Although the principle of licensing appears to be conserved across all eukaryotes, the mechanisms that control it vary, and it is not clear how licensing is regulated in plants. In this work, we demonstrate that subunits of the MCM2-7 complex are coordinately expressed during Arabidopsis (Arabidopsis thaliana) development and are abundant in proliferating and endocycling tissues, indicative of a role in DNA replication. We show that endogenous MCM5 and MCM7 proteins are localized in the nucleus during G1, S, and G2 phases of the cell cycle and are released into the cytoplasmic compartment during mitosis. We also show that MCM5 and MCM7 are topologically constrained on DNA and that the MCM complex is stable under high-salt conditions. Our results are consistent with a conserved replicative helicase function for the MCM complex in plants but not with the idea that plants resemble budding yeast by actively exporting the MCM complex from the nucleus to prevent unauthorized origin licensing and rereplication during S phase. Instead, our data show that, like other higher eukaryotes, the MCM complex in plants remains in the nucleus throughout most of the cell cycle and is only dispersed in mitotic cells.

In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research.

Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.

Gene targeting in plants: fingers on the move.

Zinc-finger endonucleases (ZFNs) make targeted double-stranded breaks in genomic DNA and, thus, stimulate recombination and repair processes at specific sites. ZFNs can now be harnessed to stimulate homologous recombination and gene targeting in plants, which represents a major step towards modifying the plant genome more precisely. ZFN-mediated gene targeting is likely to become a powerful tool for genome research and genetic engineering.

Matrix attachment regions and regulated transcription increase and stabilize transgene expression.

Transgene silencing has been shown to be associated with strong promoters, but it is not known whether the propensity for silencing is caused by the level of transcription, or some other property of the promoter. If transcriptional activity fosters silencing, then transgenes with inducible promoters may be less susceptible to silencing. To test this idea, a doxycycline-inducible luciferase transgene was transformed into an NT1 tobacco suspension culture cell line that constitutively expressed the tetracycline repressor. The inducible luciferase gene was flanked by tobacco Rb7 matrix attachment regions (MAR) or spacer control sequences in order to test the effects of MARs in conjunction with regulated transcription. Transformed lines were grown under continuous doxycycline (CI), or delayed doxycycline induction (DI) conditions. Delayed induction resulted in higher luciferase expression initially, but continued growth in the presence of doxycycline resulted in a reduction of expression to levels similar to those found in continuously induced lines. In both DI and CI treatments, the Rb7 MAR significantly reduced the percentage of silenced lines and increased transgene expression levels. These data demonstrate that active transcription increases silencing, especially in the absence of the Rb7 MAR. Importantly, the Rb7 MAR lines showed higher expression levels under both CI and DI conditions and avoided silencing that may occur in the absence of active transcription such as what would be expected as a result of condensed chromatin spreading.

Transgene integration: use of matrix attachment regions.

Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. When MARs are positioned at the 5'- and 3'-ends of a transgene higher more predictable expression of the transgene results. MARs are increasingly being applied to prevent unwanted transgene silencing, which is especially common when direct DNA transformation methods are used. This chapter describes methods for the isolation of MARs and the subsequent methods allowing the investigator to incorporate MARS into transformation strategies that can both improve transformation frequency and result in predictable, stable expression of the transgenic trait.

Analysis of trans-silencing interactions using transcriptional silencers of varying strength and targets with and without flanking nuclear matrix attachment regions.

We investigated the effect of the Rb7 matrix attachment region (MAR) on trans-silencing in tobacco plants, comparing the effects of three transgene silencer loci on ten target loci. Two of the silencer loci, C40 and C190, contain complex and rearranged transgene arrays consisting of 35S:GUS or NOS:NPTII containing plasmids. The third silencer locus, V271, was previously characterized as a complex locus containing rearranged 35S:RiN sequences. Each of these silencers can reduce 35S promoter-driven expression at other loci, albeit with varying efficiencies. The presence of MARs at a target locus does not prevent trans-silencing by the V271 silencer. However, four of seven MAR-containing loci were at least partially resistant to silencing by the C40 and C190 loci. One MAR locus was unaffected by C40, our weakest silencer, and three were silenced only when the silencer locus was maternally inherited. Silencing is progressive in the F1 and F2 generations; two days after germination there is little or no difference between seedlings derived from crosses to silencing or control lines, but seedlings containing silencer loci slowly lose expression during subsequent development. These observations are compatible with the hypothesis that a product of the silencer locus must accumulate before unlinked loci can be affected. However, our silencer loci are themselves silenced for GUS transcription, and coding region homology is not required for their effects on target loci. Our results are consistent with a model in which transcriptional silencing is triggered by transcription of sequences during the early stages of embryo or seedling development.

Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3' MAR.

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.

Differential Top10 promoter regulation by six tetracycline analogues in plant cells.

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.