Project description: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.

Project description: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. Replicating DNA in cultured Arabidopsis cells was labeled with BrdU for 1 hour. Cells were harvested, nuclei extracted, and the nuclei were sorted by FACS as Early, Mid or Late S phase based on DNA content (DAPI signal). Newly replicated DNA was isolated by immunoprecipitation to BrdU, amplified, and labeled with Cy5/Cy3. Input DNA served as a reference and was labeled with Cy3/Cy5. Equal amounts of DNA was hybridized to a spotted cDNA that covers Arabidopsis chr4. Each S phase sample has 3 biological replicates with a dye swap serving as a technical replicate for a total of 18 arrays.

Project description:Pooled CRISPR knock-out (KO) screens using live viruses are a proven and valuable approach to identify essential host factors acting across the viral life cycle. Here we describe the development of a pooled, genome-wide CRISPR KO screening approach using stable viral replicon cell lines to specifically target host genes essential for viral replication complex formation and function. We benchmarked this approach with dengue virus 2 (DENV2), a model virus with well-established replicon systems and knowledge of the host factors requirements for replication. We developed a stable fluorescent DENV2 replicon cell line to perform a pooled genome wide FACS-based CRISPR KO screen. Our benchmark DENV2 screen successfully identified previously known host factors involved in DENV replication such as components of the ER membrane complex (EMC) and oligosaccharyltransferase (OST) complex. In addition, 2 other genes identified in the screen (DOHH and ZFP36L2) were validated to impact DENV2 replication. We extended the replicon screening approach to two different classes of viruses - Chikungunya virus (CHIKV), a positive sense RNA virus that is known to replicate at the plasma membrane, and Zaire Ebola Virus (ZEBOV), a negative sense RNA virus that replicates in cytoplasmic inclusion bodies. The CHIKV replicon screen identified several important host factors involved in CHIKV replication, including CSDE1 and GOLGA7 that were successfully validated in the context of live CHIKV infection. A distinct set of host factors such as Euchromatic histone-lysine N-methyltransferase 1 and 2 (EHMT1, 2), and Ubiquitin specific peptidase 7 (USP7) were identified in the ZEBOV replicon screen and were further validated using independent transient transfection assays. Comparative analysis of the combined hits from all 3 screens reveals distinct subsets of host pathway requirements for each virus. Thus, viral replicons-based screens can be a useful tool that can be extended to diverse classes of viruses to identify host pathways essential for viral replication and to uncover potential novel targets for host-directed antiviral therapeutics.

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Replication Timing in Yeast: CGH microarrays: S vs G1 cells in various stress conditions

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