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.

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.

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.

Genome-wide analysis of the core DNA replication machinery in the higher plants Arabidopsis and rice.

Core DNA replication proteins mediate the initiation, elongation, and Okazaki fragment maturation functions of DNA replication. Although this process is generally conserved in eukaryotes, important differences in the molecular architecture of the DNA replication machine and the function of individual subunits have been reported in various model systems. We have combined genome-wide bioinformatic analyses of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) with published experimental data to provide a comprehensive view of the core DNA replication machinery in plants. Many components identified in this analysis have not been studied previously in plant systems, including the GINS (go ichi ni san) complex (PSF1, PSF2, PSF3, and SLD5), MCM8, MCM9, MCM10, NOC3, POLA2, POLA3, POLA4, POLD3, POLD4, and RNASEH2. Our results indicate that the core DNA replication machinery from plants is more similar to vertebrates than single-celled yeasts (Saccharomyces cerevisiae), suggesting that animal models may be more relevant to plant systems. However, we also uncovered some important differences between plants and vertebrate machinery. For example, we did not identify geminin or RNASEH1 genes in plants. Our analyses also indicate that plants may be unique among eukaryotes in that they have multiple copies of numerous core DNA replication genes. This finding raises the question of whether specialized functions have evolved in some cases. This analysis establishes that the core DNA replication machinery is highly conserved across plant species and displays many features in common with other eukaryotes and some characteristics that are unique to plants.